Method of detecting almond allergen

Abstract

To provide means capable of detecting an almond allergen in a sample even at a low concentration without showing cross-reactivity with other nuts or the like from a test sample such as food.SOLUTION: It is confirmed that an almond can be accurately detected by using two kinds of monoclonal antibodies which bind to almond 11S globulin and do not bind to 11S globulin of nuts and seeds other than the almond.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method and a kit for detecting almond allergens. [Background technology]

[0002] Due to various factors such as the decline of the natural environment, exhaust fumes from cars and factories, housing conditions, and changes in food, it is said that one in three people suffer from some kind of allergic disease. In particular, food allergies are harmful immune reactions caused by the ingestion of allergy-inducing substances contained in food (hereinafter referred to as "food allergens"), and are known to cause dermatitis, asthma, gastrointestinal disorders, anaphylactic shock, etc. Since these symptoms can be fatal, seven items, namely eggs, milk, wheat, shrimp, crab, peanuts, and buckwheat, are required to be labeled as specific ingredients on packaged processed foods. In addition, 21 items, namely almonds, abalone, squid, salmon roe, oranges, cashew nuts, kiwi fruit, beef, walnuts, sesame seeds, salmon, mackerel, soybeans, chicken, bananas, pork, matsutake mushrooms, peaches, yams, apples, and gelatin, are recommended to be labeled as much as possible as they are equivalent to specific ingredients.

[0003] In order to detect the above-mentioned food allergens quickly and easily, immunoassays that utilize an antigen-antibody reaction to detect a substance to be detected that consists of a specific antigen or antibody are widely used, and an immunoassay method is adopted in which an antibody or antigen labeled with a labeling substance consisting of a fluorescent substance or the like is bound to the substance to be detected in a sample through an immune reaction, and the bound labeling substance is measured. In these immunoassays, competitive reactions and sandwich reactions are widely used, and an allergen detection method using an immunochromatography method that utilizes a sandwich reaction (see, for example, Patent Document 1) has been proposed.

[0004] On the other hand, almonds (Prunus dulcis) are a type of nut that contain more than 50% by mass of lipids, of which about 70% is oleic acid, a monounsaturated fatty acid, and are also said to be rich in vitamin E and vitamin B2. In recent years, the use of almonds in confectionery and the production of almond drinks that contain almost no lactose or cholesterol has increased, and in anticipation of an increase in the number of people who develop allergies to almonds in the future, almonds were newly added to the list of specified raw materials in 2019. Therefore, appropriate detection methods for almonds are required to detect almonds that have unintentionally been mixed into raw materials, foods, food manufacturing equipment, food packaging, etc., and to confirm the consistency with food labeling.

[0005] Currently, the commercially available almond allergen detection kit "AlerTox Sticks Kit (registered trademark) Almond (KIT3030)" (manufactured by Hygiena Diagnostica Espana) is a kit using immunochromatography, but the detection limit is only 20 ppm. In addition, NEOGEN's Reveal3-D Almond Kit is also commercially available, but the instruction manual clearly states that it is designed to screen for almond components from wiping liquids and cleaning liquids, and is not necessarily suitable for detecting almond components in foods.

[0006] On the other hand, a simultaneous PCR detection method for almonds, hazelnuts, macadamia nuts, and Brazil nuts using a PCR primer set for detecting almonds has been proposed (see, for example, Patent Document 2, etc.). However, this requires operations such as PCR, and is thought to take time until a determination can be made. In addition, this method is not capable of detecting only almonds. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 5-010950 [Patent Document 2] JP 2022-160499 A Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a means for accurately detecting almond allergens in a test sample such as food at a lower concentration without showing cross-reactivity with other nuts, etc. [Means for solving the problem]

[0009] The present inventors have identified two types of monoclonal antibodies suitable for detecting almond allergens by immunochromatography and / or sandwich ELISA. Furthermore, they have continued to study how to quantitatively detect almond allergens with higher sensitivity in sandwich ELISA using these two types of monoclonal antibodies, and have confirmed that more sensitive quantification is possible by using an appropriate extract to prepare a measurement sample, thereby completing the present invention.

[0010] That is, the present invention is as follows. [1] A method for detecting an allergen from an allergen measurement sample extracted from a test sample using an extract solution by sandwich ELISA using a solid-phase monoclonal antibody and a labeled monoclonal antibody, characterized in that the allergen is heat-denatured almond 11S globulin, one of the monoclonal antibodies is the anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and the other is the anti-almond monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts. [2] The method for detecting an almond allergen according to [1] above, wherein the extract contains one or more of the following surfactants: Sodium Laureth-5 Carboxylate Sodium Cocoamphoacetate Sodium Cocoyl Glycinate Laureth-4 Potassium coconut fatty acid Sodium N-dodecanoyl sarcosinate PEG-3 coconut fatty acid amide Sodium Lauraminopropionate Lauryl glucoside PEG-8 Glyceryl Isostearate Oreth-2 [3] The method for detecting an almond allergen according to [2] above, wherein the extract further contains polyoxyethylene (20) sorbitan monolaurate and sodium thiosulfate. [4] The method for detecting an almond allergen according to any one of [1] to [3] above, wherein the allergen measurement sample is extracted by heating from the test specimen using an extraction liquid. [5] The method for detecting an almond allergen according to any one of [1] to [3] above, wherein the allergen measurement sample is extracted from the test specimen using an extraction liquid without heating. [6] The method for detecting an almond allergen according to any one of [1] to [3] above, wherein the immobilized antibody is monoclonal antibody PDAM3 and the labeled monoclonal antibody is monoclonal antibody PDAM4. [7] A kit for detecting almond allergens by sandwich ELISA, comprising: a monoclonal antibody PDAM3 that binds to almonds but not to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts; and a monoclonal antibody PDAM4 that recognizes an epitope different from that of PDAM3 and binds to almonds but not to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts. [8] The kit according to [7] above, further comprising an extract containing one or more of the following surfactants: Sodium Laureth-5 Carboxylate Sodium Cocoamphoacetate Sodium Cocoyl Glycinate Laureth-4 Potassium coconut fatty acid Sodium N-dodecanoyl sarcosinate PEG-3 coconut fatty acid amide Sodium Lauraminopropionate Lauryl glucoside PEG-8 Glyceryl Isostearate Oreth-2 [9] The almond allergen detection kit according to [7] or [8] above, characterized in that the extract further contains polyoxyethylene (20) sorbitan monolaurate, sodium thiosulfate, and / or a mixture of 2-methyl-1,2-thiazol-3(2H)-one and 5-chloro-2-methyl-1,2-thiazol-3(2H)-one.

[10] In an immunochromatographic method for detecting an allergen based on the presence or absence of gold colloid accumulation at the predetermined position, a gold colloid-labeled antibody in which a gold colloid is bound to a monoclonal antibody that recognizes an allergen, a developing support on which a monoclonal antibody that recognizes an allergen and recognizes an epitope different from that of the gold colloid-labeled antibody is fixed at a predetermined position, and a measurement sample of an allergen extracted from a test sample using an extraction solution is developed on the developing support, the allergen is detected based on the presence or absence of gold colloid accumulation at the predetermined position, The method for detecting an almond allergen by immunochromatography is characterized in that one of the monoclonal antibodies is an anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts, and the other is an anti-almond monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts.

[11] The method for detecting almond allergens according to

[10] above, wherein the extraction solution contains SDS, polyoxyethylene (20) sorbitan monolaurate, and sodium thiosulfate.

[12] The method for detecting almond allergens according to

[10] or

[11] above, wherein the allergen measurement sample is obtained by heating and extracting the allergen from the test sample using an extraction solution.

[13] The method for detecting almond allergens according to

[10] or

[11] above, wherein the allergen measurement sample is extracted from the test sample without heating using an extraction solution.

[0011] The present invention is also as follows.

[14] A kit for detecting an almond allergen, comprising: a gold colloid-labeled antibody in which gold colloid is bound to a monoclonal antibody that recognizes an allergen; and a developing support on which a monoclonal antibody that recognizes an allergen and recognizes an epitope different from that of the gold colloid-labeled antibody is fixed at a predetermined position, wherein the allergen is heat-denatured almond 11S globulin, one of the monoclonal antibodies is anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts, and the other is monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts.

[15] The almond allergen detection kit according to

[14] above, further comprising an extraction solution containing SDS, polyoxyethylene (20) sorbitan monolaurate, and sodium thiosulfate.

[16] PDAM3 monoclonal antibody produced by hybridoma (NITE P-03854).

[17] PDAM4 monoclonal antibody produced by hybridoma (NITE P-03855). Effect of the Invention

[0012] By using two types of monoclonal antibodies, monoclonal antibody PDAM3 and monoclonal antibody PDAM4, used in the present invention, almond allergens can be accurately detected quantitatively by sandwich ELISA and qualitatively by immunochromatography. [Brief description of the drawings]

[0013] [Figure 1] This graph shows the absorbance measured at the primary wavelength of 450 nm minus the absorbance measured at the secondary wavelength of 620 nm on the Y-axis for 8 wells of the antibody combination of solid-phase antibody PDAM3 and HRP-labeled antibody PDAM4, and the concentration of heat-denatured almond crude protein in each measurement sample on the X-axis. [Diagram 2] 1 is a graph showing the results of extracting almond protein using sodium dodecyl sulfate according to the prior art. [Diagram 3] 1 is a graph showing the results of extracting almond protein using sodium laureth-5 carboxylate of the present invention. [Figure 4] 1 is a graph showing the results of extracting almond protein using sodium cocoamphoacetate of the present invention. [Diagram 5] 1 is a graph showing the results of extracting almond protein using sodium cocoyl glycinate of the present invention. [Figure 6] 1 is a graph showing the results of extracting almond protein using Laureth-4 of the present invention. [Figure 7] 1 is a graph showing the results of extracting almond protein using the potassium cocoate of the present invention. [Figure 8] 1 is a graph showing the results of extracting almond protein using sodium N-dodecanoyl sarcosinate of the present invention. [Figure 9] 1 is a graph showing the results of extracting almond protein using the PEG-3 coconut oil fatty acid amide of the present invention. [Figure 10]1 is a graph showing the results of extracting almond protein using sodium lauraminopropionate of the present invention. [Figure 11] 1 is a graph showing the results of extracting almond protein using the lauryl glucoside of the present invention. [Figure 12] 1 is a graph showing the results of extracting almond protein using PEG-8 glyceryl isostearate of the present invention. [Figure 13] 1 is a graph showing the results of extracting almond protein using oleth-2 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The method for detecting almond allergens of the present invention is a method for detecting allergens by sandwich ELISA using a solid-phase monoclonal antibody and a labeled monoclonal antibody from a measurement sample obtained by heating and extracting allergens from a test sample using an extracting solution, or from a measurement sample obtained by heating and extracting allergens from a wiping solution or a cleaning solution using an extracting solution, in which the allergen is heat-denatured almond 11S globulin, and one of the monoclonal antibodies binds to almonds but not to cashew nuts, hazelnuts, pistachios, macadamia nuts, and cucumbers. a monoclonal antibody PDAM3 (PDAM3 antibody) that does not bind to almonds, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and another monoclonal antibody PDAM4 (PDAM4 antibody) that recognizes an epitope different from that of the PDAM3 antibody and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts; a detection method using a sandwich ELISA method in which a gold colloid-labeled antibody is bound to a monoclonal antibody that recognizes an allergen, and a gold colloid-labeled antibody that recognizes an allergen and binds to the gold colloid-labeled antibody; The immunochromatographic method uses a developing support on which a monoclonal antibody recognizing an epitope different from that of a specific antibody is fixed at a predetermined position, and a measurement sample of an allergen extracted from a test sample using an extraction solution, and after the measurement sample is developed on the developing support, the allergen is detected based on the presence or absence of gold colloid accumulation at the predetermined position. The allergen is heat-denatured almond 11S globulin, and one of the monoclonal antibodies binds to almonds but not to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and the other is an anti-almond monoclonal antibody PDAM4 that recognizes an epitope different from that of the PDAM3 antibody and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts. There are no particular limitations on the detection method as long as it is an immunochromatographic method. The above-mentioned almond 11S globulin is a general term for globulins, which are generally soluble globular proteins, and have a sedimentation coefficient equivalent to 11S,It is known as a storage protein in plants and is also known as one of the major allergens in almonds.

[0015] The almonds in the present invention include nuts obtained by heat-treating kernels (raw almonds) obtained by removing the flesh and seed shells from fruits harvested from almond (Prunus dulcis) trees, which belong to the deciduous tall tree of the Rosaceae Cerasus. Almonds can be broadly classified into bitter and sweet varieties, but sweet varieties are preferred because bitter varieties contain amygdalin, which causes bitterness. Many varieties of sweet varieties are known, including Nonpareil, California, Carmel, Mission, and Butte.

[0016] 1. Monoclonal antibodies One of the monoclonal antibodies, which binds to the above-mentioned heat-denatured almond 11S globulin but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, is the anti-almond monoclonal antibody PDAM3 produced by an antibody-producing cell (hybridoma) (NITE P-03854), and the other monoclonal antibody, which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, is the anti-almond monoclonal antibody PDAM4 produced by a hybridoma (NITE P-03855).

[0017] An example of a method for preparing antibody-producing cells that produce monoclonal antibodies that bind to the above-mentioned almond 11S globulin is a method in which heat-treated almond 11S globulin or a ground product thereof is administered as an immunogen, either directly or together with an appropriate adjuvant, to a mammal to immunize the mammal.

[0018] Examples of the mammals include rats, mice, rabbits, and goats, but it is preferable to use rats and mice because of the ease of preparation, and monoclonal antibodies derived from rats or mice can be preferably used. Examples of the administration site include intravenous injection, subcutaneous injection, and intraperitoneal injection. Booster immunization can be performed several days to several weeks later, more preferably 10 days to 3 weeks later. Isolation of anti-almond 11S globulin monoclonal antibody-producing cells can be performed by collecting the cells from the immunized animal 1 to 60 days, preferably 1 to 10 days after the final immunization, and the antibody-producing cells are preferably spleen cells, lymph node cells, or peripheral blood-derived cells, and more preferably mouse spleen cells or rat iliac lymph node cells.

[0019] Examples of methods for preparing monoclonal antibodies against almond 11S globulin include known methods such as the hybridoma method by Keller and Milstein (Nature 256, 495-497, 1975), in which the antibody-producing cells are fused with myeloma cells to produce hybridomas that produce monoclonal antibodies that recognize almond 11S globulin, and the hybridomas are cultured on a medium or administered intraperitoneally to an animal to grow in the ascites, and the monoclonal antibodies are then collected from the culture medium or ascites.

[0020] As the myeloma cells, any commonly available established cell line can be used, but it is preferable that the cells have the property of being unable to survive in an unfused state in HAT selection medium and being able to survive as positive hybridomas only when fused with antibody-producing cells. Specific examples of the cells include mouse myeloma cell lines such as P3-X63-Ag8-U, P3X63Ag8.653, NSI / 1-Ag4-1, and NS0 / 1, and rat myeloma cell lines such as YB2 / 0.

[0021] The above-mentioned cell fusion can be performed by mixing antibody-producing cells and myeloma cells in the presence of a fusion promoter such as polyethylene glycol. For example, after completion of cell fusion, the mixture is appropriately diluted with RPMI1640 medium or the like, centrifuged, and the precipitate is suspended in a selection medium such as HAT medium and cultured to select hybridomas, and then the culture supernatant is used to search for antibody-producing hybridomas by enzyme antibody techniques or the like, and cloning is performed by limiting dilution or the like to obtain hybridomas that produce monoclonal antibodies that recognize almond 11S globulin.

[0022] Suitable examples include the anti-almond 11S globulin monoclonal antibody PDAM3 (PDAM3 antibody) produced by the above hybridoma (NITE P-03854) and the anti-almond 11S globulin monoclonal antibody PDAM4 (PDAM4 antibody) produced by the hybridoma (NITE P-03855). The above two types of hybridomas have been entrusted to the National Institute of Technology and Evaluation (NITE) Patent Microorganism Deposit Center (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on March 14, 2023.

[0023] According to the method of the present invention using the above-mentioned PDAM3 antibody and PDAM4 antibody, it is possible to detect the activity of various nuts, such as cashew nuts (Anacardium occidentale: Anacardiaceae Anacardium), hazelnuts (Corylus avellana: Betulaceae Corylus, some of which are hybrids of common corylus and purple corylus), pistachios (Pistacia vera: Anacardiaceae Pistacia), macadamia nuts (Macadamia integrifolia: Proteaceae Macadamia), walnuts (Juglandaceae Juglans), pecan nuts (Juglandaceae Carya), and peanuts (Arachis hypogaea: Fabaceae Fabaceae). The present invention does not react with the proteins of Arachis (sometimes collectively referred to as "other nuts" in the present specification), particularly with the proteins that constitute the edible parts of the nuts.

[0024] 2. Allergen measurement samples The above-mentioned test samples may include food, wiping liquid or cleaning liquid in which allergens may be present, and the above-mentioned foods may include not only the food itself, but also raw materials or scattered material used to produce the food, residues remaining on the wrapping paper or packaging containers in which the food is packaged, and residues such as sediments from the food production process.

[0025] The wiping liquid or cleaning liquid may include a wide variety of liquids that may contain almonds, such as solutions that may contain almond allergens at food manufacturing sites, etc. Specifically, the wiping liquid or cleaning liquid may include, for convenience, cleaning water used to clean equipment used to manufacture food, etc.; rinsing liquid used to remove the cleaning water; and dried products of the cleaning water and rinsing liquid.

[0026] As the solvent in the measurement sample consisting of the wiping liquid or the cleaning liquid, water such as tap water or pure water, (physiological) saline, PBS, and other aqueous solvents can be preferably mentioned, but a mixture of two or more of these may also be used. For example, it may be dissolved in wiping water with water, it may be dissolved in wiping water with (physiological) saline, it may be dissolved in wiping water with PBS, it may be dissolved in wiping water with water and (physiological) saline, it may be dissolved in wiping water with (physiological) saline, it may be dissolved in wiping water with (physiological) saline, it may be dissolved in wiping water with PBS and (physiological) saline, it may be dissolved in wiping water with (physiological) saline, it may be dissolved in wiping PBS with PBS, it may be dissolved in wiping PBS with (physiological) saline, it may be dissolved in wiping PBS with PBS. It is also possible to use an extraction (solution) liquid to perform wiping or washing to prepare a wiping liquid or a cleaning liquid.

[0027] The above test sample can be subjected to 20-fold extraction by adding an extraction (solution) solution (for example, adding 19 mL of extraction solution to 1 mL of wiping solution or washing solution) and then subjected to heat treatment, or without heat treatment (non-heat treatment), to detect allergens as a measurement sample. When wiping or washing is performed with the above extraction (solution) solution to prepare a wiping solution or washing solution, allergens can be detected by using the wiping solution or washing solution as a measurement sample after heat treatment or without heat treatment without performing the above 20-fold extraction.

[0028] The heat treatment can be performed, for example, in a boiling water bath for 5 to 30 minutes, preferably 8 to 15 minutes.

[0029] 3. Detection of almond allergens by sandwich ELISA The method for detecting almond allergens by the sandwich ELISA (enzyme-linked immunosorbent assay) of the present invention includes a method in which two types of antibodies with different antigen recognition sites (epitopes) are used, one of the antibodies is immobilized in advance on a solid phase such as a well of a microplate to form a solid-phase antibody, a test sample which may contain the antigen to be detected is added to the well, and the other antibody which has been labeled with a labeling substance capable of producing a detectable signal is added as a labeled antibody, so that the antigen to be detected (allergen) is sandwiched between the two types of antibodies to form an antibody-antigen-antibody complex, and a reaction according to the labeling substance is carried out to detect and quantify the antigen to be detected.

[0030] The extract used in detecting almond allergens using the above sandwich ELISA method is not particularly limited as long as it is an extract that can detect the almond allergen 11S globulin by adding it to a test sample and extracting it, but is preferably an extract containing one or more selected from sodium laureth-5 carboxylate, sodium cocoamphoacetate, sodium cocoyl glycine, laureth-4, potassium coconut fatty acid, sodium N-dodecanoyl sarcosinate, PEG-3 coconut oil fatty acid amide, sodium lauraminopropionate, lauryl glucoside, PEG-8 glyceryl isostearate, and oleth-2. Examples of suitable surfactants include, more preferably, an extract containing one or more selected from sodium laureth-5 carboxylate, sodium cocoamphoacetate, sodium cocoyl glycine, laureth-4, PEG-3 coconut oil fatty acid amide, sodium lauraminopropionate, lauryl glucoside, PEG-8 glyceryl isostearate, and oleth-2, and even more preferably, an extract containing one or more selected from sodium laureth-5 carboxylate, lauryl glucoside, PEG-3 coconut fatty acid amide, and oleth-2, and preferably, an extract containing sodium laureth-5 carboxylate. By using such a surfactant, it is possible to avoid the problem that antibodies may be denatured and their reactivity may be reduced when detecting allergens in the sandwich ELISA method in which extraction was conventionally performed using sodium dodecyl sulfate.

[0031] The properties of the above surfactants relevant to the present invention are shown in Table 1.

[0032] [Table 1]

[0033] When measuring allergens by the above sandwich ELISA method, the method for preparing a measurement sample of the allergen extracted with an extract is not particularly limited as long as it is a measurement method that uses, as a measurement sample, a solution obtained by mixing 1 g of food with 19 mL of a food allergen extract containing 1% of the above surfactants (one or more selected from sodium laureth-5 carboxylate, sodium cocoamphoacetate, sodium cocoyl glycine, laureth-4, potassium coconut fatty acid, sodium N-dodecanoyl sarcosinate, PEG-3 coconut fatty acid amide, sodium lauraminopropionate, lauryl glucoside, PEG-8 glyceryl isostearate, and oleth-2, followed by heating and extracting for 10 minutes, cooling, and then filtering; or a non-heated solution; and since this method does not require further dilution of the extract, it is possible to perform quantification with 20 times higher sensitivity than the conventional method of diluting the extract 20 times with a buffer solution and measuring it.

[0034] The concentration of the surfactant in the extract is, for example, 0.1 to 3.0%, preferably 0.2 to 2.0%, more preferably 0.5 to 1.5%, and further preferably 0.8 to 1.2%.

[0035] It is preferable that the extract containing the above-mentioned surfactant further contains polyoxyethylene (20) sorbitan monolaurate (Tween (registered trademark) 20) and / or sodium thiosulfate. When such an extract is used, the allergen extraction efficiency is higher and non-specific reactions can be suppressed.

[0036] The concentration of the Tween 20 can be 0.01 to 1.0%, preferably 0.08 to 0.5%, and more preferably 0.1 to 0.3%, and the concentration of the sodium thiosulfate can be 0.01 to 5.0%, preferably 0.02 to 0.5%, and more preferably 0.05 to 0.2%.

[0037] In detecting almond allergens by the above-mentioned sandwich ELISA method, the method for solidifying the antibody to prepare the above-mentioned solid-phase antibody can be a conventionally known method, but other means such as chemical fixation, physical adsorption, covalent bonding, and ionic bonding can be used as appropriate.

[0038] Examples of the material of the carrier used in the above sandwich ELISA include polymeric compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, polysaccharide, as well as glass, metal, magnetic particles, and combinations of these. The shape of the carrier may be tray-shaped, spherical, rod-shaped, disk-shaped, container-shaped, cell, microplate, test tube, etc., with a microplate being preferred.

[0039] The labeling substance used to prepare the labeled antibody is not particularly limited as long as it is a labeling substance that can produce a detectable signal by reacting alone or with other substances, and examples of the labeling substance include enzymes, fluorescent substances, and chemiluminescent substances. Examples of the enzymes include peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, and luciferase. Examples of the fluorescent substances include fluorescein isothiocyanate, phycobiliprotein, dansyl chloride, and tetramethylrhodamine isothiocyanate. Examples of the luminescent substances include luminols, dioxetanes, and acridinium salts. When the labeling substance is an enzyme, a detectable signal is produced by reacting it with a coloring agent, a fluorescent agent, a luminescent agent, a substrate, or the like.

[0040] When the signal produced by carrying out the above-mentioned sandwich ELISA can be measured as absorbance based on enzyme activity, a calibration curve can be prepared using a sample (standard) with a known concentration of almond 11S globulin, and the absorbance of standard solutions of different concentrations can be plotted using this calibration curve to prepare a standard curve (calibration curve). By comparing the absorbance in the test sample with the calibration curve, it is possible to determine whether or not the antigen (allergen) to be detected in the test sample has been detected, and also to quantify the antigen.

[0041] In the method for detecting almond allergens by sandwich ELISA of the present invention, the reactivity (detection accuracy) of allergens extracted using the above-mentioned extract to almonds in a measurement sample can be detected in a range of 0.05 μg / mL to 1.0 μg / mL as the protein concentration of almonds (standard material) in the test sample, and can be detected in a range of 1 to 20 μg / g as the product concentration in foods, etc. assuming a 20-fold extraction.

[0042] The sandwich ELISA allergen detection kit of the present invention is not particularly limited as long as it is a kit for detecting almond allergens by sandwich ELISA, comprising monoclonal antibody PDAM3 that binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and monoclonal antibody PDAM4 that recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and may be configured as a kit that includes an attached document such as an instruction manual describing a method for preparing a measurement sample and a method for detecting almond allergens from the measurement sample. In addition, it is desirable for the kit to have sufficient accuracy and stability to withstand practical use even when stored at room temperature for more than one year from the date of manufacture, and it is preferable to add a preservative such as a mixture of 2-methyl-1,2-thiazol-3(2H)-one and 5-chloro-2-methyl-1,2-thiazol-3(2H)-one (ProClin 300). The concentration of ProClin 300 can be 0.0005 to 0.05%, preferably 0.001 to 0.01%, more preferably 0.0025 to 0.0075%, and even more preferably 0.004 to 0.006%.

[0043] The detection kit for almond allergens using the sandwich ELISA method may contain an extract containing one or more of the surfactants shown below, preferably an extract containing one or more selected from sodium laureth-5 carboxylate, PEG-3 coconut fatty acid amide, lauryl glucoside and oleth-2, and more preferably an extract containing sodium laureth-5 carboxylate. (Surfactant) Sodium Laureth-5 Carboxylate Sodium Cocoamphoacetate Sodium Cocoyl Glycinate Laureth-4 Potassium coconut fatty acid Sodium N-dodecanoyl sarcosinate PEG-3 coconut fatty acid amide Sodium Lauraminopropionate Lauryl glucoside PEG-8 Glyceryl Isostearate Oreth-2 The extract may further contain Tween® 20 and sodium thiosulfate, and may further contain ProClin 300, which has a preservative effect.

[0044] 4. Detection of almond allergens by immunochromatography In the present invention, when the above-mentioned PDAM3 antibody and PDAM4 antibody are used to detect almond allergens by immunochromatography, a combination of PDAM4 antibody as the colloidal gold-labeled antibody bound to the above-mentioned gold colloid and PDAM3 antibody as a monoclonal antibody that recognizes an epitope different from that of the colloidal gold-labeled antibody can be used; and / or a combination of PDAM3 antibody as the colloidal gold-labeled antibody bound to the above-mentioned gold colloid and PDAM4 antibody as a monoclonal antibody that recognizes an epitope different from that of the colloidal gold-labeled antibody can be used. It is more preferable to use a combination of PDAM4 antibody as the colloidal gold-labeled antibody and PDAM3 antibody as a monoclonal (membrane solid-phase) antibody that recognizes an epitope different from that of the colloidal gold-labeled antibody.

[0045] The method for producing the above-mentioned colloidal gold-labeled antibody by binding colloidal gold to the monoclonal antibody includes conventionally known methods and is not particularly limited, but for example, a method in which a solution of monoclonal antibody dissolved in 2 mM borate buffer (pH 9.0) is added to a colloidal gold solution adjusted to pH 9.0 with 0.2 M potassium carbonate solution, reacted at room temperature for 30 minutes, then a 10% BSA solution is added, reacted for another 15 minutes, and centrifuged can be mentioned. In addition, a colloidal gold-labeled antibody carrier can be produced by applying the produced colloidal gold-labeled antibody to, for example, a glass wool conjugate pad and drying it.

[0046] The above-mentioned developing support (antibody-immobilized membrane) can be prepared, for example, by linearly applying a buffer solution containing a monoclonal antibody that recognizes both denatured and native allergens and recognizes an epitope different from that of the gold colloid-labeled antibody to a nitrocellulose membrane, drying the membrane, and then performing a blocking treatment.

[0047] An example of a sample carrier part capable of supporting the above-mentioned measurement sample is a glass wool sample pad. An immunochromatographic strip can be prepared by sequentially connecting the above-mentioned glass wool sample pad, the above-mentioned gold colloid-labeled antibody carrier as necessary, a developing support, and an absorption pad to the other end of the developing support.

[0048] The extraction solution used in preparing the above-mentioned measurement sample is not particularly limited as long as it is an extraction solution that can detect the allergen almond 11S globulin by adding it to the test sample and extracting it, but preferably includes a solution containing sodium dodecyl sulfate (SDS), polyoxyethylene (20) sorbitan monolaurate (Tween 20 (registered trademark)), and sodium thiosulfate, and more preferably includes an extraction solution containing SDS, Tween 20, and sodium thiosulfate in Dulbecco's PBS solution. When such an extraction solution is used, the allergen extraction efficiency is higher and non-specific reactions can be suppressed.

[0049] The concentration of the SDS can be 0.1 to 2.0%, preferably 0.2 to 1.0%, and more preferably 0.3 to 0.7%. The concentration of the Tween 20 can be 0.01 to 1.0%, preferably 0.08 to 0.5%, and more preferably 0.1 to 0.3%. The concentration of the sodium thiosulfate can be 0.01 to 5.0%, preferably 0.02 to 0.5%, and more preferably 0.05 to 0.2%.

[0050] The above-mentioned gold colloid-labeled antibody and preferably a developing solution are added to the measurement sample of the allergen extracted with the above-mentioned extraction solution, and the sample is spotted on an immunochromatographic strip, whereby the gold colloid-labeled antibody that recognizes almond 11S globulin binds to the almond allergen in the measurement sample to form an antigen-antibody complex; the formed antigen-antibody complex moves through the developing support on the immunochromatographic strip by capillary action or the like; the above-mentioned antigen-antibody complex is captured at a predetermined position where a monoclonal antibody that recognizes an almond allergen that recognizes an epitope of 11S globulin different from the gold colloid-labeled antibody is fixed; and the allergen can be detected based on the presence or absence of coloring of a test line that appears due to the accumulation of gold colloid. The gold colloid-labeled antibody may be supported on the immunochromatographic strip in advance, in which case it is not necessarily necessary to add the gold colloid-labeled antibody to the measurement sample.

[0051] The developing solution is preferably a solution containing fetal bovine serum (FBS), and the concentration of fetal bovine serum (FBS) in this case can be 10 to 50% by weight, preferably 20 to 40% by weight, more preferably 25 to 35% by weight. If the concentration is less than 10% by weight, it may be undesirable because it may easily cause non-specific reactions, but there are cases where it is not necessary to add fetal bovine serum (FBS). In addition, the developing solution can also be prepared by suspending, emulsifying, or dissolving various additives such as other surfactants, preservatives, and inorganic salts, as necessary. When a buffer is added to the developing solution, the pH is preferably 4 to 10, particularly 6 to 8, and examples of suitable solutions include phosphate buffer (PBS) and Tris buffer (TBS). The buffer can also be included in the tip of an immunochromatographic stick such as a cotton swab.

[0052] In detecting almond allergens from the above-mentioned measurement sample by immunochromatography, the sample carrier portion of the immunochromatographic strip is immersed in the above-mentioned measurement sample, whereby almond 11S globulin in the above-mentioned measurement sample binds to the colloidal gold-labeled antibody that recognizes the almond allergen to form an antigen-antibody complex; the formed antigen-antibody complex moves through the developing support on the immunochromatographic strip by capillary action or the like; the antigen-antibody complex is captured at a predetermined position where a monoclonal antibody that recognizes the almond allergen and recognizes an epitope of 11S globulin different from the colloidal gold-labeled antibody is fixed; and the allergen can be detected based on the presence or absence of a colored line that appears due to the accumulation of colloidal gold.

[0053] In the method for detecting almond allergens using the immunochromatography method of the present invention, the reactivity of the allergens extracted using the extraction solution to almonds in the measurement sample can be detected at a protein concentration of 2 ppm to 20 ppm of almond (standard) in the test sample.

[0054] The allergen detection kit for immunochromatography of the present invention comprises a gold colloid-labeled antibody in which gold colloid is bound to a monoclonal antibody that recognizes an allergen, and a developing support on which a monoclonal antibody that recognizes an allergen and recognizes an epitope different from that of the gold colloid-labeled antibody is fixed at a predetermined position, the allergen being heat-denatured almond 11S globulin, and one of the monoclonal antibodies binding to almonds but not to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, anti-almond monoclonal antibody PD There is no particular limitation on the almond allergen detection kit, provided that the anti-almond monoclonal antibody is PDAM3, and the other is PDAM4, which recognizes an epitope different from PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and the kit may be configured to include an instruction manual describing a method for preparing a measurement sample and a method for detecting almond allergens from a measurement sample, and an attached document such as an instruction manual for each component of the reagent solution contained in the immunochromatographic strip. In addition, it is preferable that the kit has sufficient accuracy and stability to be practically usable even when stored at room temperature for more than one year from the date of manufacture.

[0055] When the above kit is a kit for measuring allergens extracted from a test sample using an extraction solution, the kit may contain SDS, Tween 20, and sodium thiosulfate as the extraction solution. In addition, FBS may be added to the developing solution. Furthermore, the instruction manual may state that the extraction solution can be used as a wiping solution or a cleaning solution.

[0056] The method for detecting an allergen of the present invention can also be a method for detecting an allergen for non-diagnostic purposes.

[0057] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0058] [Example 1] [Establishment of monoclonal antibody against almond 11S globulin] Based on the method for producing monoclonal antibodies used in the conventional immunochromatography method, an almond 11S globulin monoclonal antibody was established by the following procedure.

[0059] (Preparation of heat-denatured almond standard solution) The edible part of raw almonds (nonpareil variety) (the kernel part with the flesh and seed shell removed) was ground in a mill, defatted with acetone, and then air-dried overnight at room temperature to remove the acetone, and the defatted almond powder was prepared. 0.1 g of the defatted almond powder was weighed out, and 20 mL of Dulbecco's PBS (5913, Nissui Pharmaceutical Co., Ltd.) containing 0.5% sodium dodecyl sulfate (SDS), 0.2% Tween 20, and 0.1% sodium thiosulfate (hereinafter also referred to as "IC extraction solution") was added and stirred, and the mixture was heated in boiling water for 10 minutes for denaturation treatment, cooled, centrifuged, and each centrifugal supernatant was collected to prepare a heat-denatured almond standard solution (D-AM).

[0060] (Preparation of heat-denatured nut extract solution) The edible parts of cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts were each ground in a blender, and 1 g of each was weighed out, and 19 mL of the above extract was added and stirred, after which the nuts were heated in boiling water for 10 minutes for denaturation treatment, and after cooling, the nuts were centrifuged. The supernatants were collected and prepared as cashew nut heat-denatured protein ("D-CS") IC extract solution, hazelnut heat-denatured protein ("D-HZ") IC extract solution, pistachio heat-denatured protein ("D-PS") IC extract solution, macadamia nut heat-denatured protein ("D-MC") IC extract solution, pecan nut heat-denatured protein ("D-PE") IC extract solution, walnut heat-denatured protein IC extract solution ("D-WN"), and peanut heat-denatured protein ("D-PN") IC extract solution. The protein concentration in each solution was measured using a 2-DQuantKit (GE Healthcare Life Sciences) as necessary, and serially diluted in increments of two with the above IC extraction solution from 50 ng / mL to 3.13 ng / mL to prepare IC standard solutions.

[0061] (Purification of almond 11S globulin as an indicator of almond allergens) The defatted almond powder was purified using PrepCell 960 (Bio Rad). The 11S globulin fraction was dialyzed and then freeze-dried. Using the freeze-dried powder, a 0.1% almond 11S globulin solution was prepared in physiological saline, and 500 μL of the solution was dispensed into 1 mL tubes to prepare an almond 11S globulin antigen solution, which was then frozen and stored at −40° C. until use in immunization.

[0062] (Production of rat-derived anti-almond 11S globulin monoclonal antibody) (1) Immunization of rats One 8-week-old F344 / DuCrlCrlj rat (obtained from Charles River Japan) was used as the test animal. For the initial immunization of the rat, an equal amount of complete Freund's adjuvant (Sigma-Aldrich) was added to a tube containing 500 μL of 0.1% almond 11S globulin solution, and the mixture was stirred with a vortex mixer to prepare an emulsion, and 200 μL of this emulsion was injected into the tail base. Two weeks after the initial immunization, 100 μL of 0.1% 11S globulin solution was injected into the tail vein as a booster immunization.

[0063] (2) Generation of antibody-producing hybridomas from rats The preparation of rat-derived antibody-producing hybridomas was performed according to the method of Keller and Milstein (1975). That is, 4 days after the tail vein injection, iliac lymph nodes were aseptically removed from the test rats. The iliac lymph nodes were cut into small pieces, washed with RPMI1640 (Fujifilm Wako Pure Chemical Industries, Ltd.), and passed through a sterile nylon mesh (CellStrainer, 70 mm, Becton Dickinson Co., Ltd.) to obtain an iliac lymph node cell suspension. Iliac lymph node cells were collected by centrifugation at 1000 rpm x 10 minutes, resuspended again in RPMI1640, and the number of cells was counted. This iliac lymph node cell suspension was mixed with a mouse myeloma cell (P3X63Ag8.653) suspension, and centrifuged again at 1000 rpm x 10 minutes to obtain a pellet. 45% polyethylene glycol with an average molecular weight of 3350 was added dropwise to this pellet to perform cell fusion. The cell solution was diluted with RPMI1640 and centrifuged to form a pellet. The pellet was added with HAT selection medium containing hybridoma medium (RPMI1640 medium containing 10% fetal bovine serum, 40 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin) containing 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine, and 5 × 10 6 The cells were dispensed into a 96-well cell culture plate (Becton Dickinson) at 10 cells / well and cultured at 37° C. under 5% CO 2 .

[0064] (3) Cloning of antibody-producing hybridomas by limiting dilution The culture supernatant from each well of the cell culture plate was used as the primary antibody in ELISA to examine the presence of hybridomas producing anti-11S globulin antibodies. Hybridomas from wells that were positive for 11S globulin by ELISA were transferred to a 96-well cell culture plate at 0.9 cells / well and cloned by limiting dilution. The cloned hybridomas were cultured in RPMI1640 medium containing 10% fetal bovine serum, 40 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0065] (4) Monoclonal antibody screening The monoclonal antibodies from the above culture supernatant that were found to be reactive to D-AM containing almond 11S globulin were examined by indirect ELISA for their reactivity to heat-denatured crude proteins containing 11S globulin from cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, namely D-CS, D-HZ, D-PS, D-MC, D-WN, D-PE, and D-PN, respectively, and three types of monoclonal antibodies, PDAM3 antibody, R1 antibody, and R2 antibody, were selected as rat-derived monoclonal antibodies that are highly reactive to D-AM and have low reactivity to D-CS, D-HZ, D-PS, D-MC, D-WN, D-PE, and D-PN.

[0066] (5) Collection of mouse ascites and purification of rat-derived monoclonal antibodies According to Jones et al. (1990), CB-17 / scid mice (obtained from Japan CLEA) were first intraperitoneally injected with 200 μL of incomplete Freund's adjuvant (Sigma-Aldrich). One week later, 5 × 10 6The three types of hybridomas producing the above selected antibodies cloned into cells were inoculated. After ascites collection, the ascites was collected using a syringe. The collected ascites was purified using a Protein G column (GE Healthcare) to obtain rat-derived purified monoclonal antibodies (MAbs) specific to walnut 11S globulin, which were named PDAM3 monoclonal antibody (hereinafter also simply referred to as "PDAM3" or "PDAM3 antibody"), R1 monoclonal antibody, and R2 monoclonal antibody.

[0067] (Production of mouse-derived anti-walnut 11S globulin monoclonal antibody) (1) Immunization of mice Two 4-week-old BALB / c mice (obtained from Japan CLEA) were used as test animals. For the initial immunization of the mice, an equal amount of complete Freund's adjuvant was added to a tube containing 500 μL of 0.1% walnut 11S globulin solution, and the emulsion was prepared by stirring with a vortex mixer. 100 μL of this emulsion was injected intraperitoneally per mouse. For the booster immunization, an equal amount of incomplete Freund's adjuvant was added to a tube containing 500 μL of 0.1% 11S globulin solution, and the emulsion was prepared by stirring with a vortex mixer. 100 μL of this emulsion was injected intraperitoneally per mouse.

[0068] (2) Measurement of antibody titers in the blood One week after the initial or booster injection of 11S globulin, blood was collected from the tail vein of each BALB / c mouse. The collected blood was left at room temperature for 2 hours, then centrifuged to obtain serum. Ten-fold dilutions of these sera were prepared, and the anti-11S globulin antibody titer in the mouse blood was examined by non-competitive ELISA. An alkaline phosphatase-labeled anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch Laboratories) was used as the secondary antibody. Booster immunization was further performed five times at two-week intervals, and 100 μL of 0.1% 11S globulin solution was injected into the tail vein of mice with sufficient antibody titer.

[0069] (3) Preparation of mouse-derived antibody-producing hybridomas The mouse-derived antibody-producing hybridoma was produced according to the method of Keller and Milstein (1975). That is, 4 days after the tail vein injection of the above 11S globulin solution, the spleen was aseptically removed from the mouse. The spleen was cut into pieces, washed with RPMI1640, and passed through a sterile nylon mesh to obtain a spleen cell suspension. The spleen cells were collected by centrifugation at 1000 rpm x 10 minutes, resuspended again in RPMI1640, and the cell number was counted. This spleen cell suspension was mixed with a mouse myeloma cell (P3X63Ag8.653) suspension, and centrifuged again at 1000 rpm x 10 minutes to obtain a pellet. 45% polyethylene glycol with an average molecular weight of 3350 was dropped onto this pellet to perform cell fusion. RPMI1640 was added to the cell solution to dilute it, and then the mixture was centrifuged to pellet it. To this pellet, a hybridoma medium (RPMI1640 medium containing 10% fetal bovine serum, 40 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin) containing 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine was added, and 5 × 10 6 The cells were dispensed into a 24-well cell culture plate (Becton Dickinson) at 10 cells / well and cultured at 37° C. under 5% CO 2 .

[0070] (4) Cloning of mouse antibody-producing hybridomas by limiting dilution The culture supernatant from each well of the cell culture plate was used as the primary antibody in ELISA to examine the presence of hybridomas producing anti-11S globulin antibodies. Hybridomas from wells that were positive for 11S globulin by ELISA were transferred to a 96-well cell culture plate at 0.9 cells / well and cloned by limiting dilution. The cloned hybridomas were cultured in RPMI1640 medium containing 10% fetal bovine serum, 40 μM 2-mercaptoethanol, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0071] (5) Monoclonal antibody screening For the clones in the culture supernatant that showed reactivity to D-AM containing almond 11S globulin, the reactivity to heat-denatured proteins containing 11S globulin, namely D-CS, D-HZ, D-PS, D-MC, D-WN, D-PE, and D-PN, in seven types of nuts, namely cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, was determined by direct ELISA, and three types of mouse-derived monoclonal antibodies, PDAM4 antibody, M1 antibody, and M2 antibody, that were positive for D-AM but negative for the proteins of the other seven types of nuts were selected.

[0072] (6) Collection of mouse ascites and purification of mouse-derived monoclonal antibodies According to Jones et al. (1990), BALB / c mice were first intraperitoneally injected with 200 μL of incomplete Freund's adjuvant. One week later, 5 × 10 6 The hybridomas producing the selected antibodies obtained by cloning the cells were inoculated. After ascites collection, the ascites was collected using a syringe. The collected ascites was purified using a Protein G column (GE Healthcare) to obtain purified mouse-derived monoclonal antibodies specific to walnut 11S globulin, which were named PDAM4 monoclonal antibody (hereinafter also simply referred to as "PDAM4" or "PDAM4 antibody"), M1 monoclonal antibody, and M2 monoclonal antibody.

[0073] [Example 2] [Evaluation of antibody combinations] We decided to investigate antibody combinations suitable for detecting almond proteins by selecting two antibodies from a total of six purified monoclonal antibodies: the three purified monoclonal antibodies derived from rats, PDAM3 antibodies, R1 antibodies, and R2 antibodies, and the three purified monoclonal antibodies derived from mice, PDAM4 antibodies, M1 antibodies, and M2 antibodies.

[0074] (Immobilization of antibodies) Immobilized antibodies were prepared using a 96-well microplate (Nunc-ImmunoModule plate F8NAL, 468667). Each of the six purified antibodies was prepared as a 5 μg / mL immobilized antibody solution in phosphate buffered saline (PBS), and 100 μL / well of each antibody was dispensed into 12 wells. After leaving the plate at 37°C for 1.5 hours to allow the immobilization reaction, each well was washed five times with 250 μL of PBS containing 0.05% Tween 20 (hereinafter referred to as PBST). All subsequent washings were performed using the same procedure. 1% bovine serum albumin (BSA, Sigma-Aldrich A3059) / PBS was dispensed at 150 μL / well, left to stand at 37°C for 1 hour to allow the reaction to proceed, and then washed.

[0075] (Preparation and addition of almond protein solution for measurement) The above D-AM was prepared using PBST to give a 50 ppb D-AM solution, which was used as the almond protein solution for measurement. 100 μL of 50 ppb D-AM solution was dispensed into the 16 wells in which the above solid-phased antibodies were prepared, and the wells were left to stand at 37° C. for 2.5 hours to react the D-AM with each solid-phased antibody, and then washed. PBST was used as the blank.

[0076] (Reaction with HRP-labeled antibody) Each of the six purified monoclonal antibodies was enzyme-labeled with horseradish peroxidase (HRP) using Peroxidase Labeling Kit-SH (LK09, Dojindo Laboratories, Inc.). Twelve types of 0.1 μg / mL HRP-labeled antibodies were prepared using PBST, and 100 μL / well of each of the 12 wells was dispensed and left to react at 37°C for 0.5 hours. Each well was then washed five times with 250 μL of PBST.

[0077] (Addition of color former) A coloring agent 3,3',5,5' tetramethylbenzidine solution (TMB solution, 1-Component Microwell Peroxidase Substrate, SureBlue, 5120-0075, KPL) was dispensed at 100μL / well and allowed to stand for 10 minutes at room temperature under light shielding to allow reaction. The reaction was stopped by dispensing 1N hydrochloric acid (Wako Pure Chemical Industries, 080-01066) at 100μL / well, and the absorbance was measured at a main wavelength of 450nm and a sub-wavelength of 620nm. The actual absorbance was calculated by subtracting the absorbance at the sub-wavelength of 620nm from the absorbance at the main wavelength of 450nm in each well, as shown in Table 2 below. The sub-wavelength of 620nm was measured to cancel the absorbance difference between wells due to stains in the wells caused by samples, clouding of the wells due to cleaning operations, scratches, etc.

[0078] [Table 2]

[0079] (result) In terms of the value obtained by subtracting the absorbance measurement value at the secondary wavelength of 620 nm from the absorbance measurement value at the primary wavelength of 450 nm (net absorbance: 450 nm-620 nm), the net absorbance was higher when the PDAM3 antibody was used as an immobilized antibody and the PDAM4 antibody was used as an enzyme-labeled antibody using Peroxidase, confirming that the use of these two antibodies resulted in stronger reactivity to almond protein.

[0080] The present inventors have confirmed that a specific allergen can be detected by using a combination of two antibodies selected by the above procedure as a method for selecting a combination of antibodies suitable for immunochromatography (Patent Application No. 2023-069165, etc.). That is, almond allergens can be detected by immunochromatography using the PDAM3 antibody and PDAM4 antibody selected by the above results, and almond allergens can be detected in the range of 2 ppm to 20 ppm by immunochromatography using a combination of the PDAM4 antibody as a gold colloid-labeled antibody and the PDAM3 antibody as a monoclonal (membrane solid-phase) antibody that recognizes an epitope different from that of the gold colloid-labeled antibody.

[0081] [Example 3] [2. Consideration of antibody combinations in ELISA configuration] Using the PDAM3 and PDAM4 antibodies, which were confirmed to have strong reactivity with the above almond proteins, we investigated the combination of immobilized and labeled antibodies in the ELISA configuration. Specifically, using a 96-well microplate, we investigated the detection accuracy of the antibody combination of immobilized antibody PDAM3 and HRP-labeled antibody PDAM4 when the concentration of almond protein was changed.

[0082] (Preparation of measurement samples containing almond protein) Assuming that the sample would be diluted 20-fold with a buffer solution after extraction, the above D-AM was serially diluted in increments of 2-fold starting from 50 ng / mL, and the concentrations in the test solution were adjusted to 0.78125 ng / mL (15.625 ng / mL), 1.56250 ng / mL (31.25 ng / mL), 3.125 ng / mL (62.5 ng / mL), 6.25 ng / mL (125 ng / mL), 12.5 ng / mL (250 ng / mL), 25 ng / mL (500 ng / mL), and 50 ng / mL (1000 ng / mL) using 1.0% BSA / PBST to prepare measurement samples containing almond heat-denatured crude protein at each concentration. PBST was used as the blank (0 ng / mL). The values ​​in parentheses are the product equivalent concentrations assuming 20-fold extraction.

[0083] (Primary antibody immobilization reaction) A 5 μg / mL PDAM3 antibody solution was prepared using Dulbecco's PBS (Nissui Pharmaceutical Co., Ltd., 5913) and dispensed into 8 wells at 100 μL / well. After standing at 37°C for 1.5 hours to allow for immobilization reaction, each well to which the PDAM3 antibody had been reacted was washed five times with 250 μL of PBST. Then, 150 μL / well of 1% bovine serum albumin / PBS was dispensed and reacted at 37°C for 1 hour and 30 minutes. After the reaction, each well was washed five times with 250 μL of 0.05% PBST. All subsequent washings were performed using the same procedure. 200 μL / well of 1% BSA / PBS was dispensed into each well, and the wells were left to stand at 37°C for 1 hour to allow reaction, followed by blocking, and then washed.

[0084] (Preparation and addition of measurement samples) Measurement samples containing the eight types of almond heat-denatured crude proteins prepared at the eight concentrations described above were dispensed as standard solutions at 100 μL / well into the eight wells in which the immobilized PDAM3 antibody was prepared, and the wells were left to stand at room temperature for 1.0 hour to allow the D-AM to react with each immobilized antibody, after which the wells were washed.

[0085] (Reaction with HRP-labeled antibody) The PDAM4 antibody was enzyme-labeled with HRP using Peroxidase Labeling Kit-SH to prepare HRP-labeled PDAM4 antibody. 100ng / mL HRP-labeled PDAM4 antibody was prepared using PBST, and 100μL / well was dispensed into the 8 wells where the above-mentioned immobilized PDAM3 antibody had been prepared, and the reaction was allowed to proceed for 0.5h at room temperature. After that, each well was washed five times with 250μL of PBST, and an antigen-antibody complex was formed by the antibody combination of immobilized antibody PDAM3 and HRP-labeled antibody PDAM4.

[0086] (Addition of color former) According to the procedure described in the section on the addition of the color developer, the actual absorbance was determined by subtracting the absorbance at the secondary wavelength of 620 nm from the absorbance at the primary wavelength of 450 nm in each well.

[0087] (result) Figure 1 shows a graph of the eight wells containing the antibody combination of immobilized antibody PDAM3 and HRP-labeled antibody PDAM4, with the absorbance measurements at the primary wavelength of 450 nm to the secondary wavelength of 620 nm on the Y-axis and the concentration of heat-denatured almond crude protein in each measurement sample on the X-axis.

[0088] [Confirmation of cross-reactivity of PDAM3 and PDAM4 antibodies with nuts proteins other than almond] The IC extract solutions of D-CS, D-HZ, D-PS, D-MC, D-WN, D-PE, and D-PN were used as detection targets to evaluate the presence or absence of cross-reactivity in ELISA assays using the above antibody combinations.

[0089] The results of detection of nuts other than walnuts by indirect ELISA using the above antibody combination are shown in Table 3 below.

[0090] [Table 3] However, <1 μg / g indicates that it was not detectable.

[0091] (result) As is clear from the graph in Figure 1, the coefficient of determination (square of the correlation coefficient) was 0.99 or more, ensuring linearity. However, as is clear from Table 3 above, it was confirmed that the antibody combination using PDAM3 as the solid-phase antibody and PDAM4 as the HRP-labeled antibody had reactivity to cashew nuts and hazelnuts other than almonds.

[0092] Therefore, it was confirmed that in the sandwich ELISA method for detecting almonds using PDAM3 and PDAM4 according to the previous procedure, cross-reactivity occurs with other nuts such as cashew nuts, hazelnuts, etc. Therefore, it was determined that it is necessary to consider a means for preventing cross-reactivity with other nuts while maintaining high reactivity with almonds in the sandwich ELISA method.

[0093] [Investigation into improving the sensitivity of the almond sandwich ELISA method] Since it has been pointed out that SDS contained in the above IC extraction solution may inhibit the reaction of monoclonal antibodies in the ELISA method, we decided to study the extraction solution in order to develop a highly sensitive ELISA method that can detect almond allergens at 1 μg / mL or less. First, we decided to replace SDS with other components. We decided to use the following surfactants as replacement candidates to examine an excellent extraction solution with low cross-reactivity for use in the sandwich ELISA method.

[0094] (Preparation of extract) The following 11 types of surfactants were added to Dulbecco's phosphate buffered saline (Nissui Pharmaceutical, 5913 (hereinafter also referred to as "PBS")) containing 0.2% Tween (registered trademark) 20 (MP Biomedicals, 103168) and 0.1% sodium thiosulfate (Wako Pure Chemical Industries, 197-03605) at final concentrations of 1.0%, 5.0%, or 10.0%, instead of 0.5% sodium dodecyl sulfate (SDS). Three types of extracts were prepared for each of the 12 types of surfactants (36 types in total), and comparisons were made with SDS.

[0095] (1) SDS (2) Sodium laureth-5 carboxylate (3) Sodium cocoamphoacetate (4) Sodium cocoyl glycinate (5) Laureth-4 (6) Potassium coconut fatty acid (7) Sodium N-dodecanoyl sarcosinate (8) PEG-3 coconut fatty acid amide (9) Sodium lauraminopropionate (10) Lauryl glucoside (11) PEG-8 glyceryl isostearate (12) Oleth-2

[0096] (Preparation of heat-denatured almond standard solution for ELISA) The above 36 kinds of extracts were further added with 0.005% ProClin300 (Sigma-Aldrich) to obtain the extracts. According to the procedure in "Preparation of heat-denatured almond standard solution" above, heat-denatured almond standard solutions were prepared. The protein concentration in each solution was measured as necessary using 2-DQuantKit (GE Healthcare Life Science).

[0097] (Immobilization of antibodies) The above 36 types of heat-denatured almond standard solutions were measured using an antibody combination using PDAM3 as the immobilized antibody and PDAM4 as the HRP-labeled antibody. 100 μL of a solution adjusted to 5 μg / mL in PBS was added to each well of the microplate and reacted at 37°C for 1 hour and 30 minutes. Each well was washed five times with 250 μL of 0.05% PBST. All subsequent washings were performed using the same procedure. 200 μL of 1% BSA / PBS was dispensed into each well, left to stand at 37°C for 1 hour to react, and then washed.

[0098] (Sample reaction) The almond protein solution was added to each of the 36 types of extraction solutions so that the almond protein was 62.5ppb, 250ppb, and 1000ppb to prepare a crude measurement solution, and then the crude measurement solution was diluted 20 times with PBST containing 0.5% fish gelatin (Sigma-Aldrich, 7765) to adjust the almond protein concentration to 3.125ppb, 12.5ppb, and 50ppb to prepare each measurement solution. 100μL of each measurement solution was added to each well, reacted at 25℃ for 1 hour, and then washed. For the above 36 types of extraction solutions, the negative control was a solution without almond protein.

[0099] (Enzyme-labeled antibody reaction) 100 μL of horseradish peroxidase-labeled anti-almond mouse monoclonal antibody (NITE P-02041) was added to each well, and the wells were incubated at 25° C. for 30 minutes and then washed.

[0100] (Addition of color former) According to the procedure described in the section on adding the color developer, the actual absorbance was calculated by subtracting the absorbance at the secondary wavelength of 620 nm from the absorbance at the primary wavelength of 450 nm in each well. Graphs were created using the absorbance values ​​for the 12 types of surfactants when the almond protein concentrations were 3.125 ppb, 12.5 ppb, and 50 ppb. The results are shown in Figures 2 to 13.

[0101] (result) 1) As is clear from FIG. 2, when an extract containing sodium dodecyl sulfate (SDS) was used, no measurement was possible at any concentration. 2) Sodium laureth-5 carboxylate in Fig. 3, PEG-3 coconut fatty acid amide in Fig. 9, lauryl glucoside in Fig. 11, and oleth-2 in Fig. 13 showed very favorable results, with absorbances of 0.50 or more at 50ng / mL at concentrations of 1.0%, 5.0%, and 10.0%, and it was confirmed that measurements were possible at concentrations of 1% to 10%. In particular, sodium laureth-5 carboxylate in Fig. 3 showed particularly favorable results, with absorbances of 0.99 or more at 50ng / mL at concentrations of 1.0%, 5.0%, and 10.0%. 3) Laureth-4 in Figure 6, sodium lauraminopropionate in Figure 10, and PEG-8 glyceryl isostearate in Figure 12 showed extremely favorable results, with absorbance at 50 ng / mL of 0.50 or more at both the 1.0% and 5.0% concentrations, confirming that satisfactory measurement is possible at concentrations of 1% to 5%. 4) For sodium cocoamphoacetate in Figure 4 and sodium cocoyl glycine in Figure 5, the absorbance at 50 ng / mL was 0.40 or more at a concentration of 1.0%, confirming that measurement was possible at a concentration of 1.0%. 5) For potassium coconut fatty acid in Figure 7 and sodium N-dodecanoyl sarcosinate in Figure 8, when the concentration was 1.0%, the absorbance at 50 ng / mL was 0.10 or more, confirming that measurement was possible at a concentration of 1.0%.

[0102] [Examination of extract concentration] Assuming commercially available almond-containing processed foods, measurements were performed by sandwich ELISA using PDAM3 antibody as the immobilized antibody and PDAM4 antibody as the HRP-labeled antibody. For commercially available porridge, cookies, and orange juice that are clearly not containing almonds, 1g x 4 samples were prepared, and the above-mentioned D-AM was added so that the almond protein in each food was 10 μg / g. 19 mL of Dulbecco's PBS containing 0.5%, 1.0%, 2.5%, or 5.0% sodium laureth-5 carboxylate, 0.2% Tween 20, and 0.1% sodium thiosulfate was added as an extraction solution, and extracted in boiling water for 10 minutes to prepare each measurement sample. Using the calibration curve obtained by measuring the standard solution, the almond protein concentration in each measurement solution was quantified (calculated) by linear regression. The recovery rate was calculated as the quantified value / added amount x 100. The results are shown in Table 4.

[0103] [Table 4]

[0104] As is clear from Table 4 above, the recovery rates were good, ranging from 70 to 132%, for all foods and at all concentrations, but the recovery rate was 104 to 109%, and it was determined that the surfactant concentration of 1.0%, which was closest to 100%, was optimal for the extract.

[0105] The edible parts of cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts were each ground in a mill, and 1 g of each was weighed out, and 19 mL of Dulbecco's PBS containing 1.0% sodium laureth-5 carboxylate, 0.2% Tween 20, and 0.1% sodium thiosulfate was added as an extraction solution and stirred, and then heated in boiling water for 10 minutes for denaturation treatment. After cooling, the mixture was centrifuged, and the centrifuged supernatants were collected and analyzed to determine the cashew nut heat-denatured protein ("D The following solutions were prepared: D-CS) ELISA extraction solution, hazelnut heat-denatured protein (D-HZ) ELISA extraction solution, pistachio heat-denatured protein (D-PS) ELISA extraction solution, macadamia nut heat-denatured protein (D-MC) ELISA extraction solution, pecan nut heat-denatured protein (D-PE) ELISA extraction solution, walnut heat-denatured protein ELISA extraction solution (D-WN), and peanut heat-denatured protein (D-PN) ELISA extraction solution. The protein concentration in each solution was measured as necessary using 2-DQuantKit (GE Healthcare Life Science).

[0106] Using the above cashew nut, hazelnut, pistachio, macadamia nut, walnut, pecan nut, and peanut ELISA extraction solutions, PDAM3 was used as the immobilized antibody, and PDAM4 was used as the HRP-labeled antibody, and a sandwich ELISA assay was performed according to the procedure of the primary antibody immobilization reaction, addition of the measurement sample, reaction with the HRP-labeled antibody, and color development. The results are shown in Table 5.

[0107] [Table 5]

[0108] Furthermore, for the raw foods listed as specific raw materials that must be labeled under the Food Labeling Act (promulgated June 28, 2013), chicken eggs, milk, wheat flour, buckwheat flour, peanuts, shrimp, and crab, we ground up each of them and extracted 1 g of each with the above extract containing 1.0% sodium laureth-5 carboxylate to examine whether they cross-reacted. The results are shown in Table 6.

[0109] [Table 6]

[0110] (result) It was also confirmed that there was no cross-reactivity with foods other than nuts, such as chicken eggs, milk, wheat flour, buckwheat flour, peanuts, shrimp, and crab.

[0111] (Quantification of almond protein in wipe solution) Assuming swabbing tests at food manufacturing sites, almond milk was added to five types of commercially available buffer solutions for swabbing tests (products A to E) at concentrations of 1.0, 0.1, and 0.05 μg / mL to create model swabbing solutions. After extraction with an extraction solution containing 1.0% sodium laureth-5 carboxylate, measurements were performed by ELISA. The amount of almond protein in the model swabbing solution was quantified using the regression line obtained from the measurement results of the standard solution, and the recovery rate was calculated. The results are shown in Table 7.

[0112] [Table 7]

[0113] For the products A to E verified above, the recovery rates were 100% or more (103-136%) when the additive concentration of the products was 0.05 μg / mL, 60% or more (63-121%) when the additive concentration was 0.1 μg / mL, and 50% or more (53-59%) when the additive concentration was 1.00 μg / mL, confirming that almond allergens can be quantitatively and qualitatively detected even when they are present in the products at low concentrations of less than 0.1 μg / mL. [Industrial Applicability]

[0114] The method for detecting an almond allergen of the present invention, which can rapidly and accurately detect an almond allergen in a sample, and the allergen detection kit of the present invention that can be used therefor, are particularly useful in the food industry.

Claims

1. The method for detecting an almond allergen by sandwich ELISA using a solid-phase monoclonal antibody and a labeled monoclonal antibody from an allergen measurement sample extracted from a test specimen using an extracting solution, wherein the allergen is heat-denatured almond 11S globulin, one of the monoclonal antibodies is an anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts, and the other is an anti-almond monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts.

2. 2. The method for detecting an almond allergen according to claim 1, characterized in that the extract contains one or more of the following surfactants: ・Sodium laureth-5 carboxylate ・Sodium cocoamphoacetate ・Sodium cocoyl glycine ・Laureth-4 ・Potassium coconut fatty acid Sodium N-dodecanoyl sarcosinate ・PEG-3 coconut fatty acid amide ・Sodium lauraminopropionate ・Lauryl glucoside ・PEG-8 glyceryl isostearate ・Oles-2

3. The method for detecting an almond allergen according to claim 2, wherein the extract further contains polyoxyethylene (20) sorbitan monolaurate and sodium thiosulfate.

4. 4. The method for detecting almond allergens according to claim 1, wherein the allergen measurement sample is extracted by heating from the test specimen using an extraction liquid.

5. 4. The method for detecting almond allergens according to claim 1, wherein the allergen measurement sample is extracted from the test specimen using an extraction liquid without heating.

6. 4. The method for detecting an almond allergen according to claim 1, wherein the immobilized antibody is monoclonal antibody PDAM3, and the labeled monoclonal antibody is monoclonal antibody PDAM4.

7. A kit for detecting an almond allergen by sandwich ELISA, comprising: a monoclonal antibody PDAM3 that binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts; and a monoclonal antibody PDAM4 that recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, and peanuts.

8. The kit according to claim 7, further comprising an extract solution containing one or more of the following surfactants: ・Sodium laureth-5 carboxylate ・Sodium cocoamphoacetate ・Sodium cocoyl glycine ・Laureth-4 ・Potassium coconut fatty acid Sodium N-dodecanoyl sarcosinate ・PEG-3 coconut fatty acid amide ・Sodium lauraminopropionate ・Lauryl glucoside ・PEG-8 glyceryl isostearate ・Oles-2

9. The almond allergen detection kit according to claim 7 or 8, characterized in that the extract further contains polyoxyethylene (20) sorbitan monolaurate, sodium thiosulfate and / or a mixture of 2-methyl-1,2-thiazol-3(2H)-one and 5-chloro-2-methyl-1,2-thiazol-3(2H)-one.

10. The present invention relates to an immunochromatographic method for detecting an allergen by detecting the presence or absence of gold colloid accumulation at a predetermined position using a gold colloid-labeled antibody obtained by binding gold colloid to a monoclonal antibody that recognizes an allergen, a developing support on which a monoclonal antibody that recognizes an allergen and recognizes an epitope different from that of the gold colloid-labeled antibody is immobilized at a predetermined position, and a measurement sample of an allergen extracted from a test sample using an extraction solution, the measurement sample is developed on the developing support, and the allergen is detected by detecting the presence or absence of gold colloid accumulation at the predetermined position, the allergen being heat-denatured almond 11S globulin, The method for detecting an almond allergen by immunochromatography is characterized in that one of the monoclonal antibodies is an anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts, and the other is an anti-almond monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts.

11. The method for detecting an almond allergen according to claim 10, characterized in that the extraction solution contains SDS, polyoxyethylene (20) sorbitan monolaurate, and sodium thiosulfate.

12. 12. The method for detecting almond allergens according to claim 10 or 11, wherein the allergen measurement sample is obtained by heating and extracting the allergen from the test specimen using an extraction solution.

13. 12. The method for detecting almond allergens according to claim 10 or 11, characterized in that the allergen measurement sample is extracted from the test specimen using an extraction solution without heating.

14. A kit for detecting an almond allergen, comprising: a gold colloid-labeled antibody in which gold colloid is bound to a monoclonal antibody that recognizes an allergen; and a developing support on which a monoclonal antibody that recognizes an allergen and recognizes an epitope different from that of the gold colloid-labeled antibody is fixed at a predetermined position, wherein the allergen is heat-denatured almond 11S globulin, one of the monoclonal antibodies is anti-almond monoclonal antibody PDAM3 which binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts, and the other is monoclonal antibody PDAM4 which recognizes an epitope different from that of PDAM3 and binds to almonds but does not bind to cashew nuts, hazelnuts, pistachios, macadamia nuts, walnuts, pecan nuts, or peanuts.

15. The almond allergen detection kit according to claim 14, further comprising an extraction solution containing SDS, polyoxyethylene (20) sorbitan monolaurate, and sodium thiosulfate.

16. PDAM3 monoclonal antibody produced by hybridoma (NITE P-03854).

17. PDAM4 monoclonal antibody produced by hybridoma (NITE P-03855).

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