WO2023190098A1 - Examination kit and examination method for peanut allergen - Google Patents
Examination kit and examination method for peanut allergen Download PDFInfo
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- WO2023190098A1 WO2023190098A1 PCT/JP2023/011680 JP2023011680W WO2023190098A1 WO 2023190098 A1 WO2023190098 A1 WO 2023190098A1 JP 2023011680 W JP2023011680 W JP 2023011680W WO 2023190098 A1 WO2023190098 A1 WO 2023190098A1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Definitions
- the present invention relates to a technology for detecting peanut allergens in foods.
- Food allergy occurs when the body recognizes a specific protein (food allergen) contained in ingested food as a foreign substance, and various symptoms such as itching of the skin, hives, and cough are caused through a hypersensitive immunological mechanism. In severe cases, the symptoms can be life-threatening, such as loss of consciousness, drop in blood pressure, and shock symptoms. It is estimated that food allergy patients account for 1 to 2% of the total population (approximately 10% if limited to infants). To prevent food allergy sufferers from ingesting foods containing the food allergen that causes them, especially processed products that are difficult to distinguish visually, food hygiene requires that if a food contains ingredients that contain a food allergen, it should be labeled to that effect. Required or recommended by law.
- ⁇ Specified Ingredient'' includes ⁇ Abalone, squid, salmon roe, orange, cashew nut, kiwi fruit, beef, walnut, sesame, salmon, mackerel, soybean, chicken, banana, pork, matsutake mushroom, peach, yam, apple. , gelatin, and almonds (for which cases have been reported with a certain frequency in the past) are designated as ⁇ specified raw materials,'' which are recommended to be labeled.
- the Consumer Affairs Agency (formerly the Ministry of Health, Labor and Welfare) conducts a survey every three years to understand the actual state of food allergies nationwide, and reviews specific ingredients, etc. based on the results.
- kits to test whether food containing food allergens is included as an ingredient in processed products. has been done. Since allergic symptoms to peanuts tend to be severe, there is a need for kits that can detect peanut allergens in processed products to be even more reliable.
- ELISA methods that use antigen-antibody reactions as screening tests and PCR methods that use DNA amplification reactions as confirmatory tests as techniques for detecting specific raw materials, etc. contained in processed products.
- the ELISA method uses antibodies that recognize proteins unique to peanuts to test whether peanuts are included as a raw material in processed products.
- Peanut-specific proteins targeted for detection based on antigen-antibody reactions include peanut allergens Ara h1, Ara h2, and Ara h3, which are the main causes of peanut allergy.
- Ara h1 is a protein of the 7s globulin family with a molecular weight of 63 kDa, which accounts for approximately 12-16% of the total protein in peanut extract and causes sensitization in 35-95% of allergic patients.
- Ara h2 is a protein of the 2s globulin family with a molecular weight of 20 kDa, and in addition to causing systemic allergy, its trypsin inhibitor activity is further enhanced several times when treated at high temperatures, inhibiting the digestion of Ara h1. , further increases allergic activity.
- Ara h3 is a protein of the 11s globulin family with a molecular weight of 58 kDa, accounts for about 19% of the total protein in peanut extract, and causes systemic allergies.
- Other peanut allergens such as Ara h4, Ara h5, Ara h6, and Ara h7 are also known.
- Non-Patent Document 1 Indonesian Beauty Peanut is a special kind of peanut that does not have Ara h1, but it still contains other peanut-derived allergens (Ara h2, Ara h3, etc.), so the incidence of allergies is different from that of ordinary peanuts. It has been reported that there was no such problem (Non-Patent Document 1).
- Detection kits or detection methods using monoclonal antibodies targeting Ara h1 protein have poor detection sensitivity when the concentration of Ara h1 protein in the food sample is low, but on the other hand, the detection sensitivity is poor when the concentration of Ara h1 protein in the food sample is high. There was a problem that sometimes a false negative result could not be detected.
- the food is manufactured using Ara h1-deficient peanuts, peanut allergens cannot be detected depending on the detection kit or detection method using monoclonal antibodies targeting Ara h1 protein, and Ara h2, Ara h3 This is a big problem for people with protein allergies.
- the content of Ara h1 the main allergenic protein in peanuts, increases or decreases with heating, there is a problem in that it is difficult to detect peanuts with an Arah1 test kit.
- An object of the present invention is to provide improved means for detecting peanut allergens in foods.
- the present inventors When the present inventors used an anti-Ara h3 antibody to detect Ara h3 protein in a peanut allergen detection kit or detection method, the present inventors unexpectedly found that the Ara h1 protein was detected using an anti-Ara h1 antibody. Compared to the detection target, the detection sensitivity is superior even when the allergen protein in a food sample is at a low concentration, and even when the allergen protein is at a high concentration in a food sample, it can be detected without false negatives.
- the present inventors have discovered that there are advantages and have completed the present invention. When anti-Ara h3 antibodies are used, there is also the advantage that peanut allergens contained in foods manufactured using Ara h1-deficient peanuts can be detected.
- a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or in food processing equipment using an immunoassay method, comprising anti-Ara h3 antibody or its antigen-binding fragment comprising anti-Ara h3 antibody or its antigen-binding fragment (hereinafter referred to as " A test kit containing an anti-Ara h3 antibody, etc.).
- a test kit containing an anti-Ara h3 antibody, etc. The test according to item 1, wherein the immunoassay method is ELISA, and the anti-Ara h3 antibody, etc. includes an anti-Ara h3 antibody, etc. for immobilization, and an anti-Ara h3 antibody, etc. for enzyme labeling. kit for.
- Item 2 Item 2.
- the test kit according to item 1 wherein the immunoassay method is immunochromatography, and the anti-Ara h3 antibody and the like include an anti-Ara h3 antibody and the like for chromogenic labeling.
- the immunoassay method is immunochromatography
- the anti-Ara h3 antibody and the like include an anti-Ara h3 antibody and the like for chromogenic labeling.
- the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
- the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
- Item 6 The test kit according to any one of Items 1 to 5, further comprising an extraction reagent.
- Item 7 The test kit according to Item 6, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
- the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
- the immunoassay is ELISA, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody or the like for immobilization and an anti-Ara h3 antibody or the like for enzyme labeling.
- Item 9 The testing method according to item 8, wherein the immunoassay is immunochromatography, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody for chromogenic labeling.
- 11 The testing method according to any one of Items 8 to 10, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
- 12 12.
- Item 13 Item 13.
- Item 14 The testing method according to any one of Items 8 to 12, wherein the extraction in step (1) is performed using an extraction reagent.
- Item 14 The testing method according to any one of Items 8 to 13, wherein the extraction in step (1) is performed by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
- the present invention it becomes possible to detect peanut allergen with higher reliability than before, regardless of whether the peanut allergen in food is at a low concentration or a high concentration.
- FIG. 1 shows the results of Western blotting on SDS-PAGE as described in Example 1.
- test kit is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, and is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, fragments (referred to herein as "anti-Ara h3 antibodies, etc.”).
- the anti-Ara h3 antibody etc. may be any antibody or antigen-binding fragment thereof that recognizes and specifically binds to an epitope present on the Ara h3 protein, and its amino acid sequence (full length of heavy chain and light chain, variable The region (complementarity determining region: CDR, framework region: FR), constant region, or a fragment thereof) is not particularly limited.
- the "antigen-binding fragment” include Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody (bispecific antibody), nanobody, and the like.
- antibody in this specification will be read as “antigen-binding fragment” or “antibody, etc.” In other words, embodiments using “antibody” will be replaced with “antigen-binding fragment” or “antibody, etc.” It can be replaced depending on the embodiment used.
- the anti-Ara h3 antibody and the like may be any antibody or antigen-binding fragment thereof that can recognize and bind to the Ara h3 protein with the specificity required depending on its use. It is preferable that anti-Ara h3 antibodies etc. do not bind to various proteins contained in foods and food allergens other than peanuts (at least, the binding remains to a level that does not affect the test results according to the present invention), but depending on the use and degree of Accordingly, binding (cross-reaction) to a certain degree with proteins other than the Ara h3 protein and nonspecific adsorption to proteins or other substances that are unavoidable due to the nature of antibodies are allowed.
- Anti-Ara h3 antibodies etc. may be used alone or in combination of two or more depending on the purpose and embodiment of the test kit.
- polyclonal antibodies or their antigen binding It may be a monoclonal antibody or an antigen-binding fragment thereof (referred to herein as a "monoclonal antibody, etc.”).
- monoclonal antibody a monoclonal antibody or an antigen-binding fragment thereof.
- specificity does it react only with Ara h3 protein and do not react (cross) with other proteins?
- reproducibility does the test result differ depending on the manufacturing lot
- the preferred antibody may vary depending on the use and embodiment of the test kit because of differences in stability (whether binding to the antigen is lost due to immobilization treatment, labeling treatment, etc. of the antibody in the kit), etc. Taking this into account, it is possible to select whether to use polyclonal antibodies or monoclonal antibodies, or to use a combination of both.
- the Ara h3 protein in foods such as processed foods is determined by the manufacturing process and storage process of the food (e.g., the presence or absence of kneading, molding, pressurization, heating, drying, enzyme treatment, freezing, thawing, etc., and the processing conditions).
- the Ara h3 proteins may contain Ara h3 proteins that have been variously denatured due to the extraction process from food (sample) (e.g., the presence or absence of extraction treatment using an extraction reagent containing a solubilizing agent and the treatment conditions). It may be a collection of denatured Ara h3 protein and variously denatured Ara h3 proteins. Therefore, the anti-Ara h3 antibodies etc. of the present invention have a certain detection sensitivity for aggregates of various Ara h3 proteins in foods (preventing detection sensitivity from decreasing depending on the food). Preferred embodiments are those capable of binding to h3 protein.
- a test kit such as an anti-Ara h3 antibody
- an anti-Ara h3 antibody is specific for both native Ara h3 protein and denatured Ara h3 protein (e.g., heat-denatured Ara h3 protein).
- denatured Ara h3 protein e.g., heat-denatured Ara h3 protein
- the test kit includes a polyclonal antibody or the like as the anti-Ara h3 antibody or the like.
- the detection kit includes both polyclonal antibodies and monoclonal antibodies as anti-Ara h3 antibodies and the like.
- a monoclonal antibody or the like is used as a capture antibody (immobilization antibody in ELISA, immobilization antibody in immunochromatography) against Ara h3 protein
- the captured A polyclonal antibody or the like can be used as a detection antibody (enzyme labeling antibody in ELISA, gold colloid labeling or latex particle labeling antibody in immunochromatography) against the Ara h3 protein.
- the anti-Ara h3 antibodies used in the present invention are generally extracted from foods according to conventional methods and are usually further purified from food allergens (in the present invention, Ara h3 protein ) can be used to produce polyclonal antibodies or monoclonal antibodies.
- Common methods for producing polyclonal antibodies include methods using immunized animals and phage display methods.
- the outline of the procedure using the immunized animal is as follows. An appropriate amount of purified target protein (Ara h3 protein in the present invention) is mixed with an adjuvant as necessary to prepare an immunogen, and the immunogen is used to infect animals (rabbits, guinea pigs, goats, sheep, rats, mice, chickens). etc.), antibodies can be produced in the blood of that animal. After repeated immunization at appropriate intervals and times, blood (plasma, serum) is collected and the antibodies contained therein are purified. For example, affinity using an affinity column on which a target protein (Ara h3 protein in the present invention) is immobilized.
- a polyclonal antibody is obtained by purification by chromatography and, if necessary, further gel filtration chromatography. Moreover, the outline of the procedure of the phage display method is as follows. An antibody gene is introduced into a bacteriophage, and a protein in which the H chain and L chain variable regions are linked is expressed (displayed) on the coat protein of the bacteriophage. Using the obtained antibody phage library, antibodies having affinity for the target protein (Ara h3 protein in the present invention) are selected. Polyclonal antibodies can be obtained by infecting Escherichia coli with bacteriophages that produce the antibodies, multiplying them, collecting them, and purifying the antibodies contained therein.
- a common method for producing monoclonal antibodies is to use hybridomas.
- the outline of the method using hybridoma is as follows. In the same manner as when producing polyclonal antibodies, an immunogen is injected into an animal and antibodies are produced. B cells are collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells). Among the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein (Ara h3 protein in the present invention) are selected (screened). The hybridoma is cultured to produce a single antibody in the culture supernatant. Monoclonal antibodies can be obtained by collecting the culture supernatant and purifying the antibodies contained therein.
- an anti-Ara h3 antibody (polyclonal or monoclonal) is obtained, its amino acid sequence can be determined, and various antigen-binding fragments (polyclonal or monoclonal) can be produced according to conventional methods.
- the anti-Ara h3 antibody etc. may be in a form suitable for quantitatively or qualitatively detecting the Ara h3 protein by immunoassay, for example, it may be subjected to various modifications or alterations. can.
- Immunological assay is particularly limited if it is a method that makes it possible to quantitatively or qualitatively detect Ara h3 protein by utilizing the reaction between Ara h3 protein and anti-Ara h3 antibodies, etc.
- Various known methods can be used.
- Test kits compatible with various immunoassay methods are also known and commercially available, and the test kit of the present invention can also be modified to use the specific one of the present invention as an extraction reagent. Other than this, it can be manufactured according to known test kits.
- the immunoassay is ELISA (Enzyme-Linked Immuno Sorbent Assay).
- ELISA is capable of quantitatively detecting various proteins with high accuracy, is a suitable measurement method for detecting food allergens from processed products such as heating, and complies with the Consumer Affairs Agency guidelines.
- Anti-Ara h3 antibodies for ELISA include, for example, anti-Ara h3 antibodies for immobilization and anti-Ara h3 antibodies for enzyme labeling.
- the anti-Ara h3 antibody or the like for immobilization is intended to be immobilized on the surface of a member for performing ELISA, for example, on the bottom of a plate (well).
- Anti-Ara h3 antibodies for enzyme labeling bind to Ara h3 protein captured by immobilized antibodies, and then react with a substrate to develop color with an intensity that corresponds to the amount of Ara h3 protein. It is something. Anti-Ara h3 antibodies etc.
- H3 antibody etc. may be used, but anti-Ara H3 antibody etc. which will be indirectly labeled with an enzyme, for example, an enzyme bound to streptavidin as exemplified in the procedure described later in relation to the "test method". It may also be an anti-Ara h3 antibody bound to biotin, which can be further reacted to complete an enzyme-labeled anti-Ara h3 antibody.
- the immunoassay method is immunochromatography.
- Immunochromatography is a measurement method that allows qualitative detection of various proteins in a relatively short time using simple operations.
- Anti-Ara h3 antibodies for immunochromatography include, for example, anti-Ara h3 antibodies for immobilization and anti-Ara h3 antibodies for chromogenic labels (e.g., colloidal gold labels, latex particle labels, platinum particle labels).
- Anti-Ara h3 antibodies and the like for chromogenic labeling are generally anti-Ara h3 antibodies and the like that are pre-bound to a chromogenic label and are contained in a predetermined position (sample dropping part) of a test strip, and are used in combination with Ara h3 protein.
- the anti-Ara h3 antibody for immobilization is generally included in a predetermined position (test line) of the test strip, and is used to bind the Ara h3 protein that has migrated by capillary action and the coloring label. This is an antibody for capturing complexes with anti-Ara h3 antibodies, etc.
- the test kit of the present invention may contain antibodies other than anti-Ara h3 antibodies, etc., as necessary or within a range that does not inhibit the effects of the present invention.
- the test kit of the present invention targets proteins other than Ara h3 (e.g., peanut allergens other than Ara h3, or food allergens other than peanut allergen) as the above-mentioned capture antibodies and detection antibodies. It may further contain antibodies and the like.
- the test kit of the present invention contains at least an anti-Ara h3 antibody and the like, but can also contain arbitrary contents such as an extraction reagent, other reagents, and members as necessary depending on the embodiment. If it is a kit for ELISA, 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, and a kit for stopping the enzyme reaction in ELISA. BSA to protect the extracted proteins from degradation, instruction manuals that describe the procedure for testing by ELISA, etc., as well as anti-Ara h3 antibodies for immobilization and enzyme labeling, and extraction reagents. It can also be included in the contents of the kit.
- a kit for ELISA 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, and a kit for stopping the enzyme reaction in ELISA.
- a kit for immunochromatography includes, for example, a test strip that can develop various solutions and reagents by capillary action, a diluent for preparing a target protein solution, and instructions for testing by immunochromatography. Instruction manuals and the like can be included in the contents of the kit, together with anti-Ara h3 antibodies for immobilization and color labeling, and extraction reagents.
- Extraction reagent refers to a reagent (solution) that is commonly used to extract various proteins in foods, such as food allergens.
- extraction reagents Various types of extraction reagents are known, and similar extraction reagents can be used in the present invention.
- An extraction reagent is generally a solution prepared by adding a "solubilizer" as an agent for extracting various proteins in foods to a buffer solution having an appropriate pH.
- Examples of the buffer for preparing the extraction reagent include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer.
- Tris buffer and phosphate buffer are preferable.
- the pH of the buffer solution is generally 4.5 to 8.0 (pH within the range that can occur in vivo), for example 6.0 to 8.0.
- Those skilled in the art can prepare a buffer solution having a desired concentration and a desired pH by using appropriate amounts of appropriate compounds (adding and dissolving appropriate amounts of multiple compounds in water). .
- solubilizing agents for preparing extraction reagents include surfactants, chaotropic agents, and reducing agents.
- the extraction reagent may contain any one kind alone, or may contain a combination of a plurality of kinds.
- surfactant examples include anionic surfactants, cationic surfactants, and nonionic surfactants.
- anionic surfactants include alkyl sulfates and alkylbenzenesulfonates.
- Alkyl in alkyl sulfates, alkylbenzenesulfonates, etc. is, for example, dodecyl, decyl, nonyl, octyl.
- Salts such as alkyl sulfates and alkylbenzenesulfonates are, for example, sodium salts, potassium salts, and ammonium salts.
- alkyl sulfates include sodium dodecyl sulfate (SDS), and specific examples of alkylbenzenesulfonates include sodium dodecylbenzenesulfonate.
- alkyl sulfates include sodium dodecyl sulfate (SDS), and specific examples of alkylbenzenesulfonates include sodium dodecylbenzenesulfonate.
- Specific examples of cationic surfactants include hexadecylpyridinium chloride, hexadecyltrimethylammonium bromide, and hexadecyltrimethylammonium chloride.
- 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).
- the concentration of the surfactant depends on the type of surfactant and its effects, as well as other components (e.g. chaotropic agent, reducing agent) in the extraction reagent as necessary. It can be adjusted as appropriate by considering the presence or absence, type, concentration, etc.
- the extraction reagent contains an anionic surfactant such as SDS or other surfactant as a surfactant
- the lower limit of the concentration is, for example, 0.005, 0.01 or 0.05% ( (w/v)
- the upper limit can be, for example, 5.0, 3.0 or 2.0% (w/v), and these upper and lower limits can be arbitrarily combined. be able to.
- chaotropic agents include urea, formamide, salts containing anions or cations with a salt-soluble effect (e.g., guanidine hydrochloride containing guanidinium ions, sodium chloride containing sodium ions, potassium chloride containing potassium ions). etc.).
- the concentration of the chaotropic agent depends on the type of chaotropic agent and its effects (influence on the effects of the present invention), and, if necessary, other components in the extraction reagent (e.g. It can be adjusted as appropriate by considering the presence or absence, type, and concentration of surfactants and reducing agents.
- the lower limit of the concentration can be, for example, 0.01, 0.05, or 0.1 (w/v)
- the upper limit is, for example, 10 , 5.0 or 3.0% (w/v), and these upper and lower limits can be arbitrarily combined.
- Examples of the reducing agent include mercaptoalkanol and/or sulfite.
- Examples of the mercaptoalkanol include thioglycerol, 2-mercaptoethanol, and dithiothreitol.
- Examples of sulfites include alkali metal sulfites, and examples of the alkali metals include sodium and potassium. Specific examples of sulfites include sodium sulfite.
- the concentration of the reducing agent depends on the type of reducing agent and its effects, as well as the presence or absence of other components (e.g. surfactants, chaotropic agents) in the extraction reagent as necessary. It can be adjusted as appropriate by considering the type, concentration, etc.
- 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)
- 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 arbitrarily combined.
- the lower limit of its concentration can be, for example, 0.05, 0.1 or 0.5% (w/v)
- 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.
- antibody or “antibody etc.” (antibodies and antigen-binding fragments thereof) is further expanded to include various known “detection molecules” capable of binding to proteins (embodiment). ) is possible.
- detection molecules other than antibodies and antigen-binding fragments (antibodies, etc.) include aptamers, receptors, antibacterial peptides, and other peptides.
- the present invention can also be practiced by embodiments based on specific reactions using detection molecules.
- the "inspection method” of the present invention is a method for testing peanut allergens remaining in food or food processing equipment, and includes (1) a step of extracting peanut protein remaining in food or food processing equipment ( (herein referred to as “extraction step”), and (2) quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunoassay method using anti-Ara h3 antibodies etc. (referred to herein as a “detection step").
- Food may be peanuts themselves as raw materials (for example, fresh peanuts, dried peanuts, roasted peanuts, boiled peanuts), or processed products that contain peanuts as raw materials (which are subject to inspection). Good too.
- the form of the food (processed product) is not particularly limited, but for example, allergenic proteins are generally insolubilized and difficult to extract by being manufactured through a process that is performed under heating and/or pressurized conditions. It may also be a processed product that tends to be in a state.
- the food may be, for example, solid, semi-solid, jelly, liquid, or emulsified.
- the "immunological assay” is not particularly limited as long as it can detect proteins such as food allergens extracted from foods, and can be selected from various known methods.
- the immunoassay in the detection step is ELISA.
- ELISA can be carried out using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto, for example, according to the following procedure. 1) Add a solution of the target protein to the wells of the plate containing the immobilized antibody, bring the immobilized antibody etc. into contact with the target protein, and bind by antigen-antibody reaction to form the first complex. Form.
- a substrate solution (coloring agent) is added to cause the enzyme in the third complex to react with the substrate to develop color.
- a substrate solution coloring agent
- the immunoassay in the detection step is immunochromatography.
- Immunochromatography can be performed, for example, by the following procedure using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto. 1) Drop a sample solution containing the target protein onto a predetermined area (sample dropping part) on the test strip that contains the color-labeled antibody, etc., bring the color-labeled antibody, etc. into contact with the target protein, and cause an antigen-antibody reaction. A first complex is formed by binding the first complex. 2) The sample solution containing the first complex, unreacted color-labeled antibody, etc.
- the first complex, etc. is also spread due to capillary action.
- a line colored by a coloring label for example, a gold A reddish-purple line due to colloid. If a test line appears, it indicates that the target protein was contained in the sample solution.
- a coloring label for example, a gold A reddish-purple line due to colloid
- control line area downstream of the test line
- anti-immunoglobulin antibodies etc.
- a reddish-purple line appears due to colloidal gold. If the control line does not appear, it is suggested that there was an abnormality in the development of the sample solution, regardless of whether the sample contained the target protein (retesting is required).
- extraction process can be performed in the same way as a general extraction process, which is usually performed as a process to extract various proteins in foods using extraction reagents. It can be modified as appropriate to suit the present invention.
- the food sample to be subjected to the extraction process must be micronized or emulsified into a homogeneous state by high-speed shearing and stirring using a food cutter, miller, mixer, homogenizer, etc., depending on the form of the food (high-speed shearing and stirring).
- treatment method Such treatment such as micronization may be performed on the food, etc. in advance before the extraction process, and the resulting micronized food and extraction reagent may be mixed, or the food and extraction reagent may be mixed.
- the extraction process may be performed on a mixture of reagents for use, and the extraction process may be performed simultaneously with the process such as micronization.
- Processing conditions such as time, temperature, number of revolutions (rpm), centrifugal force (xg), etc. will vary depending on the selected processing method for micro-refining etc. and the processing equipment used, and will also affect the effects of the present invention. It can be adjusted accordingly.
- the food to be subjected to the extraction step may be further subjected to a treatment, such as a defatting treatment, in consideration of extraction and/or post-extraction detection of the Ara h3 protein, if necessary.
- the extraction process can also be performed by mixing the food that has been or has not been micronized as described above and the extraction reagent and then shaking the mixture (shaking method).
- the shaking processing conditions time, temperature, shaking number, rotation speed (rpm), etc.
- the duration of the shaking treatment is usually 12 hours or more and less than 24 hours (for example, overnight).
- the temperature of the shaking treatment is usually room temperature, but may be heated if necessary. In the present invention, it is possible to detect Ara h3 protein at a low concentration with high sensitivity and to detect a high concentration Ara h3 protein without causing a false negative, even without heating in the extraction process (shaking process). can.
- Example 1 Production of immunochromatography kit for detecting peanut allergen (Ara h3 protein) [1-1] Production of anti-Ara h3 monoclonal antibody Raw peanuts were ground into powder and prepared for defatting. Stirred in hexane and then in acetone. After repeating the stirring in hexane and acetone three more times (four times in total), the mixture was air-dried to obtain a sample of raw peanut powder (specimen A).
- a common method for producing monoclonal antibodies is to use hybridomas.
- the outline of the method for obtaining the monoclonal antibody of this example using a hybridoma is as follows.
- an immunizing antigen in this example, a mixture of equal amounts of the above-mentioned unheated specimen and heated specimen
- B cells were collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells).
- the anti-Ara h3 monoclonal antibody obtained in [1-1] above includes both purified undenatured Ara h3 protein and purified heat-denatured Ara h3 protein. This included those that were confirmed to specifically bind to (data not shown).
- One type of such specific anti-Ara h3 antibodies was used as an anti-Ara h3 antibody for immobilization to create a test strip, and another type was used as a coloring label to create a colloidal gold-labeled antibody. It was used as an anti-Ara h3 antibody.
- an antibody (anti-mouse IgG antibody) against anti-Ara h3 antibody for chromogenic labeling which was prepared according to a conventional method, was also used.
- a colloidal gold-labeled antibody was produced using a color-labeling anti-Ara h3 antibody according to a conventional method. Using this colloidal gold-labeled antibody, anti-Ara h3 antibody for immobilization, and common parts for test strips including a sample dropping part, developing part, absorbent pad, etc., the reagent-containing part is coated with gold according to a conventional method.
- a test strip was prepared in which a colloid labeled antibody was placed, an anti-Ara h3 antibody for immobilization was placed at the position where the test line appeared, and an antibody against the anti-Ara h3 antibody for chromogenic labeling was placed at the position where the control line appeared.
- a colloidal gold-labeled antibody was prepared by binding an anti-Ara h3 antibody for chromogenic labeling to colloidal gold according to a conventional method.
- These preparations constituted an immunochromatography kit for detecting peanut protein (allergen) targeting Ara h3 protein, and was used in Example 2.
- Example 2 Detection of peanut allergen (Ara h3 protein) by immunochromatography
- two specific types obtained in [1-1] of Example 1 were used as antibodies for detection of peanut allergen. (for immobilization and enzyme labeling) included in a commercially available kit using an anti-Ara h3 monoclonal antibody (hereinafter referred to as the "antibody of the present invention") and an anti-Arah1 antibody (hereinafter referred to as the "control product").
- An anti-Ara h1 monoclonal antibody hereinafter referred to as "control antibody” is used.
- test sample a 1 g each of raw peanut powder sample (sample a) and peanut butter sample (sample b) was mixed with the sample extract contained in the control product (8 volumes of purified water). To the mixture, 19 mL (prepared by adding and stirring 1 volume each of the first extract and the second extract) was added, thoroughly stirred, and then heated in a boiling water bath for 10 minutes. Each solution was filtered through a filter paper, and the obtained filtrate (undiluted solution) was used as a test sample (test sample a1, test sample b1).
- Test sample a1 and test sample b1 were each dropped onto the sample dropping portion of the test strip of the kit produced in Example 1 (hereinafter referred to as "the product of the present invention"). After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
- test sample a1 and test sample b1 were each dropped onto the sample dropping portion of a control test strip. After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
- test sample a2 with an Ara h3 protein concentration of 500 ng/mL, a test sample a3 with an Ara h3 protein concentration of 100 ng/mL, etc. were prepared by serial dilution.
- Test sample a2 and test sample a3 were dropped onto the sample dropping portions of the test strips of the present invention product and the control product, respectively. After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
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Abstract
An examination kit according to the present invention, which provides an improved means for detecting a peanut allergen remaining in a food product or on a food product processing device, is for quantitatively or qualitatively detecting a peanut allergen remaining in a food product or on a food product processing device by an immunoassay method and includes an anti-Ara h 3 antibody or an antigen binding fragment (anti-Ara h 3 antibody or the like) therefor. An examination method for a peanut allergen in a food product according to the present invention includes: (1) a step for extracting peanut proteins remaining in a food product or on a food product processing device; and (2) a step for quantitatively or qualitatively detecting the Ara h 3 protein in the obtained extract by an immunoassay method using an anti-Ara h 3 antibody or the like.
Description
本発明は、食品中の落花生アレルゲンを検出するための技術に関する。
The present invention relates to a technology for detecting peanut allergens in foods.
食物アレルギーは、身体が摂取した食物に含まれる特定のタンパク質（食物アレルゲン）を異物として認識し、過敏な免疫学的機序を介して、皮膚のかゆみ、じんましん、せきなど様々な症状が引き起こされる症状であり、重症の場合は、意識の喪失、血圧低下・ショック症状など、生命にかかわる深刻なものとなる。食物アレルギーの患者は、全人口の１～２％（乳児に限定すると約１０％）になると推定されている。食物アレルギーの患者が、原因となる食物アレルゲンを含む食品、特に外観的に判別しにくい加工品を摂取しないよう、食物アレルゲンを含む原材料を食品が含む場合は、その旨を表示することが食品衛生法により義務または推奨とされている。現在、「卵、乳、小麦、えび、かに」（発症件数が多いもの）および「そば、落花生」（症状が重くなることが多く、生命に関わるもの）の７品目は、表示義務がある「特定原材料」に指定されており、「あわび、いか、いくら、オレンジ、カシューナッツ、キウイフルーツ、牛肉、くるみ、ごま、さけ、さば、大豆、鶏肉、バナナ、豚肉、まつたけ、もも、やまいも、りんご、ゼラチン、アーモンド」（過去に一定の頻度で発症が報告されたもの）の２１品目は、表示が推奨されている「特定原材料に準ずるもの」に指定されている。消費者庁（以前は厚生労働省）は、食物アレルギーの全国的な実態把握のために３年毎に調査を行っており、その結果を踏まえて特定原材料等の見直しを行っている。
Food allergy occurs when the body recognizes a specific protein (food allergen) contained in ingested food as a foreign substance, and various symptoms such as itching of the skin, hives, and cough are caused through a hypersensitive immunological mechanism. In severe cases, the symptoms can be life-threatening, such as loss of consciousness, drop in blood pressure, and shock symptoms. It is estimated that food allergy patients account for 1 to 2% of the total population (approximately 10% if limited to infants). To prevent food allergy sufferers from ingesting foods containing the food allergen that causes them, especially processed products that are difficult to distinguish visually, food hygiene requires that if a food contains ingredients that contain a food allergen, it should be labeled to that effect. Required or recommended by law. Currently, seven items are required to be labeled: "eggs, milk, wheat, shrimp, and crab" (those that cause a large number of cases) and "buckwheat and peanuts" (those that often cause severe symptoms and are life-threatening). It is designated as a ``Specified Ingredient'' and includes ``Abalone, squid, salmon roe, orange, cashew nut, kiwi fruit, beef, walnut, sesame, salmon, mackerel, soybean, chicken, banana, pork, matsutake mushroom, peach, yam, apple. , gelatin, and almonds (for which cases have been reported with a certain frequency in the past) are designated as ``specified raw materials,'' which are recommended to be labeled. The Consumer Affairs Agency (formerly the Ministry of Health, Labor and Welfare) conducts a survey every three years to understand the actual state of food allergies nationwide, and reviews specific ingredients, etc. based on the results.
上記のような食物アレルゲンに関する制度の運用のために、食品メーカーや公的な検査機関等において、食物アレルゲンを含む食品が加工品中に原材料として含まれていないか、キット等を使用して検査されている。落花生に対するアレルギー症状は重篤なものとなる傾向にあることから、加工品中の落花生アレルゲンを検出することのできるキットにとって、より一層の信頼性の向上などが要求されている。
In order to operate the food allergen-related system mentioned above, food manufacturers and public inspection institutions use kits to test whether food containing food allergens is included as an ingredient in processed products. has been done. Since allergic symptoms to peanuts tend to be severe, there is a need for kits that can detect peanut allergens in processed products to be even more reliable.
加工品中に含まれる特定原材料等の検出技術としては、一般的に、スクリーニング検査として抗原抗体反応を用いたＥＬＩＳＡ法、および確定検査としてＤＮＡの増幅反応を用いたＰＣＲ法が存在している。このうちＥＬＩＳＡ法は、落花生特有のタンパク質を認識する抗体を用いることで、加工品中に落花生が原材料として含まれているかどうかを検査する。
Generally speaking, there are ELISA methods that use antigen-antibody reactions as screening tests and PCR methods that use DNA amplification reactions as confirmatory tests as techniques for detecting specific raw materials, etc. contained in processed products. Among these, the ELISA method uses antibodies that recognize proteins unique to peanuts to test whether peanuts are included as a raw material in processed products.
抗原抗体反応に基づく検出の標的とする落花生特有のタンパク質としては、落花生アレルギーの主な原因である、落花生アレルゲンＡｒａ ｈ１、Ａｒａ ｈ２、Ａｒａ ｈ３などが知られている。Ａｒａ ｈ１は、分子量６３ｋＤａの、７ｓグロブリンファミリーのタンパク質であり、ピーナッツ抽出物の総タンパク質の約１２～１６％を占め、このアレルギー患者の３５～９５％で感作を引き起こす。Ａｒａ ｈ２は、分子量２０ｋＤａの、２ｓグロブリンファミリーのタンパク質であり、全身アレルギーを引き起こすことに加えて、高温で処理されたときにはトリプシンインヒビター活性がさらに数倍増強され、Ａｒａ ｈ１の消化を阻害することで、さらにアレルギー活性が高まる。Ａｒａ ｈ３は、分子量５８ｋＤａの、１１ｓグロブリンファミリーのタンパク質であり、ピーナッツ抽出物の総タンパク質の約１９％を占め、全身アレルギーを引き起こす。その他、Ａｒａ ｈ４、Ａｒａ ｈ５、Ａｒａ ｈ６、Ａｒａ ｈ７などの落花生アレルゲンも知られている。
Peanut-specific proteins targeted for detection based on antigen-antibody reactions include peanut allergens Ara h1, Ara h2, and Ara h3, which are the main causes of peanut allergy. Ara h1 is a protein of the 7s globulin family with a molecular weight of 63 kDa, which accounts for approximately 12-16% of the total protein in peanut extract and causes sensitization in 35-95% of allergic patients. Ara h2 is a protein of the 2s globulin family with a molecular weight of 20 kDa, and in addition to causing systemic allergy, its trypsin inhibitor activity is further enhanced several times when treated at high temperatures, inhibiting the digestion of Ara h1. , further increases allergic activity. Ara h3 is a protein of the 11s globulin family with a molecular weight of 58 kDa, accounts for about 19% of the total protein in peanut extract, and causes systemic allergies. Other peanut allergens such as Ara h4, Ara h5, Ara h6, and Ara h7 are also known.
そのような落花生特有のタンパク質を対象として、抗原抗体反応により食品中の落花生を検出するための手段としては、例えば、落花生アレルゲン未変性Ａｒａ ｈ１タンパク質および加熱変性Ａｒａ ｈ１タンパク質を認識する抗Ａｒａ ｈ１タンパク質モノクローナル抗体を用いて、ＥＬＩＳＡ等により検出する方法およびそのためのキットが提案されている（特許文献１）。
As a means for detecting peanuts in foods by antigen-antibody reaction targeting such peanut-specific proteins, for example, anti-Ara h1 protein that recognizes the peanut allergen undenatured Ara h1 protein and heat-denatured Ara h1 protein is used. A method of detection by ELISA or the like using a monoclonal antibody and a kit therefor have been proposed (Patent Document 1).
また、インドネシア産Ｂａｌｉ ＰｅａｎｕｔはＡｒａ ｈ１を持っていない特種な落花生であるが、その他落花生由来アレルゲン（Ａｒａ ｈ２、Ａｒａ ｈ３など）が依然に含まれているため、アレルギーの発症状況は普通落花生と変わりがなかったことが報告されている（非特許文献１）。
In addition, Indonesian Bali Peanut is a special kind of peanut that does not have Ara h1, but it still contains other peanut-derived allergens (Ara h2, Ara h3, etc.), so the incidence of allergies is different from that of ordinary peanuts. It has been reported that there was no such problem (Non-Patent Document 1).
Ａｒａ ｈ１タンパク質を標的とするモノクローナル抗体を用いた検出用キットまたは検出方法は、食品検体中のＡｒａ ｈ１タンパク質の濃度が低いときには検出感度が悪く、一方で食品検体中のＡｒａ ｈ１タンパク質の濃度が高いときには偽陰性となって検出できないといった問題点があった。また、食品がＡｒａ ｈ１欠損落花生を用いて製造されている場合は、Ａｒａ ｈ１タンパク質を標的とするモノクローナル抗体を用いた検出用キットまたは検出方法によっては落花生アレルゲンを検出できず、Ａｒａ ｈ２、Ａｒａ ｈ３タンパク質のアレルギー患者にとって大きな問題となる。また、落花生中の主要アレルゲンタンパク質であるＡｒａ ｈ１の含有量は加熱よって増減するため、Ａｒａｈ１の検査キットで落花生を検出しにくい問題がある。
Detection kits or detection methods using monoclonal antibodies targeting Ara h1 protein have poor detection sensitivity when the concentration of Ara h1 protein in the food sample is low, but on the other hand, the detection sensitivity is poor when the concentration of Ara h1 protein in the food sample is high. There was a problem that sometimes a false negative result could not be detected. In addition, if the food is manufactured using Ara h1-deficient peanuts, peanut allergens cannot be detected depending on the detection kit or detection method using monoclonal antibodies targeting Ara h1 protein, and Ara h2, Ara h3 This is a big problem for people with protein allergies. Furthermore, since the content of Ara h1, the main allergenic protein in peanuts, increases or decreases with heating, there is a problem in that it is difficult to detect peanuts with an Arah1 test kit.
本発明は、食品中の落花生アレルゲンを検出するための改良された手段を提供することを課題とする。
An object of the present invention is to provide improved means for detecting peanut allergens in foods.
本発明者らは、落花生アレルゲンの検出用キットまたは検出方法において、抗Ａｒａ ｈ３抗体を用いてＡｒａ ｈ３タンパク質を検出対象とする場合、意外なことに、抗Ａｒａ ｈ１抗体を用いてＡｒａ ｈ１タンパク質を検出対象とする場合よりも、食品検体中のアレルゲンタンパク質が低濃度であっても検出感度に優れ、また食品検体中のアレルゲンタンパク質が高濃度であっても偽陰性とならずに検出できるなどの利点があることを見出し、本発明を完成させるに至った。抗Ａｒａ ｈ３抗体を用いた場合は、Ａｒａ ｈ１欠損落花生を用いて製造された食品であっても、そこに含まれる落花生アレルゲンを検出することができるという利点もある。
When the present inventors used an anti-Ara h3 antibody to detect Ara h3 protein in a peanut allergen detection kit or detection method, the present inventors unexpectedly found that the Ara h1 protein was detected using an anti-Ara h1 antibody. Compared to the detection target, the detection sensitivity is superior even when the allergen protein in a food sample is at a low concentration, and even when the allergen protein is at a high concentration in a food sample, it can be detected without false negatives. The present inventors have discovered that there are advantages and have completed the present invention. When anti-Ara h3 antibodies are used, there is also the advantage that peanut allergens contained in foods manufactured using Ara h1-deficient peanuts can be detected.
すなわち、本発明には少なくとも下記の事項が含まれる。
［１］
食品中又は食品加工装置に残留している落花生アレルゲンを免疫学的測定法により定量的または定性的に検出するための検査用キットであって、抗Ａｒａ ｈ３抗体またはその抗原結合性断片（以下「抗Ａｒａ ｈ３抗体等」と呼ぶ。）を含む、検査用キット。
［２］
前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等が、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを含む、項１に記載の検査用キット。
［３］
前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等が、発色標識用の抗Ａｒａ ｈ３抗体等を含む、項１に記載の検査用キット。
［４］
前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、項１～３のいずれか一項に記載の検査用キット。
［５］
前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、項１～４のいずれか一項に記載の検査用キット。
［６］
さらに抽出用試薬を含む、項１～５のいずれか一項に記載の検査用キット。
［７］
前記抽出用試薬が、加熱を伴わない振盪法または高速剪断・撹拌処理法による抽出用試薬である、項６に記載の検査用キット。
［８］
（１）食品中又は食品加工装置に残留している落花生タンパク質を抽出する工程、および
（２）得られた抽出物中のＡｒａ ｈ３タンパク質を抗Ａｒａ ｈ３抗体等を用いた免疫学的測定法により定量的または定性的に検出する工程
を含む、食品中の落花生アレルゲンの検査方法。
［９］
前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等として、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを用いる、項８に記載の検査方法。
［１０］
前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等として、発色標識用の抗Ａｒａ ｈ３抗体等を用いる、項８に記載の検査方法。
［１１］
前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、項８～１０のいずれか一項に記載の検査方法。
［１２］
前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、項８～１１のいずれか一項に記載の検査方法。
［１３］
前記工程（１）における抽出が、抽出用試薬を用いて行われる、項８～１２のいずれか一項に記載の検査方法。
［１４］
前記工程（１）における抽出が、加熱を伴わない振盪法または高速剪断・撹拌処理法により行われる、項８～１３のいずれか一項に記載の検査方法。 That is, the present invention includes at least the following matters.
[1]
A test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or in food processing equipment using an immunoassay method, comprising anti-Ara h3 antibody or its antigen-binding fragment (hereinafter referred to as " A test kit containing an anti-Ara h3 antibody, etc.).
[2]
The test according to item 1, wherein the immunoassay method is ELISA, and the anti-Ara h3 antibody, etc. includes an anti-Ara h3 antibody, etc. for immobilization, and an anti-Ara h3 antibody, etc. for enzyme labeling. kit for.
[3]
Item 2. The test kit according to item 1, wherein the immunoassay method is immunochromatography, and the anti-Ara h3 antibody and the like include an anti-Ara h3 antibody and the like for chromogenic labeling.
[4]
4. The test kit according to any one of Items 1 to 3, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
[5]
5. The test kit according to any one of Items 1 to 4, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
[6]
Item 6. The test kit according to any one of Items 1 to 5, further comprising an extraction reagent.
[7]
Item 7. The test kit according to Item 6, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
[8]
(1) Extracting peanut protein remaining in food or food processing equipment, and (2) Ara h3 protein in the obtained extract by immunoassay using anti-Ara h3 antibody etc. A method for testing peanut allergens in food, including a quantitative or qualitative detection step.
[9]
The test according to item 8, wherein the immunoassay is ELISA, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody or the like for immobilization and an anti-Ara h3 antibody or the like for enzyme labeling. Method.
[10]
Item 9. The testing method according to item 8, wherein the immunoassay is immunochromatography, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody for chromogenic labeling.
[11]
11. The testing method according to any one of Items 8 to 10, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
[12]
12. The testing method according to any one of Items 8 to 11, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
[13]
Item 13. The testing method according to any one of Items 8 to 12, wherein the extraction in step (1) is performed using an extraction reagent.
[14]
Item 14. The testing method according to any one of Items 8 to 13, wherein the extraction in step (1) is performed by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
［１］
食品中又は食品加工装置に残留している落花生アレルゲンを免疫学的測定法により定量的または定性的に検出するための検査用キットであって、抗Ａｒａ ｈ３抗体またはその抗原結合性断片（以下「抗Ａｒａ ｈ３抗体等」と呼ぶ。）を含む、検査用キット。
［２］
前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等が、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを含む、項１に記載の検査用キット。
［３］
前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等が、発色標識用の抗Ａｒａ ｈ３抗体等を含む、項１に記載の検査用キット。
［４］
前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、項１～３のいずれか一項に記載の検査用キット。
［５］
前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、項１～４のいずれか一項に記載の検査用キット。
［６］
さらに抽出用試薬を含む、項１～５のいずれか一項に記載の検査用キット。
［７］
前記抽出用試薬が、加熱を伴わない振盪法または高速剪断・撹拌処理法による抽出用試薬である、項６に記載の検査用キット。
［８］
（１）食品中又は食品加工装置に残留している落花生タンパク質を抽出する工程、および
（２）得られた抽出物中のＡｒａ ｈ３タンパク質を抗Ａｒａ ｈ３抗体等を用いた免疫学的測定法により定量的または定性的に検出する工程
を含む、食品中の落花生アレルゲンの検査方法。
［９］
前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等として、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを用いる、項８に記載の検査方法。
［１０］
前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等として、発色標識用の抗Ａｒａ ｈ３抗体等を用いる、項８に記載の検査方法。
［１１］
前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、項８～１０のいずれか一項に記載の検査方法。
［１２］
前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、項８～１１のいずれか一項に記載の検査方法。
［１３］
前記工程（１）における抽出が、抽出用試薬を用いて行われる、項８～１２のいずれか一項に記載の検査方法。
［１４］
前記工程（１）における抽出が、加熱を伴わない振盪法または高速剪断・撹拌処理法により行われる、項８～１３のいずれか一項に記載の検査方法。 That is, the present invention includes at least the following matters.
[1]
A test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or in food processing equipment using an immunoassay method, comprising anti-Ara h3 antibody or its antigen-binding fragment (hereinafter referred to as " A test kit containing an anti-Ara h3 antibody, etc.).
[2]
The test according to item 1, wherein the immunoassay method is ELISA, and the anti-Ara h3 antibody, etc. includes an anti-Ara h3 antibody, etc. for immobilization, and an anti-Ara h3 antibody, etc. for enzyme labeling. kit for.
[3]
Item 2. The test kit according to item 1, wherein the immunoassay method is immunochromatography, and the anti-Ara h3 antibody and the like include an anti-Ara h3 antibody and the like for chromogenic labeling.
[4]
4. The test kit according to any one of Items 1 to 3, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
[5]
5. The test kit according to any one of Items 1 to 4, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
[6]
Item 6. The test kit according to any one of Items 1 to 5, further comprising an extraction reagent.
[7]
Item 7. The test kit according to Item 6, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
[8]
(1) Extracting peanut protein remaining in food or food processing equipment, and (2) Ara h3 protein in the obtained extract by immunoassay using anti-Ara h3 antibody etc. A method for testing peanut allergens in food, including a quantitative or qualitative detection step.
[9]
The test according to item 8, wherein the immunoassay is ELISA, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody or the like for immobilization and an anti-Ara h3 antibody or the like for enzyme labeling. Method.
[10]
Item 9. The testing method according to item 8, wherein the immunoassay is immunochromatography, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody for chromogenic labeling.
[11]
11. The testing method according to any one of Items 8 to 10, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
[12]
12. The testing method according to any one of Items 8 to 11, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
[13]
Item 13. The testing method according to any one of Items 8 to 12, wherein the extraction in step (1) is performed using an extraction reagent.
[14]
Item 14. The testing method according to any one of Items 8 to 13, wherein the extraction in step (1) is performed by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
なお、当業者であれば、本発明の技術的思想、本明細書の記載事項および技術常識に基づき、上記の各項に記載の発明に関する事項を、他のカテゴリーの発明に関する事項に変換する（読み替える）ことが可能である。
Note that a person skilled in the art would convert the matters related to the invention described in each of the above sections into matters related to inventions in other categories based on the technical idea of the present invention, the matters described in this specification, and common general knowledge. It is possible to read it differently.
本発明により、食品中の落花生アレルゲンが低濃度である場合も高濃度である場合も、従来よりも高い信頼性で落花生アレルゲンを検出することが可能となる。
According to the present invention, it becomes possible to detect peanut allergen with higher reliability than before, regardless of whether the peanut allergen in food is at a low concentration or a high concentration.
―検査用キット―
本発明の「検査用キット」は、食品中又は食品加工装置に残留している落花生アレルゲンを定量的または定性的に検出するための検査用キットであって、抗Ａｒａ ｈ３抗体またはその抗原結合性断片（本明細書において「抗Ａｒａ ｈ３抗体等」と呼ぶ。）を含む。 -Test kit-
The "test kit" of the present invention is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, and is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, fragments (referred to herein as "anti-Ara h3 antibodies, etc.").
本発明の「検査用キット」は、食品中又は食品加工装置に残留している落花生アレルゲンを定量的または定性的に検出するための検査用キットであって、抗Ａｒａ ｈ３抗体またはその抗原結合性断片（本明細書において「抗Ａｒａ ｈ３抗体等」と呼ぶ。）を含む。 -Test kit-
The "test kit" of the present invention is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, and is a test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment, fragments (referred to herein as "anti-Ara h3 antibodies, etc.").
抗Ａｒａ ｈ３抗体等は、Ａｒａ ｈ３タンパク質上に存在するエピトープを認識して特異的に結合する抗体またはその抗原結合性断片であればよく、そのアミノ酸配列（重鎖および軽鎖それぞれの全長、可変領域（相補性決定領域：ＣＤＲ、フレームワーク領域：ＦＲ）、定常領域、またはそれらの断片）は特に限定されるものではない。「抗原結合性断片」としては、例えば、Ｆａｂ、Ｆａｂ’、Ｆ（ａｂ’）２、Ｆｖ、ｓｃＦｖ、ｄｓＦｖ、ダイアボディー（二重特異性抗体）、ナノボディーなどが挙げられる。なお、特に断らない限り、本明細書における「抗体」を「抗原結合性断片」または「抗体等」に読み替える、つまり「抗体」を用いる実施形態を「抗原結合性断片」または「抗体等」を用いる実施形態に置き換えることができる。
The anti-Ara h3 antibody etc. may be any antibody or antigen-binding fragment thereof that recognizes and specifically binds to an epitope present on the Ara h3 protein, and its amino acid sequence (full length of heavy chain and light chain, variable The region (complementarity determining region: CDR, framework region: FR), constant region, or a fragment thereof) is not particularly limited. Examples of the "antigen-binding fragment" include Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody (bispecific antibody), nanobody, and the like. Unless otherwise specified, "antibody" in this specification will be read as "antigen-binding fragment" or "antibody, etc." In other words, embodiments using "antibody" will be replaced with "antigen-binding fragment" or "antibody, etc." It can be replaced depending on the embodiment used.
抗Ａｒａ ｈ３抗体等は、その用途に応じて必要な特異性でもって、Ａｒａ ｈ３タンパク質を認識して結合できる抗体またはその抗原結合性断片であればよい。抗Ａｒａ ｈ３抗体等は、食品に含まれる各種のタンパク質および落花生以外の食物アレルゲンと結合しないこと（少なくとも、本発明による検査結果に影響しない程度の結合に留まること）が好ましいが、用途や程度に応じて、Ａｒａ ｈ３タンパク質以外のタンパク質と一定程度結合する（交差反応する）ことや、抗体の性質上不可避的なタンパク質またはその他の物質に対して非特異的に吸着することは許容される。
The anti-Ara h3 antibody and the like may be any antibody or antigen-binding fragment thereof that can recognize and bind to the Ara h3 protein with the specificity required depending on its use. It is preferable that anti-Ara h3 antibodies etc. do not bind to various proteins contained in foods and food allergens other than peanuts (at least, the binding remains to a level that does not affect the test results according to the present invention), but depending on the use and degree of Accordingly, binding (cross-reaction) to a certain degree with proteins other than the Ara h3 protein and nonspecific adsorption to proteins or other substances that are unavoidable due to the nature of antibodies are allowed.
抗Ａｒａ ｈ３抗体等は、検査用キットの用途や実施形態に応じて、いずれか１種類のみを用いてもよいし、２種類以上を組み合わせて用いてもよく、例えば、ポリクローナル抗体またはその抗原結合性断片（本明細書において「ポリクローナル抗体等」と呼ぶ。）であってもよいし、モノクローナル抗体またはその抗原結合性断片（本明細書において「モノクローナル抗体等」と呼ぶ。）であってもよい。ポリクローナル抗体等とモノクローナル抗体等とでは、特異性（Ａｒａ ｈ３タンパク質のみに反応し、それ以外のタンパク質とは反応（交差）しないか）、再現性（製造ロットにより検査結果に差が生じないか）、安定性（キットにおける抗体の固相化処理、標識処理等により、抗原への結合性が失われないか）などが異なるため、検査用キットの用途や実施形態によっては好ましい抗体が変動する場合もあるので、そのことを考慮してポリクローナル抗体等とモノクローナル抗体等のどちらを用いるか、またはその両方を組み合わせて用いるかを選択することができる。
Anti-Ara h3 antibodies etc. may be used alone or in combination of two or more depending on the purpose and embodiment of the test kit. For example, polyclonal antibodies or their antigen binding It may be a monoclonal antibody or an antigen-binding fragment thereof (referred to herein as a "monoclonal antibody, etc."). . For polyclonal antibodies and monoclonal antibodies, etc., specificity (does it react only with Ara h3 protein and do not react (cross) with other proteins?), reproducibility (does the test result differ depending on the manufacturing lot?) The preferred antibody may vary depending on the use and embodiment of the test kit because of differences in stability (whether binding to the antigen is lost due to immobilization treatment, labeling treatment, etc. of the antibody in the kit), etc. Taking this into account, it is possible to select whether to use polyclonal antibodies or monoclonal antibodies, or to use a combination of both.
ここで、加工食品等の食品中のＡｒａ ｈ３タンパク質は、食品の製造工程や保存工程（例えば、混捏、成形、加圧、加熱、乾燥、酵素処理、冷凍、解凍などの有無や処理条件）により、また食品（検体）からの抽出工程（例えば、可溶化剤が添加された抽出用試薬を用いた抽出処理の有無や処理条件）により、様々に変性したＡｒａ ｈ３タンパク質を含んでいる、例えば未変性のＡｒａ ｈ３タンパク質と様々に変性したＡｒａ ｈ３タンパク質との集合物になっている場合がある。したがって、本発明における抗Ａｒａ ｈ３抗体等は、食品中の様々なＡｒａ ｈ３タンパク質の集合物に対して一定の検出感度を有する（食品によって検出感度が低下することを防止できる）よう、様々なＡｒａ ｈ３タンパク質に対して結合できる実施形態であることが好ましい。
Here, the Ara h3 protein in foods such as processed foods is determined by the manufacturing process and storage process of the food (e.g., the presence or absence of kneading, molding, pressurization, heating, drying, enzyme treatment, freezing, thawing, etc., and the processing conditions). , and may contain Ara h3 proteins that have been variously denatured due to the extraction process from food (sample) (e.g., the presence or absence of extraction treatment using an extraction reagent containing a solubilizing agent and the treatment conditions). It may be a collection of denatured Ara h3 protein and variously denatured Ara h3 proteins. Therefore, the anti-Ara h3 antibodies etc. of the present invention have a certain detection sensitivity for aggregates of various Ara h3 proteins in foods (preventing detection sensitivity from decreasing depending on the food). Preferred embodiments are those capable of binding to h3 protein.
そのような観点から、本発明の一実施形態において、検査用キットは、抗Ａｒａ ｈ３抗体等として、未変性Ａｒａ ｈ３タンパク質および変性Ａｒａ ｈ３タンパク質（例えば加熱変性Ａｒａ ｈ３タンパク質）の両方に対して特異的に結合可能なものを含むことができる。
From such a point of view, in one embodiment of the present invention, a test kit, such as an anti-Ara h3 antibody, is specific for both native Ara h3 protein and denatured Ara h3 protein (e.g., heat-denatured Ara h3 protein). can include those that can be combined with each other.
また、本発明の別の一実施形態において、検査用キットは、抗Ａｒａ ｈ３抗体等としてポリクローナル抗体等を含む。本発明のさらなる一実施形態において、検出用キットは、抗Ａｒａ ｈ３抗体等として、ポリクローナル抗体等と、モノクローナル抗体等の両方を含む。例えば、後述するような実施形態の免疫学的測定法において、Ａｒａ ｈ３タンパク質に対する捕捉用抗体（ＥＬＩＳＡにおける固相化用抗体、イムノクロマトグラフィーにおける固定化用抗体）としてモノクローナル抗体等を使用し、捕捉されたＡｒａ ｈ３タンパク質に対する検出用抗体（ＥＬＩＳＡにおける酵素標識用抗体、イムノクロマトグラフィーにおける金コロイド標識用もしくはラテックス粒子標識用抗体）としてポリクローナル抗体等を使用することができる。捕捉用抗体としてモノクローナル抗体等を用いることでＡｒａ ｈ３タンパク質に対する特異性を高め、検出用抗体としてポリクローナル抗体等を用いることで捕捉したＡｒａ ｈ３タンパク質を高感度で検出することができる。
In another embodiment of the present invention, the test kit includes a polyclonal antibody or the like as the anti-Ara h3 antibody or the like. In a further embodiment of the present invention, the detection kit includes both polyclonal antibodies and monoclonal antibodies as anti-Ara h3 antibodies and the like. For example, in the immunoassay method of the embodiment described below, a monoclonal antibody or the like is used as a capture antibody (immobilization antibody in ELISA, immobilization antibody in immunochromatography) against Ara h3 protein, and the captured A polyclonal antibody or the like can be used as a detection antibody (enzyme labeling antibody in ELISA, gold colloid labeling or latex particle labeling antibody in immunochromatography) against the Ara h3 protein. By using a monoclonal antibody or the like as a capture antibody, the specificity for the Ara h3 protein can be increased, and by using a polyclonal antibody or the like as a detection antibody, the captured Ara h3 protein can be detected with high sensitivity.
本発明で用いる抗Ａｒａ ｈ３抗体等は、他の食物アレルゲンに対する抗体と同様に、常法に従って、一般的には、食品から抽出され、通常はさらに精製された食物アレルゲン（本発明ではＡｒａ ｈ３タンパク質）を用いて、ポリクローナル抗体等またはモノクローナル抗体等として作製することができる。
The anti-Ara h3 antibodies used in the present invention, like antibodies against other food allergens, are generally extracted from foods according to conventional methods and are usually further purified from food allergens (in the present invention, Ara h3 protein ) can be used to produce polyclonal antibodies or monoclonal antibodies.
ポリクローナル抗体の作製方法としては、免疫動物を用いる方法や、ファージディスプレイ法が一般的である。免疫動物を用いる方法の手順の概略は次の通りである。適量の標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）の精製物を、必要に応じてアジュバンドと混合して免疫原とし、その免疫原を動物（ウサギ、モルモット、ヤギ、ヒツジ、ラット、マウス、ニワトリ等）に注射する（免疫する）ことにより、その動物の血液中に抗体を産生させることができる。適当な間隔および回数で繰り返し免疫した後、血液（血漿、血清）を回収し、そこに含まれる抗体を精製する、例えば標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）を固定化したアフィニティーカラムを用いるアフィニティークロマトグラフィー、および必要に応じてさらにゲル濾過クロマトグラフィーにより精製することで、ポリクローナル抗体が得られる。また、ファージディスプレイ法の手順の概略は次の通りである。バクテリオファージに抗体の遺伝子を導入し、Ｈ鎖およびＬ鎖の可変領域が連結したタンパク質をバクテリオファージのコートタンパク質上に発現させる（提示する）ようにする。得られた抗体ファージライブラリーを用いて、標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）に対して親和性を有する抗体を選択する。その抗体を産生するバクテリオファージを大腸菌に感染させて増殖させた後に回収し、そこに含まれる抗体を精製することで、ポリクローナル抗体が得られる。
Common methods for producing polyclonal antibodies include methods using immunized animals and phage display methods. The outline of the procedure using the immunized animal is as follows. An appropriate amount of purified target protein (Ara h3 protein in the present invention) is mixed with an adjuvant as necessary to prepare an immunogen, and the immunogen is used to infect animals (rabbits, guinea pigs, goats, sheep, rats, mice, chickens). etc.), antibodies can be produced in the blood of that animal. After repeated immunization at appropriate intervals and times, blood (plasma, serum) is collected and the antibodies contained therein are purified. For example, affinity using an affinity column on which a target protein (Ara h3 protein in the present invention) is immobilized. A polyclonal antibody is obtained by purification by chromatography and, if necessary, further gel filtration chromatography. Moreover, the outline of the procedure of the phage display method is as follows. An antibody gene is introduced into a bacteriophage, and a protein in which the H chain and L chain variable regions are linked is expressed (displayed) on the coat protein of the bacteriophage. Using the obtained antibody phage library, antibodies having affinity for the target protein (Ara h3 protein in the present invention) are selected. Polyclonal antibodies can be obtained by infecting Escherichia coli with bacteriophages that produce the antibodies, multiplying them, collecting them, and purifying the antibodies contained therein.
モノクローナル抗体の作製方法としては、ハイブリドーマを用いる方法が一般的である。ハイブリドーマを用いる方法の概略は次の通りである。ポリクローナル抗体を作製する場合と同様にして、免疫原を動物に注射し、抗体を産生させる。その免疫動物の脾臓からＢ細胞を採取し、ミエローマ細胞（不死化したがん細胞）と融合して、ハイブリドーマ（融合細胞）を作製する。ハイブリドーマの中から、標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）への結合親和性や特異性に優れた抗体を産生するものを選択（スクリーニング）する。そのハイブリドーマを培養し、培養上清中に単一の抗体を産生させる。培養上清を回収し、そこに含まれる抗体を精製することで、モノクローナル抗体が得られる。
A common method for producing monoclonal antibodies is to use hybridomas. The outline of the method using hybridoma is as follows. In the same manner as when producing polyclonal antibodies, an immunogen is injected into an animal and antibodies are produced. B cells are collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells). Among the hybridomas, those that produce antibodies with excellent binding affinity and specificity to the target protein (Ara h3 protein in the present invention) are selected (screened). The hybridoma is cultured to produce a single antibody in the culture supernatant. Monoclonal antibodies can be obtained by collecting the culture supernatant and purifying the antibodies contained therein.
また、抗Ａｒａ ｈ３抗体（ポリクローナルまたはモノクローナル）が得られた後は、そのアミノ酸配列を決定したり、常法に従って各種の抗原結合性断片（ポリクローナルまたはモノクローナル）を作製したりすることができる。
Furthermore, after an anti-Ara h3 antibody (polyclonal or monoclonal) is obtained, its amino acid sequence can be determined, and various antigen-binding fragments (polyclonal or monoclonal) can be produced according to conventional methods.
抗Ａｒａ ｈ３抗体等は、Ａｒａ ｈ３タンパク質を免疫学的測定法により定量的または定性的に検出するのに適した形態のものとする、例えば各種の修飾をしたり、改変をしたりすることができる。
The anti-Ara h3 antibody etc. may be in a form suitable for quantitatively or qualitatively detecting the Ara h3 protein by immunoassay, for example, it may be subjected to various modifications or alterations. can.
「免疫学的測定法」は、Ａｒａ ｈ３タンパク質と抗Ａｒａ ｈ３抗体等との反応を利用することにより、Ａｒａ ｈ３タンパク質を定量的または定性的に検出することを可能とする方法であればとくに限定されず、公知の様々な方法を利用することができる。また、各種の免疫学的測定法に対応した検査用キットも公知で、市販もされており、本発明の検査用キットも、抽出用試薬として本発明の特定のものを使用するよう変更すること以外は、公知の検査用キットに準じて製造することができる。
"Immunological assay" is particularly limited if it is a method that makes it possible to quantitatively or qualitatively detect Ara h3 protein by utilizing the reaction between Ara h3 protein and anti-Ara h3 antibodies, etc. Various known methods can be used. Test kits compatible with various immunoassay methods are also known and commercially available, and the test kit of the present invention can also be modified to use the specific one of the present invention as an extraction reagent. Other than this, it can be manufactured according to known test kits.
本発明の一実施形態において、免疫学的測定法は、ＥＬＩＳＡ（Enzyme-Linked Immuno Sorbent Assay、エライザ法）である。ＥＬＩＳＡは、各種のタンパク質を高い精度で定量的に検出することが可能で、加熱等した加工品から食物アレルゲンを検出するために好適な測定法であり、消費者庁ガイドラインに準拠している。
In one embodiment of the present invention, the immunoassay is ELISA (Enzyme-Linked Immuno Sorbent Assay). ELISA is capable of quantitatively detecting various proteins with high accuracy, is a suitable measurement method for detecting food allergens from processed products such as heating, and complies with the Consumer Affairs Agency guidelines.
ＥＬＩＳＡ用の抗Ａｒａ ｈ３抗体等は、例えば、固相化用の抗Ａｒａ ｈ３抗体等および酵素標識用の抗Ａｒａ ｈ３抗体等を含む。固相化用の抗Ａｒａ ｈ３抗体等は、ＥＬＩＳＡを行う部材の表面、例えばプレート（ウェル）の底部に固相化するためのものである。酵素標識用の抗Ａｒａ ｈ３抗体等は、固相化された抗体に補足されたＡｒａ ｈ３タンパク質に結合し、その後基質と反応することで、Ａｒａ ｈ３タンパク質の量に応じた強度で発色させるためのものである。酵素標識用の抗Ａｒａ ｈ３抗体等は、直接的に酵素で標識された抗Ａｒａ ｈ３抗体等、すなわち当該抗体等自体に共有結合により（例えばリンカーを介して）あらかじめ酵素が結合している抗Ａｒａ ｈ３抗体等であってもよいが、間接的に酵素で標識されることとなる抗Ａｒａ ｈ３抗体等、例えば「検査方法」との関係で後述する手順に例示した、ストレプトアビジンと結合した酵素とさらに反応することによって酵素標識された抗Ａｒａ ｈ３抗体等を完成させることができる、ビオチンが結合した抗Ａｒａ ｈ３抗体等のようなものであってもよい。
Anti-Ara h3 antibodies for ELISA include, for example, anti-Ara h3 antibodies for immobilization and anti-Ara h3 antibodies for enzyme labeling. The anti-Ara h3 antibody or the like for immobilization is intended to be immobilized on the surface of a member for performing ELISA, for example, on the bottom of a plate (well). Anti-Ara h3 antibodies for enzyme labeling bind to Ara h3 protein captured by immobilized antibodies, and then react with a substrate to develop color with an intensity that corresponds to the amount of Ara h3 protein. It is something. Anti-Ara h3 antibodies etc. for enzyme labeling are directly enzyme-labeled anti-Ara h3 antibodies, etc., that is, anti-Ara h3 antibodies etc. that have an enzyme already bound to them by a covalent bond (for example, via a linker). H3 antibody etc. may be used, but anti-Ara H3 antibody etc. which will be indirectly labeled with an enzyme, for example, an enzyme bound to streptavidin as exemplified in the procedure described later in relation to the "test method". It may also be an anti-Ara h3 antibody bound to biotin, which can be further reacted to complete an enzyme-labeled anti-Ara h3 antibody.
本発明の一実施形態において、免疫学的測定法はイムノクロマトグラフィー（immunochromatography、イムノクロマト法）である。イムノクロマトグラフィーは、簡便な操作により、比較的短時間で、各種のタンパク質を定性的に検出することができる測定法である。
In one embodiment of the present invention, the immunoassay method is immunochromatography. Immunochromatography is a measurement method that allows qualitative detection of various proteins in a relatively short time using simple operations.
イムノクロマトグラフィー用の抗Ａｒａ ｈ３抗体等は、例えば、固定化用の抗Ａｒａ ｈ３抗体等および発色標識（例、金コロイド標識、ラテックス粒子標識、白金粒子標識）用の抗Ａｒａ ｈ３抗体等を含む。発色標識用の抗Ａｒａ ｈ３抗体等は、一般的に、テストストリップの所定の位置（試料滴下部）に含ませる、あらかじめ発色標識と結合している抗Ａｒａ ｈ３抗体等であり、Ａｒａ ｈ３タンパク質と複合体を形成した状態で固定化抗体等に捕捉された後、基質と反応することで、標的タンパク質の存在を示すよう発色させるための抗体である。固定化用の抗Ａｒａ ｈ３抗体等は、一般的に、テストストリップの所定の位置（テストライン）に含ませる抗Ａｒａ ｈ３抗体等であり、毛細管現象により移動してきたＡｒａ ｈ３タンパク質と発色標識用の抗Ａｒａ ｈ３抗体等との複合体を捕捉するための抗体である。
Anti-Ara h3 antibodies for immunochromatography include, for example, anti-Ara h3 antibodies for immobilization and anti-Ara h3 antibodies for chromogenic labels (e.g., colloidal gold labels, latex particle labels, platinum particle labels). Anti-Ara h3 antibodies and the like for chromogenic labeling are generally anti-Ara h3 antibodies and the like that are pre-bound to a chromogenic label and are contained in a predetermined position (sample dropping part) of a test strip, and are used in combination with Ara h3 protein. This is an antibody that is captured in a complexed state by an immobilized antibody, etc., and then reacts with a substrate to develop a color to indicate the presence of the target protein. The anti-Ara h3 antibody for immobilization is generally included in a predetermined position (test line) of the test strip, and is used to bind the Ara h3 protein that has migrated by capillary action and the coloring label. This is an antibody for capturing complexes with anti-Ara h3 antibodies, etc.
本発明の検査用キットは、必要に応じて、または本発明の作用効果を阻害しない範囲で、抗Ａｒａ ｈ３抗体等以外の抗体等を含むことができる。例えば、本発明の検査用キットは、上述したような捕捉用抗体等および検出用抗体等として、Ａｒａ ｈ３以外のタンパク質（例：Ａｒａ ｈ３以外の落花生アレルゲン、または落花生アレルゲン以外の食物アレルゲン）を標的とする抗体等を、さらに含んでいてもよい。
The test kit of the present invention may contain antibodies other than anti-Ara h3 antibodies, etc., as necessary or within a range that does not inhibit the effects of the present invention. For example, the test kit of the present invention targets proteins other than Ara h3 (e.g., peanut allergens other than Ara h3, or food allergens other than peanut allergen) as the above-mentioned capture antibodies and detection antibodies. It may further contain antibodies and the like.
本発明の検査用キットは、少なくとも抗Ａｒａ ｈ３抗体等を含むが、実施形態に応じて必要により、抽出用試薬、さらにその他の試薬、部材等の任意の内容物を含むことができる。ＥＬＩＳＡ用のキットであれば、例えば、ウェルを備えたプレート、標的タンパク質の溶液を調製するための希釈液、各工程を行った後にプレート（ウェル）を洗浄するための洗浄液、ＥＬＩＳＡにおける酵素反応停止液、抽出されたタンパク質を分解から保護するためのＢＳＡ、ＥＬＩＳＡによる検査方法の手順を記載した取扱説明書などが、固相化用、酵素標識用それぞれの抗Ａｒａ ｈ３抗体等や、抽出用試薬とともに、キットの内容物に含めることができる。イムノクロマトグラフィー用のキットであれば、例えば、毛細管現象により各種の溶液および試薬を展開させることができるテストストリップ、標的タンパク質の溶液を調製するための希釈液、イムノクロマトグラフィーによる検査方法の手順を記載した取扱説明書などが、固定化用、発色標識用それぞれの抗Ａｒａ ｈ３抗体等や、抽出用試薬とともに、キットの内容物に含めることができる。
The test kit of the present invention contains at least an anti-Ara h3 antibody and the like, but can also contain arbitrary contents such as an extraction reagent, other reagents, and members as necessary depending on the embodiment. If it is a kit for ELISA, 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, and a kit for stopping the enzyme reaction in ELISA. BSA to protect the extracted proteins from degradation, instruction manuals that describe the procedure for testing by ELISA, etc., as well as anti-Ara h3 antibodies for immobilization and enzyme labeling, and extraction reagents. It can also be included in the contents of the kit. A kit for immunochromatography includes, for example, a test strip that can develop various solutions and reagents by capillary action, a diluent for preparing a target protein solution, and instructions for testing by immunochromatography. Instruction manuals and the like can be included in the contents of the kit, together with anti-Ara h3 antibodies for immobilization and color labeling, and extraction reagents.
「抽出用試薬」は、食品中の各種のタンパク質、例えば食物アレルゲンを抽出するために一般的に用いられている試薬（溶液）を指す。抽出用試薬としては様々な種類のものが公知となっており、本発明でも同様の抽出用試薬を用いることができる。
"Extraction reagent" refers to a reagent (solution) that is commonly used to extract various proteins in foods, such as food allergens. Various types of extraction reagents are known, and similar extraction reagents can be used in the present invention.
抽出用試薬は一般的に、適切なｐＨを有する緩衝液に、食品中の各種のタンパク質を抽出するための剤として「可溶化剤」を添加することにより調製される溶液である。
An extraction reagent is generally a solution prepared by adding a "solubilizer" as an agent for extracting various proteins in foods to a buffer solution having an appropriate pH.
抽出用試薬を調製するための緩衝液としては、例えば、Ｔｒｉｓ緩衝液、リン酸緩衝液、クエン酸緩衝液、ＥＤＴＡ緩衝液、ＨＥＰＥＳ緩衝液、酢酸緩衝液が挙げられる。本発明における緩衝液としては、例えば、Ｔｒｉｓ緩衝液およびリン酸緩衝液が好ましい。緩衝液のｐＨは、一般的には４．５～８．０（生体内で起こりうる範囲のｐＨ）、例えば６．０～８．０である。当業者であれば、適切な化合物を適量用いる（複数の化合物を、それぞれ適量、水に添加して溶解する）ことにより、所望の濃度の、所望のｐＨを有する緩衝液を調製することができる。
Examples of the buffer for preparing the extraction reagent include Tris buffer, phosphate buffer, citrate buffer, EDTA buffer, HEPES buffer, and acetate buffer. As the buffer in the present invention, for example, Tris buffer and phosphate buffer are preferable. The pH of the buffer solution is generally 4.5 to 8.0 (pH within the range that can occur in vivo), for example 6.0 to 8.0. Those skilled in the art can prepare a buffer solution having a desired concentration and a desired pH by using appropriate amounts of appropriate compounds (adding and dissolving appropriate amounts of multiple compounds in water). .
抽出用試薬を調製するための可溶化剤としては、例えば、界面活性剤、カオトロピック剤および還元剤が挙げられる。抽出用試薬は、いずれか１つの種類を単独で含んでいてもよいし、複数の種類を組み合わせて含んでいてもよい。
Examples of solubilizing agents for preparing extraction reagents include surfactants, chaotropic agents, and reducing agents. The extraction reagent may contain any one kind alone, or may contain a combination of a plurality of kinds.
界面活性剤としては、例えば、陰イオン性（アニオン性）界面活性剤、陽イオン性（カチオン性）界面活性剤、非イオン性（ノニオン性）界面活性剤が挙げられる。陰イオン性界面活性剤としては、例えば、アルキル硫酸塩およびアルキルベンゼンスルホン酸塩が挙げられる。アルキル硫酸塩、アルキルベンゼンスルホン酸塩等の「アルキル」は、例えば、ドデシル、デシル、ノニル、オクチルである。アルキル硫酸塩、アルキルベンゼンスルホン酸塩等の「塩」は、例えば、ナトリウム塩、カリウム塩、アンモニウム塩である。アルキル硫酸塩の具体例としては、ドデシル硫酸ナトリウム（ＳＤＳ）が挙げられ、アルキルベンゼンスルホン酸塩の具体例としては、ドデシルベンゼンスルホン酸ナトリウムが挙げられる。陽イオン性界面活性剤の具体例としては、塩化ヘキサデシルピリジニウム、臭化ヘキサデシルトリメチルアンモニウム、塩化ヘキサデシルトリメチルアンモニウムが挙げられる。非イオン性界面活性剤の具体例としては、「Tween（登録商標）20」（ポリオキシエチレンソルビタンモノララウレート）、「Tween（登録商標）40」（ポリオキシエチレンソルビタンモノパルミテート）、「Tween（登録商標）60」（ポリオキシエチレンソルビタンモノステアレート）、「Tween（登録商標）80」（ポリオキシエチレンソルビタンモノオレエート）が挙げられる。
Examples of the surfactant include anionic surfactants, cationic surfactants, and nonionic surfactants. Examples of anionic surfactants include alkyl sulfates and alkylbenzenesulfonates. "Alkyl" in alkyl sulfates, alkylbenzenesulfonates, etc. is, for example, dodecyl, decyl, nonyl, octyl. "Salts" such as alkyl sulfates and alkylbenzenesulfonates are, for example, sodium salts, potassium salts, and ammonium salts. Specific examples of alkyl sulfates include sodium dodecyl sulfate (SDS), and specific examples of alkylbenzenesulfonates include sodium dodecylbenzenesulfonate. 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).
抽出用試薬が界面活性剤を含む場合、界面活性剤の濃度は、界面活性剤の種類およびその作用効果や、必要に応じて抽出用試薬中のその他の成分（例えばカオトロピック剤、還元剤）の有無、種類および濃度などを考慮して、適宜調節することができる。例えば、抽出用試薬が界面活性剤としてＳＤＳ等の陰イオン性界面活性剤またはその他の界面活性剤を含む場合、その濃度の下限値は、例えば０．００５、０．０１または０．０５％（ｗ／ｖ）とすることができ、上限値は、例えば５．０、３．０または２．０％（ｗ／ｖ）とすることができ、これらの上限値および下限値は任意に組みあわせることができる。
When the extraction reagent contains a surfactant, the concentration of the surfactant depends on the type of surfactant and its effects, as well as other components (e.g. chaotropic agent, reducing agent) in the extraction reagent as necessary. It can be adjusted as appropriate by considering the presence or absence, type, concentration, etc. 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 is, 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 arbitrarily combined. be able to.
「カオトロピック剤」としては、例えば、尿素、ホルムアミド、塩溶効果のある陰イオンまたは陽イオンを含む塩（例：グアニジニウムイオンを含む塩酸グアニジン、ナトリウムイオンを含む塩化ナトリウム、カリウムイオンを含む塩化カリウム等）が挙げられる。
Examples of "chaotropic agents" include urea, formamide, salts containing anions or cations with a salt-soluble effect (e.g., guanidine hydrochloride containing guanidinium ions, sodium chloride containing sodium ions, potassium chloride containing potassium ions). etc.).
抽出用試薬がカオトロピック剤を含む場合、カオトロピック剤の濃度は、カオトロピック剤の種類およびその作用効果（本発明の作用効果への影響）や、必要に応じて抽出用試薬中のその他の成分（例えば界面活性剤、還元剤）の有無、種類および濃度などを考慮して、適宜調節することができる。例えば、抽出用試薬がカオトロピック剤として尿素を含む場合、その濃度の下限値は、例えば０．０１、０．０５または０．１（ｗ／ｖ）とすることができ、上限値は、例えば１０、５．０または３．０％（ｗ／ｖ）とすることができ、これらの上限値および下限値は任意に組みあわせることができる。
When the extraction reagent contains a chaotropic agent, the concentration of the chaotropic agent depends on the type of chaotropic agent and its effects (influence on the effects of the present invention), and, if necessary, other components in the extraction reagent (e.g. It can be adjusted as appropriate by considering the presence or absence, type, and concentration of surfactants and reducing agents. For example, when the extraction reagent contains urea as a chaotropic agent, the lower limit of the concentration can be, for example, 0.01, 0.05, or 0.1 (w/v), and the upper limit is, for example, 10 , 5.0 or 3.0% (w/v), and these upper and lower limits can be arbitrarily combined.
還元剤としては、例えば、メルカプトアルカノールおよび／または亜硫酸塩が挙げられる。メルカプトアルカノールとしては、例えば、チオグリセロール、２－メルカプトエタノール、ジチオトレイトールが挙げられる。亜硫酸塩としては、例えば、アルカリ金属亜硫酸塩が挙げられ、そのアルカリ金属としては、例えば、ナトリウム、カリウムが挙げられる。亜硫酸塩の具体例としては、亜硫酸ナトリウムが挙げられる。
Examples of the reducing agent include mercaptoalkanol and/or sulfite. Examples of the mercaptoalkanol include thioglycerol, 2-mercaptoethanol, and dithiothreitol. Examples of sulfites include alkali metal sulfites, and examples of the alkali metals include sodium and potassium. Specific examples of sulfites include sodium sulfite.
抽出用試薬が還元剤を含む場合、還元剤の濃度は、還元剤の種類およびその作用効果や、必要に応じて抽出用試薬中のその他の成分（例えば界面活性剤、カオトロピック剤）の有無、種類および濃度などを考慮して、適宜調節することができる。抽出用試薬が還元剤としてチオグリセロールを含む場合、その濃度の下限値は、例えば０．００１、０．００５、０．０１または０．０２％（ｗ／ｖ）とすることができ、上限値は、例えば１０、５．０、３．０、２．０または１．０％（ｗ／ｖ）とすることができ、これらの上限値および下限値は任意に組みあわせることができる。抽出用試薬が還元剤として２－メルカプトエタノールまたはジチオトレイトールを含む場合、その濃度の下限値は、例えば０．０５、０．１または０．５％（ｗ／ｖ）とすることができ、上限値は、例えば５．０、２．０または１．０％（ｗ／ｖ）とすることができ、これらの上限値および下限値は任意に組みあわせることができる。
When the extraction reagent contains a reducing agent, the concentration of the reducing agent depends on the type of reducing agent and its effects, as well as the presence or absence of other components (e.g. surfactants, chaotropic agents) in the extraction reagent as necessary. It can be adjusted as appropriate by considering the type, concentration, etc. 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 arbitrarily combined. 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), 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.
本明細書の記載において、「抗体」または「抗体等」（抗体およびその抗原結合性断片）はさらに、タンパク質に結合することのできる公知の各種の「検出分子」に拡張して読み替える（実施形態において置き換える）ことが可能である。抗体および抗原結合性断片（抗体等）以外の「検出分子」としては、例えば、アプタマー、レセプター、抗菌ペプチドまたはその他のペプチドなどが挙げられる。抗体等を用いる抗原抗体反応に基づく実施形態と同様に、検出分子を用いる特異的な反応に基づく実施形態によっても、本発明を実施することが可能である。
In the description of this specification, "antibody" or "antibody etc." (antibodies and antigen-binding fragments thereof) is further expanded to include various known "detection molecules" capable of binding to proteins (embodiment). ) is possible. Examples of "detection molecules" other than antibodies and antigen-binding fragments (antibodies, etc.) include aptamers, receptors, antibacterial peptides, and other peptides. As well as embodiments based on antigen-antibody reactions using antibodies and the like, the present invention can also be practiced by embodiments based on specific reactions using detection molecules.
―検査方法―
本発明の「検査方法」は、食品中又は食品加工装置に残留している落花生アレルゲンの検査方法であって、（１）食品中又は食品加工装置に残留している落花生タンパク質を抽出する工程（本明細書において「抽出工程」と呼ぶ。）、および（２）得られた抽出物中のＡｒａ ｈ３タンパク質を抗Ａｒａ ｈ３抗体等を用いた免疫学的測定法により定量的または定性的に検出する工程（本明細書において「検出工程」と呼ぶ。）を含む。 -Inspection method-
The "inspection method" of the present invention is a method for testing peanut allergens remaining in food or food processing equipment, and includes (1) a step of extracting peanut protein remaining in food or food processing equipment ( (herein referred to as "extraction step"), and (2) quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunoassay method using anti-Ara h3 antibodies etc. (referred to herein as a "detection step").
本発明の「検査方法」は、食品中又は食品加工装置に残留している落花生アレルゲンの検査方法であって、（１）食品中又は食品加工装置に残留している落花生タンパク質を抽出する工程（本明細書において「抽出工程」と呼ぶ。）、および（２）得られた抽出物中のＡｒａ ｈ３タンパク質を抗Ａｒａ ｈ３抗体等を用いた免疫学的測定法により定量的または定性的に検出する工程（本明細書において「検出工程」と呼ぶ。）を含む。 -Inspection method-
The "inspection method" of the present invention is a method for testing peanut allergens remaining in food or food processing equipment, and includes (1) a step of extracting peanut protein remaining in food or food processing equipment ( (herein referred to as "extraction step"), and (2) quantitatively or qualitatively detecting Ara h3 protein in the obtained extract by an immunoassay method using anti-Ara h3 antibodies etc. (referred to herein as a "detection step").
「食品」は、原材料としての落花生自体（例えば生落花生、乾燥落花生、煎り落花生、茹で落花生）であってもよいし、原材料として落花生を含む（かどうかの検査対象とする）加工品であってもよい。食品（加工品）の形態は特に限定されるものではないが、例えば、加熱および／または加圧条件下で行われる工程を経て製造されることにより、一般的にアレルゲンタンパク質が不溶化して難抽出状態となる傾向にある加工品であってもよい。食品は、例えば、固形状、半固形状、ゼリー状、液状、乳化液状のいずれであってもよい。
"Food" may be peanuts themselves as raw materials (for example, fresh peanuts, dried peanuts, roasted peanuts, boiled peanuts), or processed products that contain peanuts as raw materials (which are subject to inspection). Good too. The form of the food (processed product) is not particularly limited, but for example, allergenic proteins are generally insolubilized and difficult to extract by being manufactured through a process that is performed under heating and/or pressurized conditions. It may also be a processed product that tends to be in a state. The food may be, for example, solid, semi-solid, jelly, liquid, or emulsified.
・検出工程
「免疫学的測定法」は、食品から抽出された食物アレルゲン等のタンパク質を検出できるものであれば、特に限定されるものではなく、公知の各種の方法から選択することができる。 - Detection step The "immunological assay" is not particularly limited as long as it can detect proteins such as food allergens extracted from foods, and can be selected from various known methods.
「免疫学的測定法」は、食品から抽出された食物アレルゲン等のタンパク質を検出できるものであれば、特に限定されるものではなく、公知の各種の方法から選択することができる。 - Detection step The "immunological assay" is not particularly limited as long as it can detect proteins such as food allergens extracted from foods, and can be selected from various known methods.
本発明の一実施形態において、検出工程における免疫学的測定法は、ＥＬＩＳＡである。ＥＬＩＳＡは、例えば下記のような手順により、食品（検体）中の標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）およびそれと結合する抗体等を用いて行うことができる。
１）固相化抗体を備えたプレートのウェルに、標的タンパク質の溶液を添加し、固相化抗体等と標的タンパク質とを接触させ、抗原抗体反応により結合させることで、第１の複合体を形成する。
２）上記ウェルを洗浄後、ビオチン結合抗体の溶液を添加し、第１の複合体中の標的タンパク質とビオチン結合抗体等とを接触させ、抗原抗体反応により結合させることで、第２の複合体を形成する。
３）上記ウェルを洗浄後、ストレプトアビジン結合酵素の溶液を添加し、第２の複合体中のビオチン結合抗体等とストレプトアビジン結合酵素とを接触させ、ビオチン－ストレプトアビジン反応により結合させることで、第３の複合体を形成する。
４）上記ウェルを洗浄後、基質の溶液（発色剤）を添加し、第３の複合体中の酵素と基質とを反応させ、発色させる。
５）発色反応を停止させた後、プレートリーダーで所定の波長における吸光度を測定する。
６）別途標準溶液を測定して得られた吸光度から標準曲線グラフを作成しておき、５）で測定した吸光度および標準曲線グラフから、標的タンパク質の量（溶液の濃度）を読み取り、溶液の希釈倍率を乗じて、検体中の標的タンパク質の量を算出する。 In one embodiment of the invention, the immunoassay in the detection step is ELISA. ELISA can be carried out using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto, for example, according to the following procedure.
1) Add a solution of the target protein to the wells of the plate containing the immobilized antibody, bring the immobilized antibody etc. into contact with the target protein, and bind by antigen-antibody reaction to form the first complex. Form.
2) After washing the above well, a solution of biotin-conjugated antibody is added, the target protein in the first complex is brought into contact with the biotin-conjugated antibody, etc., and the target protein in the first complex is bonded by an antigen-antibody reaction, thereby forming a second complex. form.
3) After washing the above-mentioned 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, so that they are bound by a biotin-streptavidin reaction. A third complex is formed.
4) After washing the well, a substrate solution (coloring agent) is added to cause the enzyme in the third complex to react with the substrate to develop color.
5) After stopping the color reaction, measure the absorbance at a predetermined wavelength using a plate reader.
6) Create a standard curve graph from the absorbance obtained by measuring the standard solution separately, read the amount of target protein (concentration of the solution) from the absorbance measured in 5) and the standard curve graph, and dilute the solution. Calculate the amount of target protein in the sample by multiplying by the magnification.
１）固相化抗体を備えたプレートのウェルに、標的タンパク質の溶液を添加し、固相化抗体等と標的タンパク質とを接触させ、抗原抗体反応により結合させることで、第１の複合体を形成する。
２）上記ウェルを洗浄後、ビオチン結合抗体の溶液を添加し、第１の複合体中の標的タンパク質とビオチン結合抗体等とを接触させ、抗原抗体反応により結合させることで、第２の複合体を形成する。
３）上記ウェルを洗浄後、ストレプトアビジン結合酵素の溶液を添加し、第２の複合体中のビオチン結合抗体等とストレプトアビジン結合酵素とを接触させ、ビオチン－ストレプトアビジン反応により結合させることで、第３の複合体を形成する。
４）上記ウェルを洗浄後、基質の溶液（発色剤）を添加し、第３の複合体中の酵素と基質とを反応させ、発色させる。
５）発色反応を停止させた後、プレートリーダーで所定の波長における吸光度を測定する。
６）別途標準溶液を測定して得られた吸光度から標準曲線グラフを作成しておき、５）で測定した吸光度および標準曲線グラフから、標的タンパク質の量（溶液の濃度）を読み取り、溶液の希釈倍率を乗じて、検体中の標的タンパク質の量を算出する。 In one embodiment of the invention, the immunoassay in the detection step is ELISA. ELISA can be carried out using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto, for example, according to the following procedure.
1) Add a solution of the target protein to the wells of the plate containing the immobilized antibody, bring the immobilized antibody etc. into contact with the target protein, and bind by antigen-antibody reaction to form the first complex. Form.
2) After washing the above well, a solution of biotin-conjugated antibody is added, the target protein in the first complex is brought into contact with the biotin-conjugated antibody, etc., and the target protein in the first complex is bonded by an antigen-antibody reaction, thereby forming a second complex. form.
3) After washing the above-mentioned 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, so that they are bound by a biotin-streptavidin reaction. A third complex is formed.
4) After washing the well, a substrate solution (coloring agent) is added to cause the enzyme in the third complex to react with the substrate to develop color.
5) After stopping the color reaction, measure the absorbance at a predetermined wavelength using a plate reader.
6) Create a standard curve graph from the absorbance obtained by measuring the standard solution separately, read the amount of target protein (concentration of the solution) from the absorbance measured in 5) and the standard curve graph, and dilute the solution. Calculate the amount of target protein in the sample by multiplying by the magnification.
本発明の一実施形態において、検出工程における免疫学的測定法は、イムノクロマトグラフィーである。イムノクロマトグラフィーは、例えば下記の手順により、食品（検体）中の標的タンパク質（本発明ではＡｒａ ｈ３タンパク質）およびそれと結合する抗体等を用いて行うことができる。
１）発色標識抗体等が含まれているテストストリップ上の所定の部位（試料滴下部）に、標的タンパク質を含む試料溶液を滴下し、発色標識抗体等と標的タンパク質とを接触させ、抗原抗体反応により結合させることで、第１の複合体を形成する。
２）第１の複合体や未反応の発色標識抗体等を含む試料溶液は、テストストリップ上の所定の部位（展開部）を展開していき、それに伴い第１の複合体等も毛細管現象によって移動する。
３）第１の複合体が、固定化抗体等が含まれている所定の部位（テストライン部）に到達し、固定化抗体等に捕捉されると、発色標識により発色したライン（例えば、金コロイドによる赤紫色のライン）が出現する。テストラインが出現した場合、試料溶液中に標的タンパク質が含まれていたことを示す。
４）未反応の金コロイド標識抗体等が、抗免疫グロブリン抗体が含まれている所定の部位（コントロールライン部、テストラインより下流側）に到達し、抗免疫グロブリン抗体等に捕捉されると、金コロイドによる赤紫色のラインが出現する。コントロールラインが出現しなかった場合、試料中に標的タンパク質が含まれていたか否かにかかわらず、試料溶液の展開に異常があったことが示唆される（再検査が必要となる）。 In one embodiment of the invention, the immunoassay in the detection step is immunochromatography. Immunochromatography can be performed, for example, by the following procedure using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto.
1) Drop a sample solution containing the target protein onto a predetermined area (sample dropping part) on the test strip that contains the color-labeled antibody, etc., bring the color-labeled antibody, etc. into contact with the target protein, and cause an antigen-antibody reaction. A first complex is formed by binding the first complex.
2) The sample solution containing the first complex, unreacted color-labeled antibody, etc. is spread over a predetermined area (deployment area) on the test strip, and the first complex, etc. is also spread due to capillary action. Moving.
3) When the first complex reaches a predetermined site (test line area) containing an immobilized antibody, etc. and is captured by the immobilized antibody, etc., a line colored by a coloring label (for example, a gold A reddish-purple line due to colloid) appears. If a test line appears, it indicates that the target protein was contained in the sample solution.
4) When unreacted colloidal gold-labeled antibodies, etc. reach a predetermined site containing anti-immunoglobulin antibodies (control line area, downstream of the test line) and are captured by anti-immunoglobulin antibodies, etc. A reddish-purple line appears due to colloidal gold. If the control line does not appear, it is suggested that there was an abnormality in the development of the sample solution, regardless of whether the sample contained the target protein (retesting is required).
１）発色標識抗体等が含まれているテストストリップ上の所定の部位（試料滴下部）に、標的タンパク質を含む試料溶液を滴下し、発色標識抗体等と標的タンパク質とを接触させ、抗原抗体反応により結合させることで、第１の複合体を形成する。
２）第１の複合体や未反応の発色標識抗体等を含む試料溶液は、テストストリップ上の所定の部位（展開部）を展開していき、それに伴い第１の複合体等も毛細管現象によって移動する。
３）第１の複合体が、固定化抗体等が含まれている所定の部位（テストライン部）に到達し、固定化抗体等に捕捉されると、発色標識により発色したライン（例えば、金コロイドによる赤紫色のライン）が出現する。テストラインが出現した場合、試料溶液中に標的タンパク質が含まれていたことを示す。
４）未反応の金コロイド標識抗体等が、抗免疫グロブリン抗体が含まれている所定の部位（コントロールライン部、テストラインより下流側）に到達し、抗免疫グロブリン抗体等に捕捉されると、金コロイドによる赤紫色のラインが出現する。コントロールラインが出現しなかった場合、試料中に標的タンパク質が含まれていたか否かにかかわらず、試料溶液の展開に異常があったことが示唆される（再検査が必要となる）。 In one embodiment of the invention, the immunoassay in the detection step is immunochromatography. Immunochromatography can be performed, for example, by the following procedure using a target protein (Ara h3 protein in the present invention) in a food (sample) and an antibody that binds thereto.
1) Drop a sample solution containing the target protein onto a predetermined area (sample dropping part) on the test strip that contains the color-labeled antibody, etc., bring the color-labeled antibody, etc. into contact with the target protein, and cause an antigen-antibody reaction. A first complex is formed by binding the first complex.
2) The sample solution containing the first complex, unreacted color-labeled antibody, etc. is spread over a predetermined area (deployment area) on the test strip, and the first complex, etc. is also spread due to capillary action. Moving.
3) When the first complex reaches a predetermined site (test line area) containing an immobilized antibody, etc. and is captured by the immobilized antibody, etc., a line colored by a coloring label (for example, a gold A reddish-purple line due to colloid) appears. If a test line appears, it indicates that the target protein was contained in the sample solution.
4) When unreacted colloidal gold-labeled antibodies, etc. reach a predetermined site containing anti-immunoglobulin antibodies (control line area, downstream of the test line) and are captured by anti-immunoglobulin antibodies, etc. A reddish-purple line appears due to colloidal gold. If the control line does not appear, it is suggested that there was an abnormality in the development of the sample solution, regardless of whether the sample contained the target protein (retesting is required).
・抽出工程
「抽出工程」は、通常は抽出用試薬を用いて、食品中の各種のタンパク質を抽出する処理として行われている、一般的な抽出工程と同様に行うことができ、さらに必要に応じて本発明に適合するよう適宜改変することができる。 ・Extraction process The "extraction process" can be performed in the same way as a general extraction process, which is usually performed as a process to extract various proteins in foods using extraction reagents. It can be modified as appropriate to suit the present invention.
「抽出工程」は、通常は抽出用試薬を用いて、食品中の各種のタンパク質を抽出する処理として行われている、一般的な抽出工程と同様に行うことができ、さらに必要に応じて本発明に適合するよう適宜改変することができる。 ・Extraction process The "extraction process" can be performed in the same way as a general extraction process, which is usually performed as a process to extract various proteins in foods using extraction reagents. It can be modified as appropriate to suit the present invention.
抽出工程に供する食品の検体は、食品の形態に応じて、フードカッター、ミルサー、ミキサー、ホモジナイザー等を用いた高速剪断・撹拌処理により、均質な状態に微細化または乳化すること（高速剪断・撹拌処理法）によって調製することができる。このような微細化等の処理は、抽出工程の前にあらかじめ食品等に対して行い、得られた微細化等された食品と抽出用試薬とを混合するようにしてもよいし、食品と抽出用試薬の混合物に対して行い、微細化等の処理と同時に抽出処理が行われるようにしてもよい。微細化等の処理条件（時間、温度、回転数（ｒｐｍ）または遠心力（×ｇ）等）は、選択した微細化等の処理方法や用いる処理装置に応じて、また本発明の作用効果を考慮して、適宜調節することができる。また、抽出工程に供する食品は、必要に応じて、例えば脱脂処理など、Ａｒａ ｈ３タンパク質の抽出および／または抽出後の検出などを考慮した処理がさらに行われていてもよい。
The food sample to be subjected to the extraction process must be micronized or emulsified into a homogeneous state by high-speed shearing and stirring using a food cutter, miller, mixer, homogenizer, etc., depending on the form of the food (high-speed shearing and stirring). (treatment method). Such treatment such as micronization may be performed on the food, etc. in advance before the extraction process, and the resulting micronized food and extraction reagent may be mixed, or the food and extraction reagent may be mixed. The extraction process may be performed on a mixture of reagents for use, and the extraction process may be performed simultaneously with the process such as micronization. Processing conditions such as time, temperature, number of revolutions (rpm), centrifugal force (xg), etc. for micro-refining etc. will vary depending on the selected processing method for micro-refining etc. and the processing equipment used, and will also affect the effects of the present invention. It can be adjusted accordingly. Furthermore, the food to be subjected to the extraction step may be further subjected to a treatment, such as a defatting treatment, in consideration of extraction and/or post-extraction detection of the Ara h3 protein, if necessary.
上記のように微細化等した食品、またはしていない食品と、抽出用試薬とを混合した後、その混合物を振盪処理することにより、抽出処理を行うこと（振盪法）もできる。振盪の処理条件（時間、温度、振盪数もしくは回転数（ｒｐｍ）等）は、選択した振盪処理方法や用いる処理装置に応じて、また本発明の作用効果を考慮して、適宜調節することができる。振盪処理の時間は、通常１２時間以上、２４時間未満（例えば一晩）である。振盪処理の温度は、通常は室温であるが、必要であれば加熱した温度であってもよい。本発明では、抽出工程（振盪処理）において加熱をしなくても、低濃度のＡｒａ ｈ３タンパク質を高感度で検出したり、高濃度のＡｒａ ｈ３タンパク質を偽陰性とせずに検出したりすることができる。
The extraction process can also be performed by mixing the food that has been or has not been micronized as described above and the extraction reagent and then shaking the mixture (shaking method). The shaking processing conditions (time, temperature, shaking number, rotation speed (rpm), etc.) can be adjusted as appropriate depending on the selected shaking processing method and the processing equipment used, and in consideration of the effects of the present invention. can. The duration of the shaking treatment is usually 12 hours or more and less than 24 hours (for example, overnight). The temperature of the shaking treatment is usually room temperature, but may be heated if necessary. In the present invention, it is possible to detect Ara h3 protein at a low concentration with high sensitivity and to detect a high concentration Ara h3 protein without causing a false negative, even without heating in the extraction process (shaking process). can.
食品加工装置に残留するアレルゲンを検査する場合は、例えば、ＰＢＳまたは生理食塩水で湿らせた綿棒を用意し、食品加工装置の所定の拭き取り箇所を綿棒で拭き取り、予め試験管等に分注（例：１ｍＬ）した希釈緩衝液で拭き取った綿棒に付着した汚れを懸濁すれば、その懸濁液を「抽出物」に相当する試料溶液として利用することができる。
When testing allergens remaining in food processing equipment, for example, prepare a cotton swab moistened with PBS or physiological saline, wipe the designated area of the food processing equipment with the cotton swab, and dispense it into test tubes ( For example, if dirt attached to a wiped cotton swab is suspended in a diluted buffer (1 mL), the suspension can be used as a sample solution corresponding to an "extract."
以下、実施例を通じて、本発明の実施形態をより具体的に開示するが、本発明の技術的範囲は実施例として開示した実施形態に限定されるものではない。当業者であれば、目的とする本発明の用途や作用効果に適応するよう、本発明の技術的思想ならびに本明細書および図面の内容を全体的に考慮して、実施例として開示した実施形態を拡張したり、他の様々な実施形態に改変したりすること、あるいは必要に応じて、従来技術（公知の発明）が備える技術的特徴をさらに組み合わせたりできることを、当業者は理解することができる。本明細書に記載したもの以外の、本発明を実施するために必要な事項は、本発明の属する技術分野における技術常識や従来技術を適宜参酌することができる。
Hereinafter, embodiments of the present invention will be disclosed more specifically 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 be able to understand the embodiments disclosed as examples by taking into consideration the technical idea of the present invention as well as the contents of this specification and drawings as a whole, so as to adapt to the intended uses and effects of the present invention. Those skilled in the art will understand that the invention can be extended or modified into various other embodiments, or technical features of the prior art (known inventions) can be further combined as necessary. can. For matters necessary to carry out the present invention other than those described in this specification, common general knowledge and prior art in the technical field to which the present invention pertains can be appropriately referred to.
［実施例１］落花生アレルゲン（Ａｒａ ｈ３タンパク質）を検出するためのイムノクロマト用キットの製造
［１－１］抗Ａｒａ ｈ３モノクローナル抗体の作製
生落花生を粉砕して粉末状としたものを、脱脂のためにヘキサン中で撹拌し、次いでアセトン中で撹拌した。ヘキサン中での撹拌およびアセトン中での撹拌をさらに３回繰り返した（合計４回行った）後、風乾して、生落花生粉末の検体（検体Ａ）を得た。 [Example 1] Production of immunochromatography kit for detecting peanut allergen (Ara h3 protein) [1-1] Production of anti-Ara h3 monoclonal antibody Raw peanuts were ground into powder and prepared for defatting. Stirred in hexane and then in acetone. After repeating the stirring in hexane and acetone three more times (four times in total), the mixture was air-dried to obtain a sample of raw peanut powder (specimen A).
［１－１］抗Ａｒａ ｈ３モノクローナル抗体の作製
生落花生を粉砕して粉末状としたものを、脱脂のためにヘキサン中で撹拌し、次いでアセトン中で撹拌した。ヘキサン中での撹拌およびアセトン中での撹拌をさらに３回繰り返した（合計４回行った）後、風乾して、生落花生粉末の検体（検体Ａ）を得た。 [Example 1] Production of immunochromatography kit for detecting peanut allergen (Ara h3 protein) [1-1] Production of anti-Ara h3 monoclonal antibody Raw peanuts were ground into powder and prepared for defatting. Stirred in hexane and then in acetone. After repeating the stirring in hexane and acetone three more times (four times in total), the mixture was air-dried to obtain a sample of raw peanut powder (specimen A).
１ｇの生落花生粉末と５０ｍｌのＴｒｉｓ－ＨＣｌ緩衝液と混合し、３０分振盪して、抽出液を得た。抽出液を４℃で遠心分離し（３０００×ｇ、２０分）、上澄みを濾過し、濾液を得た。濾液を陰イオンで精製し、Ａｒａ ｈ３タンパク質溶液を得た。一部のＡｒａ ｈ３タンパク質溶液は未加熱検体として使用し、一部のＡｒａ ｈ３タンパク質溶液は１００℃で１０分加熱して、加熱検体として使用した。未加熱検体と加熱検体を同量混合し、免疫抗原として使用した。
1 g of raw peanut powder and 50 ml of Tris-HCl buffer were mixed and shaken for 30 minutes to obtain an extract. The extract was centrifuged at 4° C. (3000×g, 20 minutes), and the supernatant was filtered to obtain a filtrate. The filtrate was anion-purified to obtain an Ara h3 protein solution. Some of the Ara h3 protein solutions were used as unheated samples, and some of the Ara h3 protein solutions were heated at 100° C. for 10 minutes and used as heated samples. Equal amounts of unheated and heated samples were mixed and used as an immunizing antigen.
モノクローナル抗体の作製方法としては、ハイブリドーマを用いる方法が一般的である。ハイブリドーマを用いて本実施例のモノクローナル抗体を得た方法の概略は次の通りである。ポリクローナル抗体を作製する場合と同様にして、免疫抗原（本実施例では上記の未加熱検体および加熱検体の同量混合物）を動物に注射し、抗体を産生した。その免疫動物の脾臓からＢ細胞を採取し、ミエローマ細胞（不死化したがん細胞）と融合して、ハイブリドーマ（融合細胞）を作製した。ハイブリドーマの中から、標的タンパク質（Ａｒａ ｈ３タンパク質、本実施例では未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方）への結合親和性や特異性に優れた抗体を産生するものを選択（スクリーニング）した。そのハイブリドーマを培養し、培養上清中に単一の抗体を産生させた。培養上清を回収し、そこに含まれる抗体を精製することで、モノクローナル抗体が得られた。
A common method for producing monoclonal antibodies is to use hybridomas. The outline of the method for obtaining the monoclonal antibody of this example using a hybridoma is as follows. In the same manner as when producing polyclonal antibodies, an immunizing antigen (in this example, a mixture of equal amounts of the above-mentioned unheated specimen and heated specimen) was injected into animals to produce antibodies. B cells were collected from the spleen of the immunized animal and fused with myeloma cells (immortalized cancer cells) to produce hybridomas (fused cells). Select (screen) hybridomas that produce antibodies with excellent binding affinity and specificity to the target protein (Ara h3 protein, in this example, both native Ara h3 protein and heat-denatured Ara h3 protein). )did. The hybridoma was cultured and produced a single antibody in the culture supernatant. Monoclonal antibodies were obtained by collecting the culture supernatant and purifying the antibodies contained therein.
［１－２］テストストリップおよび金コロイド標識抗体の作製
上記［１－１］で得られた抗Ａｒａ ｈ３モノクローナル抗体には、未変性Ａｒａ ｈ３タンパク質精製物および加熱変性Ａｒａ ｈ３タンパク質精製物の両方に対して特異的に結合することが確認されたもの（データ示さず）が含まれていた。そのような特定の抗Ａｒａ ｈ３抗体のうちの１種類を、テストストリップを作製するための固定化用抗Ａｒａ ｈ３抗体として用い、別の１種類を、金コロイド標識抗体を作製するための発色標識用抗Ａｒａ ｈ３抗体として用いた。さらに、常法に従って作製した、発色標識用抗Ａｒａ ｈ３抗体に対する抗体（抗マウスＩｇＧ抗体）も用いた。 [1-2] Preparation of test strip and colloidal gold-labeled antibody The anti-Ara h3 monoclonal antibody obtained in [1-1] above includes both purified undenatured Ara h3 protein and purified heat-denatured Ara h3 protein. This included those that were confirmed to specifically bind to (data not shown). One type of such specific anti-Ara h3 antibodies was used as an anti-Ara h3 antibody for immobilization to create a test strip, and another type was used as a coloring label to create a colloidal gold-labeled antibody. It was used as an anti-Ara h3 antibody. Furthermore, an antibody (anti-mouse IgG antibody) against anti-Ara h3 antibody for chromogenic labeling, which was prepared according to a conventional method, was also used.
上記［１－１］で得られた抗Ａｒａ ｈ３モノクローナル抗体には、未変性Ａｒａ ｈ３タンパク質精製物および加熱変性Ａｒａ ｈ３タンパク質精製物の両方に対して特異的に結合することが確認されたもの（データ示さず）が含まれていた。そのような特定の抗Ａｒａ ｈ３抗体のうちの１種類を、テストストリップを作製するための固定化用抗Ａｒａ ｈ３抗体として用い、別の１種類を、金コロイド標識抗体を作製するための発色標識用抗Ａｒａ ｈ３抗体として用いた。さらに、常法に従って作製した、発色標識用抗Ａｒａ ｈ３抗体に対する抗体（抗マウスＩｇＧ抗体）も用いた。 [1-2] Preparation of test strip and colloidal gold-labeled antibody The anti-Ara h3 monoclonal antibody obtained in [1-1] above includes both purified undenatured Ara h3 protein and purified heat-denatured Ara h3 protein. This included those that were confirmed to specifically bind to (data not shown). One type of such specific anti-Ara h3 antibodies was used as an anti-Ara h3 antibody for immobilization to create a test strip, and another type was used as a coloring label to create a colloidal gold-labeled antibody. It was used as an anti-Ara h3 antibody. Furthermore, an antibody (anti-mouse IgG antibody) against anti-Ara h3 antibody for chromogenic labeling, which was prepared according to a conventional method, was also used.
発色標識用抗Ａｒａ ｈ３抗体を用いて、常法に従って、金コロイド標識抗体を作製した。この金コロイド標識抗体と、固相化用抗Ａｒａ ｈ３抗体、ならびに試料滴下部、展開部、吸収パッドなどを含むテストストリップ用の一般的な部材を用いて、常法に従って、試薬含有部に金コロイド標識抗体を配置し、テストライン出現位置に固相化用抗Ａｒａ ｈ３抗体を配置し、コントロールライン出現位置に発色標識用抗Ａｒａ ｈ３抗体に対する抗体を配置したテストストリップを作製した。また、常法に従って、発色標識用抗Ａｒａ ｈ３抗体と金コロイドとを結合させることにより、金コロイド標識抗体を作製した。これらの作製物により、Ａｒａ ｈ３タンパク質を標的として落花生タンパク質（アレルゲン）を検出するためのイムノクロマト用キットを構成し、実施例２で用いた。
A colloidal gold-labeled antibody was produced using a color-labeling anti-Ara h3 antibody according to a conventional method. Using this colloidal gold-labeled antibody, anti-Ara h3 antibody for immobilization, and common parts for test strips including a sample dropping part, developing part, absorbent pad, etc., the reagent-containing part is coated with gold according to a conventional method. A test strip was prepared in which a colloid labeled antibody was placed, an anti-Ara h3 antibody for immobilization was placed at the position where the test line appeared, and an antibody against the anti-Ara h3 antibody for chromogenic labeling was placed at the position where the control line appeared. In addition, a colloidal gold-labeled antibody was prepared by binding an anti-Ara h3 antibody for chromogenic labeling to colloidal gold according to a conventional method. These preparations constituted an immunochromatography kit for detecting peanut protein (allergen) targeting Ara h3 protein, and was used in Example 2.
［実施例２］イムノクロマト法による落花生アレルゲン（Ａｒａ ｈ３タンパク質）の検出
本実施例では、落花生アレルゲンの検出のための抗体として、前記実施例１の［１－１］により得られた特定の２種類（固定化用、酵素標識用）の抗Ａｒａ ｈ３モノクローナル抗体（以下「本発明抗体」と呼ぶ。）と、坑Ａｒａｈ１抗体を用いた市販キット（以下「対照品」と呼ぶ。）に含まれている抗Ａｒａ ｈ１モノクローナル抗体（以下「対照抗体」と呼ぶ。）を使用している。 [Example 2] Detection of peanut allergen (Ara h3 protein) by immunochromatography In this example, two specific types obtained in [1-1] of Example 1 were used as antibodies for detection of peanut allergen. (for immobilization and enzyme labeling) included in a commercially available kit using an anti-Ara h3 monoclonal antibody (hereinafter referred to as the "antibody of the present invention") and an anti-Arah1 antibody (hereinafter referred to as the "control product"). An anti-Ara h1 monoclonal antibody (hereinafter referred to as "control antibody") is used.
本実施例では、落花生アレルゲンの検出のための抗体として、前記実施例１の［１－１］により得られた特定の２種類（固定化用、酵素標識用）の抗Ａｒａ ｈ３モノクローナル抗体（以下「本発明抗体」と呼ぶ。）と、坑Ａｒａｈ１抗体を用いた市販キット（以下「対照品」と呼ぶ。）に含まれている抗Ａｒａ ｈ１モノクローナル抗体（以下「対照抗体」と呼ぶ。）を使用している。 [Example 2] Detection of peanut allergen (Ara h3 protein) by immunochromatography In this example, two specific types obtained in [1-1] of Example 1 were used as antibodies for detection of peanut allergen. (for immobilization and enzyme labeling) included in a commercially available kit using an anti-Ara h3 monoclonal antibody (hereinafter referred to as the "antibody of the present invention") and an anti-Arah1 antibody (hereinafter referred to as the "control product"). An anti-Ara h1 monoclonal antibody (hereinafter referred to as "control antibody") is used.
本発明抗体がＡｒａ ｈ３タンパク質に結合してＡｒａ ｈ１タンパク質には結合しないこと、および対照抗体がＡｒａ ｈ１タンパク質に結合してＡｒａ ｈ３タンパク質には結合しないことを、前記実施例１の［１－１］と同様にして行ったＳＤＳ－ＰＡＧＥに対するウェスタンブロッティングにより確認した（図１参照）。なお、本発明抗体が、その他の食物アレルゲン（牛乳、小麦、そば、卵、甲殻類、大豆、くるみ、マカデミアナッツ、カシューナッツ、ヘーゼルナッツ、ピーカンナッツ、ピスタチオ、ココナッツ）に対して結合しないことも確認した。
[1-1 in Example 1 above] that the antibody of the present invention binds to Ara h3 protein but not to Ara h1 protein, and that the control antibody binds to Ara h1 protein but not to Ara h3 protein. ] This was confirmed by Western blotting on SDS-PAGE performed in the same manner as described above (see Figure 1). It was also confirmed that the antibody of the present invention does not bind to other food allergens (milk, wheat, buckwheat, eggs, crustaceans, soybeans, walnuts, macadamia nuts, cashew nuts, hazelnuts, pecan nuts, pistachios, coconuts).
［２－１］高濃度試料を用いた試験
生落花生粉末の検体（検体ａ）およびピーナッツバターの検体（検体ｂ）それぞれ１ｇに、対照製品に含まれている検体抽出液（精製水８容に対して、第１抽出液および第２抽出液各１容を加えて攪拌し、調製したもの）１９ｍＬを加え、十分に攪拌した後、沸騰水浴中で１０分間加熱した。それぞれの溶液を濾紙で濾過し、得られた濾液（原液）を検査試料（検査試料ａ１、検査試料ｂ１）とした。 [2-1] Test using high concentration samples 1 g each of raw peanut powder sample (sample a) and peanut butter sample (sample b) was mixed with the sample extract contained in the control product (8 volumes of purified water). To the mixture, 19 mL (prepared by adding and stirring 1 volume each of the first extract and the second extract) was added, thoroughly stirred, and then heated in a boiling water bath for 10 minutes. Each solution was filtered through a filter paper, and the obtained filtrate (undiluted solution) was used as a test sample (test sample a1, test sample b1).
生落花生粉末の検体（検体ａ）およびピーナッツバターの検体（検体ｂ）それぞれ１ｇに、対照製品に含まれている検体抽出液（精製水８容に対して、第１抽出液および第２抽出液各１容を加えて攪拌し、調製したもの）１９ｍＬを加え、十分に攪拌した後、沸騰水浴中で１０分間加熱した。それぞれの溶液を濾紙で濾過し、得られた濾液（原液）を検査試料（検査試料ａ１、検査試料ｂ１）とした。 [2-1] Test using high concentration samples 1 g each of raw peanut powder sample (sample a) and peanut butter sample (sample b) was mixed with the sample extract contained in the control product (8 volumes of purified water). To the mixture, 19 mL (prepared by adding and stirring 1 volume each of the first extract and the second extract) was added, thoroughly stirred, and then heated in a boiling water bath for 10 minutes. Each solution was filtered through a filter paper, and the obtained filtrate (undiluted solution) was used as a test sample (test sample a1, test sample b1).
実施例１で作製したキット（以下「本発明品」と呼ぶ。）のテストストリップの試料滴下部に、検査試料ａ１、検査試料ｂ１それぞれを滴下した。１５分後に、テストライン出現位置およびコントロールライン出現位置に赤紫色のラインが観察された場合は陽性、テストライン出現位置には赤紫色のラインが観察されず、コントロールライン出現位置にのみ赤紫色のラインが観察された場合は陰性、と判定した。
Test sample a1 and test sample b1 were each dropped onto the sample dropping portion of the test strip of the kit produced in Example 1 (hereinafter referred to as "the product of the present invention"). After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
同様に、対照品のテストストリップの試料滴下部に、検査試料ａ１、検査試料ｂ１それぞれを滴下した。１５分後に、テストライン出現位置およびコントロールライン出現位置に赤紫色のラインが観察された場合は陽性、テストライン出現位置には赤紫色のラインが観察されず、コントロールライン出現位置にのみ赤紫色のラインが観察された場合は陰性、と判定した。
Similarly, test sample a1 and test sample b1 were each dropped onto the sample dropping portion of a control test strip. After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
結果を下記表に示す。対照品による判定では、試料ａ１、ａ２どちらについても陰性（－）との結果となったが、タンパク質が高濃度であることによる偽陰性であると解される。これに対して本発明品は、これらの試料のようにタンパク質が高濃度であっても陽性（＋）と判定できることが分かる。
The results are shown in the table below. In the determination using the control product, both samples a1 and a2 were negative (-), but this is understood to be a false negative result due to the high protein concentration. In contrast, it can be seen that the product of the present invention can be determined to be positive (+) even when the protein concentration is high as in these samples.
［２－２］低濃度試料を用いた試験
前記［１－１］で得られた検査試料ａ１（濾液の原液）に含まれるＡｒａ ｈ３タンパク質の濃度を、ＢＣＡ法により定量したのち、当該試料を段階希釈し、Ａｒａ ｈ３タンパク質の濃度が５００ｎｇ／ｍＬである検査試料ａ２、１００ｎｇ／ｍＬである検査試料ａ３などを調製した。 [2-2] Test using a low concentration sample After quantifying the concentration of Ara h3 protein contained in the test sample a1 (undiluted filtrate) obtained in [1-1] above using the BCA method, the sample was A test sample a2 with an Ara h3 protein concentration of 500 ng/mL, a test sample a3 with an Ara h3 protein concentration of 100 ng/mL, etc. were prepared by serial dilution.
前記［１－１］で得られた検査試料ａ１（濾液の原液）に含まれるＡｒａ ｈ３タンパク質の濃度を、ＢＣＡ法により定量したのち、当該試料を段階希釈し、Ａｒａ ｈ３タンパク質の濃度が５００ｎｇ／ｍＬである検査試料ａ２、１００ｎｇ／ｍＬである検査試料ａ３などを調製した。 [2-2] Test using a low concentration sample After quantifying the concentration of Ara h3 protein contained in the test sample a1 (undiluted filtrate) obtained in [1-1] above using the BCA method, the sample was A test sample a2 with an Ara h3 protein concentration of 500 ng/mL, a test sample a3 with an Ara h3 protein concentration of 100 ng/mL, etc. were prepared by serial dilution.
本発明品、対照品それぞれのテストストリップの試料滴下部に、検査試料ａ２、検査試料ａ３それぞれを滴下した。１５分後に、テストライン出現位置およびコントロールライン出現位置に赤紫色のラインが観察された場合は陽性、テストライン出現位置には赤紫色のラインが観察されず、コントロールライン出現位置にのみ赤紫色のラインが観察された場合は陰性、と判定した。
Test sample a2 and test sample a3 were dropped onto the sample dropping portions of the test strips of the present invention product and the control product, respectively. After 15 minutes, if a reddish-purple line is observed at the test line appearance position and the control line appearance position, it is positive. No reddish-purple line is observed at the test line appearance position, and a reddish-purple line is observed only at the control line appearance position If a line was observed, it was judged as negative.
結果を下記表に示す。対照品による判定では、試料ａ２については陽性（＋）であるもののラインが薄く、試料ａ３については陰性（－）との結果となった。これに対して本発明品は、これらの試料のようにタンパク質が低濃度であっても感度が良好であり、陽性（＋）と判定できることが分かる。
The results are shown in the table below. In the determination using the control product, sample a2 was positive (+) but the line was thin, and sample a3 was negative (-). In contrast, it can be seen that the product of the present invention has good sensitivity and can be determined as positive (+) even when the protein concentration is low as in these samples.
［実施例３］加熱を伴わない撹拌処理による抽出試料を用いた試験
［３－１］粉末検体の作製
生落花生を粉砕して粉末状としたものを、脱脂のためにヘキサン中で撹拌し、次いでアセトン中で撹拌した。ヘキサン中での撹拌およびアセトン中での撹拌をさらに３回繰り返した（合計４回行った）後、風乾して、生落花生粉末の検体を得た。 [Example 3] Test using extracted sample by stirring treatment without heating [3-1] Preparation of powder sample Raw peanuts were ground into powder and stirred in hexane for defatting. It was then stirred in acetone. After repeating the stirring in hexane and acetone three more times (four times in total), the mixture was air-dried to obtain a sample of raw peanut powder.
［３－１］粉末検体の作製
生落花生を粉砕して粉末状としたものを、脱脂のためにヘキサン中で撹拌し、次いでアセトン中で撹拌した。ヘキサン中での撹拌およびアセトン中での撹拌をさらに３回繰り返した（合計４回行った）後、風乾して、生落花生粉末の検体を得た。 [Example 3] Test using extracted sample by stirring treatment without heating [3-1] Preparation of powder sample Raw peanuts were ground into powder and stirred in hexane for defatting. It was then stirred in acetone. After repeating the stirring in hexane and acetone three more times (four times in total), the mixture was air-dried to obtain a sample of raw peanut powder.
［３－２］抽出工程
生落花生粉末２ｇに対して予め調製した抽出用緩衝液（ＦＡＳＴＫＩＴスリム、日本ハム株式会社）３８ｍＬを加え、室温でホモジナイザー等で３０～６０秒×３回操作を繰り返し、得られた試料を３０００×ｇ以上、４℃、２０分間遠心分離を行い、上清をろ過した。ろ液を希釈用緩衝液（ＦＡＳＴＫＩＴスリム、日本ハム株式会社）で１０倍希釈したものを、加熱を伴わない高濃度試料ｃ１とした。高濃度試料ｃ１を希釈用緩衝液でＡｒａ ｈ３タンパク質の濃度が２５ｎｇ/ｍＬになるように希釈した試料を、加熱を伴わない低濃度試料ｃ２とした。 [3-2] Extraction process Add 38 mL of a previously prepared extraction buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) to 2 g of raw peanut powder, repeat the operation 3 times for 30 to 60 seconds using a homogenizer, etc. at room temperature, The obtained sample was centrifuged at 3000×g or more at 4° C. for 20 minutes, and the supernatant was filtered. A 10-fold dilution of the filtrate with a dilution buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) was used as a high-concentration sample c1 without heating. A sample obtained by diluting the high concentration sample c1 with a dilution buffer so that the concentration of Ara h3 protein was 25 ng/mL was designated as a low concentration sample c2 without heating.
生落花生粉末２ｇに対して予め調製した抽出用緩衝液（ＦＡＳＴＫＩＴスリム、日本ハム株式会社）３８ｍＬを加え、室温でホモジナイザー等で３０～６０秒×３回操作を繰り返し、得られた試料を３０００×ｇ以上、４℃、２０分間遠心分離を行い、上清をろ過した。ろ液を希釈用緩衝液（ＦＡＳＴＫＩＴスリム、日本ハム株式会社）で１０倍希釈したものを、加熱を伴わない高濃度試料ｃ１とした。高濃度試料ｃ１を希釈用緩衝液でＡｒａ ｈ３タンパク質の濃度が２５ｎｇ/ｍＬになるように希釈した試料を、加熱を伴わない低濃度試料ｃ２とした。 [3-2] Extraction process Add 38 mL of a previously prepared extraction buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) to 2 g of raw peanut powder, repeat the operation 3 times for 30 to 60 seconds using a homogenizer, etc. at room temperature, The obtained sample was centrifuged at 3000×g or more at 4° C. for 20 minutes, and the supernatant was filtered. A 10-fold dilution of the filtrate with a dilution buffer (FASTKIT Slim, Nippon Ham Co., Ltd.) was used as a high-concentration sample c1 without heating. A sample obtained by diluting the high concentration sample c1 with a dilution buffer so that the concentration of Ara h3 protein was 25 ng/mL was designated as a low concentration sample c2 without heating.
［３－３］評価
テストストリップ（本発明品）の試料滴下部に得られた高濃度ｃ１及び低濃度試料ｃ２１００μＬを滴下し、評価した。結果を下記表に示す。 [3-3] Evaluation 100 μL of the obtained high concentration sample c1 and low concentration sample c2 were dropped onto the sample dropping portion of the test strip (product of the present invention) and evaluated. The results are shown in the table below.
テストストリップ（本発明品）の試料滴下部に得られた高濃度ｃ１及び低濃度試料ｃ２１００μＬを滴下し、評価した。結果を下記表に示す。 [3-3] Evaluation 100 μL of the obtained high concentration sample c1 and low concentration sample c2 were dropped onto the sample dropping portion of the test strip (product of the present invention) and evaluated. The results are shown in the table below.
Claims (14)
- 食品中又は食品加工装置に残留している落花生アレルゲンを免疫学的測定法により定量的または定性的に検出するための検査用キットであって、抗Ａｒａ ｈ３抗体またはその抗原結合性断片（以下「抗Ａｒａ ｈ３抗体等」と呼ぶ。）を含む、検査用キット。 A test kit for quantitatively or qualitatively detecting peanut allergen remaining in food or food processing equipment using an immunoassay method, which contains anti-Ara h3 antibody or its antigen-binding fragment (hereinafter referred to as " A test kit containing an anti-Ara h3 antibody, etc.).
- 前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等が、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを含む、請求項１に記載の検査用キット。 2. The method according to claim 1, wherein the immunoassay method is ELISA, and the anti-Ara h3 antibody, etc. includes an anti-Ara h3 antibody, etc. for immobilization, and an anti-Ara h3 antibody, etc. for enzyme labeling. Test kit.
- 前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等が、発色標識用の抗Ａｒａ ｈ３抗体等を含む、請求項１に記載の検査用キット。 The test kit according to claim 1, wherein the immunoassay method is immunochromatography, and the anti-Ara h3 antibody, etc. includes an anti-Ara h3 antibody, etc. for chromogenic labeling.
- 前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、請求項１～３のいずれか一項に記載の検査用キット。 The test kit according to any one of claims 1 to 3, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
- 前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、請求項１～４のいずれか一項に記載の検査用キット。 The test kit according to any one of claims 1 to 4, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
- さらに抽出用試薬を含む、請求項１～５のいずれか一項に記載の検査用キット。 The test kit according to any one of claims 1 to 5, further comprising an extraction reagent.
- 前記抽出用試薬が、加熱を伴わない振盪法または高速剪断・撹拌処理法による抽出用試薬である、請求項６に記載の検査用キット。 The test kit according to claim 6, wherein the extraction reagent is an extraction reagent obtained by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
- （１）食品中又は食品加工装置に残留している落花生タンパク質を抽出する工程、および
（２）得られた抽出物中のＡｒａ ｈ３タンパク質を抗Ａｒａ ｈ３抗体等を用いた免疫学的測定法により定量的または定性的に検出する工程
を含む、食品中の落花生アレルゲンの検査方法。 (1) Extracting peanut protein remaining in food or food processing equipment, and (2) Ara h3 protein in the obtained extract by immunoassay using anti-Ara h3 antibody etc. A method for testing peanut allergens in food, including a quantitative or qualitative detection step. - 前記免疫学的測定法がＥＬＩＳＡであり、前記抗Ａｒａ ｈ３抗体等として、固相化用の抗Ａｒａ ｈ３抗体等と、酵素標識用の抗Ａｒａ ｈ３抗体等とを用いる、請求項８に記載の検査方法。 9. The method according to claim 8, wherein the immunoassay is ELISA, and the anti-Ara h3 antibody or the like is an anti-Ara h3 antibody for immobilization and an anti-Ara h3 antibody or the like for enzyme labeling. Inspection method.
- 前記免疫学的測定法がイムノクロマトグラフィーであり、前記抗Ａｒａ ｈ３抗体等として、発色標識用の抗Ａｒａ ｈ３抗体等を用いる、請求項８に記載の検査方法。 9. The testing method according to claim 8, wherein the immunoassay is immunochromatography, and the anti-Ara h3 antibody or the like uses an anti-Ara h3 antibody or the like for chromogenic labeling.
- 前記抗Ａｒａ ｈ３抗体等が、未変性Ａｒａ ｈ３タンパク質および加熱変性Ａｒａ ｈ３タンパク質の両方に対して特異的なものである、請求項８～１０のいずれか一項に記載の検査方法。 The testing method according to any one of claims 8 to 10, wherein the anti-Ara h3 antibody or the like is specific for both native Ara h3 protein and heat-denatured Ara h3 protein.
- 前記抗Ａｒａ ｈ３抗体等が、モノクローナル抗体またはその抗原結合性断片を含む、請求項８～１１のいずれか一項に記載の検査方法。 The testing method according to any one of claims 8 to 11, wherein the anti-Ara h3 antibody or the like comprises a monoclonal antibody or an antigen-binding fragment thereof.
- 前記工程（１）における抽出が、抽出用試薬を用いて行われる、請求項８～１２のいずれか一項に記載の検査方法。 The testing method according to any one of claims 8 to 12, wherein the extraction in step (1) is performed using an extraction reagent.
- 前記工程（１）における抽出が、加熱を伴わない振盪法または高速剪断・撹拌処理法により行われる、請求項８～１３のいずれか一項に記載の検査方法。 The testing method according to any one of claims 8 to 13, wherein the extraction in step (1) is performed by a shaking method or a high-speed shearing/stirring treatment method that does not involve heating.
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