Methods for extracting allergens from food

Abstract

To develop an ELISA method having higher safety and excellent detection sensitivity.SOLUTION: An allergen in a food product is measured by a sandwich ELISA method by using an extract including a surfactant (sodium laureth-5 carboxylate, lauryl glucoside, sodium PEG-3 cocamide sulfate and the like) selected by a screening method in which a measurement result in a method for detecting an allergen in a food product by the sandwich ELISA method, the method including mixing 1 g of a food product and 19 mL of an allergen extract in the food product including 1% of surfactant, performing heating extraction in boiled water for 10 minutes, cooling the solution before filtration, and using the solution as a measurement solution without dilution, selects a surfactant in which an absorbance value of 50 ppb of the allergen in a wavelengths of 450-620 nm indicates 0.5 or more.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for screening surfactants used in a method for extracting allergens in food by immunochromatography, a method for extracting allergens in food in allergen detection using a surfactant obtained by the screening method, a method for measuring allergens in food using a surfactant obtained by the screening method, and a kit for measuring allergens in food containing the surfactant obtained by the screening method. [Background technology]

[0002] The present inventors have proposed a method for detecting allergens by immunochromatography, which comprises a developing support on which a colloidal gold-labeled antibody, which is a monoclonal antibody against denatured and native allergens bound to a gold colloid, and a monoclonal antibody against denatured and native allergens that recognizes an epitope different from that of the colloidal gold-labeled antibody, is immobilized at a predetermined position, and a developing solution containing a measurement sample of denatured and native allergens extracted from a test food sample using an anionic surfactant and a thiosulfate salt, or an anionic surfactant and a nonionic surfactant, is used to develop the allergen on the developing support and detect the allergen based on the presence or absence of gold colloid accumulation, characterized in that the developing solution contains at least 10% by weight of fetal bovine serum (FBS) (Patent Document 1). The extraction method in this method is extraction in boiling water for 10 minutes.

[0003] The present inventors have also proposed a method for detecting allergens by immunochromatography, which uses a developing support on which a colloidal gold-labeled antibody, which is a monoclonal antibody that recognizes both denatured and native allergens, is bound to a colloidal gold, a monoclonal antibody that recognizes both denatured and native allergens and recognizes an epitope different from that of the colloidal gold-labeled antibody, is immobilized at a predetermined position, a measurement sample of an allergen extracted from a test sample using an extract, and a developing solution containing at least 10% by weight of fetal bovine serum (FBS), and a mixture of the colloidal gold-labeled antibody, the measurement sample, and the developing solution is developed on the developing support, and the allergen is detected based on the presence or absence of gold colloid accumulation at the predetermined position, characterized in that the allergen is soybean 7S globulin or sesame 11S globulin, and the extracting solution contains an anionic surfactant, a thiosulfate, and a nonionic surfactant (Patent Document 2). The extraction method in this method is also extraction in boiling water for 10 minutes.

[0004] According to the Food Labeling Act (promulgated on June 28, 2013), quantitative testing of allergens in specific raw materials (items subject to mandatory labeling: eggs, milk, wheat, buckwheat, peanuts, shrimp, and crab) is carried out using the notified ELISA method. This notified method stipulates that the quantitative analysis of allergens is performed by the ELISA method using an extract (0.5% sodium dodecyl sulfate and 1M Na2SO3) described in Patent No. 5451854 (patent holder: Morinaga & Co., Ltd., Patent Document 3), and it takes about two days for the test results to be available. The extraction method in this method involves extraction at room temperature for 16 hours.

[0005] Similarly, according to the Food Labeling Act (promulgated on June 28, 2013), quantitative testing of allergens in substances equivalent to specific raw materials (21 items recommended for labeling) is currently being carried out using kits made overseas, as there are no domestically produced ELISA kits. There are also testing methods such as the PCR method, but these are qualitative methods and have the problem of not being able to quantify them. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5735411 [Patent Document 2] Patent No. 6510307 [Patent Document 3] Patent No. 5451854 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, quantitative testing of allergens in items equivalent to specific raw materials (21 items recommended for labeling) is carried out using kits manufactured overseas, but there are problems with this, such as the sensitivity not matching that of Japan's labeling system and the accuracy of the testing being unclear.

[0008] Quantitative testing of allergens in specific raw materials (7 items that are required to be labeled) is carried out using the ELISA method, which is a notified method. In this notified method, the extraction solution (0.5% sodium dodecyl sulfate and 1M Na2SO3) described in Patent Document 3 is used. The sodium dodecyl sulfate contained in this extraction solution is an anionic surfactant widely used in biochemistry experiments, and has a high ability to solubilize proteins, but if the concentration of sodium dodecyl sulfate in the test solution is high, there is a problem that the antibody is denatured and the reactivity is reduced. In the test procedure of the notified method using sodium dodecyl sulfate, 19 mL of the extraction solution is mixed with 1 g of food (20-fold extraction), and the food is shaken and extracted overnight (12 hours or more) at room temperature, and the allergens are quantitatively tested using the test solution that is 20-fold diluted with a buffer solution (20-fold extraction x 20-fold dilution = 400-fold diluted test solution). Furthermore, sodium dodecyl sulfate cannot be said to be without problems in that it falls under the Act on Reporting, etc. of Releases and Transfer Registers of Chemical Substances, which is a law concerning the release and movement of chemical substances that may be harmful to human health and the ecosystem. An object of the present invention is to develop an ELISA method which is safer and has excellent detection sensitivity. [Means for solving the problem]

[0009] The present inventors focused on surfactants that are used in cosmetics and high-end shampoos and have high safety and low environmental impact, and evaluated whether they can be applied to the ELISA method. As a result, they selected sodium laureth-5 carboxylate, lauryl glucoside, and sodium PEG-3 coconut fatty acid amide MEA (monoethanolamine) sulfate as surfactants that can be measured at a surfactant concentration of 1 to 10% by the new ELISA method and can be measured with high sensitivity without dilution, and completed the present invention.

[0010] That is, the present invention is specified by the following items. (1) A method for extracting allergens in food for use in a method for detecting allergens in food using an ELISA method, comprising mixing the food with an extraction liquid containing one or more of the following surfactants, and extracting the allergens in the food: (Surfactant) Sodium Laureth-5 Carboxylate Lauryl glucoside ·PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium Oleate Sodium N-dodecanoyl sarcosinate Potassium coconut fatty acid 2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine PEG-8 Glyceryl Isostearate Sodium Cocoyl Glycinate Polyoxyethylene oleyl ether Sodium Lauraminopropionate (2) The method for extracting allergens in food according to (1) above, characterized in that the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate. (3) A method for measuring allergens in food by sandwich ELISA, which comprises mixing 1 g of food with 19 mL of a food allergen extract containing 1% of any of the following surfactants, heating the mixture in boiling water for 10 minutes to extract the allergens, cooling the mixture, and then filtering the mixture without diluting it, to use as the measurement solution. (Surfactant) Sodium Laureth-5 Carboxylate Lauryl glucoside ·PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium N-dodecanoyl sarcosinate Potassium coconut fatty acid 2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine PEG-8 Glyceryl Isostearate Sodium Cocoyl Glycinate Polyoxyethylene oleyl ether Sodium Lauraminopropionate (4) The method for measuring allergens in food according to (3) above, characterized in that the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate. (5) A kit for measuring allergens in food by ELISA, comprising an extract containing the following surfactant and a monoclonal antibody that specifically recognizes the allergen to be measured: (Surfactant) Sodium Laureth-5 Carboxylate Lauryl glucoside ·PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium N-dodecanoyl sarcosinate Potassium coconut fatty acid 2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine PEG-8 Glyceryl Isostearate Sodium Cocoyl Glycinate Polyoxyethylene oleyl ether Sodium Lauraminopropionate (6) The allergen measuring kit according to (5) above, characterized in that the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate. (7) A method for screening surfactants to be used in a method for extracting allergens in food, comprising mixing 1 g of food with 19 mL of a food allergen extract containing 1% surfactant, heating and extracting in boiling water for 10 minutes, cooling, filtering, and using the filtered solution without dilution as a measurement solution to detect allergens in food by sandwich ELISA, and selecting a surfactant that shows an absorbance value of 0.5 or more at 50 ppb of allergen. (8) The following surfactant for allergen measurement obtained by the screening method described in (7) above: (Surfactant) Sodium Laureth-5 Carboxylate Lauryl glucoside ·PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium N-dodecanoyl sarcosinate Potassium coconut fatty acid 2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine PEG-8 Glyceryl Isostearate Sodium Cocoyl Glycinate Polyoxyethylene oleyl ether Sodium Lauraminopropionate Effect of the Invention

[0011] According to the present invention, it is possible to select a surfactant that can be measured at a surfactant concentration of 1 to 10% and can be measured with high sensitivity without dilution, and the selected surfactant can be quantified without being affected by food components. [Brief description of the drawings]

[0012] [Figure 1]1 is a graph showing the results of extracting sesame protein using sodium dodecyl sulfate according to the conventional method. [Diagram 2] 1 is a graph showing the results of extracting sesame protein using sodium laureth-5 carboxylate of the present invention. [Diagram 3] 1 is a graph showing the results of extracting sesame protein using the lauryl glucoside of the present invention. [Figure 4] 1 is a graph showing the results of extracting sesame protein using PEG-3 coconut fatty acid amide MEA sodium sulfate of the present invention. [Diagram 5] 1 is a graph showing the results of extracting sesame protein using sodium oleate of the present invention. [Figure 6] 1 is a graph showing the results of extracting sesame protein using sodium N-dodecanoyl sarcosinate of the present invention. [Figure 7] 1 is a graph showing the results of extracting sesame protein using potassium coconut fatty acid of the present invention. [Figure 8] 1 is a graph showing the results of extracting sesame protein using 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine of the present invention. [Figure 9] 1 is a graph showing the results of extracting sesame protein using PEG-8 glyceryl isostearate of the present invention. [Figure 10] 1 is a graph showing the results of extracting sesame protein using sodium cocoyl glycinate of the present invention. [Figure 11] 1 is a graph showing the results of extracting sesame protein using the polyoxyethylene oleyl ether of the present invention. [Figure 12] 1 is a graph showing the results of extracting sesame protein using sodium lauraminopropionate of the present invention. [Figure 13] 1 is a graph showing the results of extracting almond protein using sodium dodecyl sulfate according to the prior art. [Figure 14] 1 is a graph showing the results of extracting almond protein using sodium laureth-5 carboxylate of the present invention. [Figure 15] 1 is a graph showing the results of extracting almond protein using the lauryl glucoside of the present invention. [Figure 16] 1 is a graph showing the results of extracting almond protein using PEG-3 coconut fatty acid amide MEA sodium sulfate of the present invention. [Figure 17] 1 is a graph showing the results of extracting almond protein using sodium oleate of the present invention. [Figure 18] 1 is a graph showing the results of extracting almond protein using sodium N-dodecanoyl sarcosinate of the present invention. [Figure 19] 1 is a graph showing the results of extracting almond protein using the potassium cocoate of the present invention. [Figure 20] 1 is a graph showing the results of extracting almond protein using 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine of the present invention. [Figure 21] 1 is a graph showing the results of extracting almond protein using PEG-8 glyceryl isostearate of the present invention. [Figure 22] 1 is a graph showing the results of extracting almond protein using sodium cocoyl glycinate of the present invention. [Figure 23] 1 is a graph showing the results of extracting almond protein using the polyoxyethylene oleyl ether of the present invention. [Figure 24] 1 is a graph showing the results of extracting almond protein using sodium lauraminopropionate of the present invention. [Diagram 25] An outline of the notified method and the novel ELISA method of the present invention is shown in FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The screening method for surfactants to be used in the method for extracting allergens in food of the present invention is not particularly limited as long as it is a method in which a surfactant that shows an absorbance value of 0.5 or more at a wavelength of 450-620 nm at 50 ppb of allergens is selected in a sandwich ELISA method by mixing 1 g of food with 19 mL of a food allergen extract containing 1% surfactant, heating and extracting in boiling water for 10 minutes, cooling and filtering the solution without diluting it as a measurement solution, and detecting allergens in food. Sodium laureth-5 carboxylate, lauryl glucoside, PEG-3 coconut fatty acid amide MEA sodium sulfate, sodium N-dodecanoyl sarcosinate, potassium coconut fatty acid, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, PEG-8 glyceryl isostearate, sodium cocoyl glycine, polyoxyethylene oleyl ether, and sodium lauraminopropionate can be selected as surfactants for allergen measurement, with sodium laureth-5 carboxylate, lauryl glucoside, and PEG-3 coconut fatty acid amide MEA sodium sulfate being particularly suitable.

[0014] The method for extracting allergens in food of the present invention is a method for extracting allergens in food for a method for detecting allergens in food using an ELISA method, which involves mixing a food with an extracting solution containing one or more of surfactants (sodium laureth-5 carboxylate, lauryl glucoside, PEG-3 coconut fatty acid amide MEA sodium sulfate, sodium oleate, sodium N-dodecanoyl sarcosinate, potassium coconut fatty acid, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, PEG-8 glyceryl isostearate, sodium cocoyl glycine, polyoxyethylene oleyl ether, and sodium lauraminopropionate can be selected as surfactants for allergen measurement, and among these, sodium laureth-5 carboxylate, lauryl glucoside, or PEG-3 coconut fatty acid amide MEA sodium sulfate) and extracting the allergens in the food, without any particular limitation. Among the above-mentioned surfactants, sodium laureth-5 carboxylate, lauryl glucoside, and PEG-3 coconut fatty acid amide MEA sodium sulfate can be preferably used.

[0015] The method for measuring allergens in food by the sandwich ELISA method of the present invention is not particularly limited as long as it involves mixing 1 g of food with 19 mL of an allergen extract in the food containing 1% of a surfactant (one or more selected from sodium laureth-5 carboxylate, lauryl glucoside, PEG-3 coconut fatty acid amide MEA sodium sulfate, sodium N-dodecanoyl sarcosinate, potassium coconut fatty acid, 2-alkyl-N-carboxymethyl-N-hydroxyethyl imidazolinium betaine, PEG-8 glyceryl isostearate, sodium cocoyl glycine, polyoxyethylene oleyl ether, and sodium lauraminopropionate), heating and extracting with boiling water for 10 minutes, cooling, and then filtering the solution, which is then used as a measurement solution without dilution. Of the above-mentioned surfactants, sodium laureth-5 carboxylate, lauryl glucoside, and PEG-3 coconut fatty acid amide MEA sodium sulfate can be preferably used.

[0016] In the measurement method of the present invention, as shown in Table 1, the measurement is performed without diluting 20-fold with a buffer solution, thereby enabling quantification with 20-fold higher sensitivity.

[0017] [Table 1]

[0018] The allergen measurement kit of the present invention is not particularly limited as long as it is a kit for measuring allergens in food by ELISA, and contains an extract containing a surfactant (one or more selected from sodium laureth-5 carboxylate, lauryl glucoside, PEG-3 coconut fatty acid amide MEA sodium sulfate, sodium N-dodecanoyl sarcosinate, potassium coconut fatty acid, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, PEG-8 glyceryl isostearate, sodium cocoyl glycine, polyoxyethylene oleyl ether, and sodium lauraminopropionate) and a monoclonal antibody that specifically recognizes the allergen to be measured. Among the above-mentioned surfactants, an extract containing sodium laureth-5 carboxylate, lauryl glucoside, or sodium PEG-3 coconut fatty acid amide MEA sulfate can be preferably exemplified.

[0019] The properties of sodium dodecyl sulfate and the above surfactants related to the present invention are shown in Table 2 below.

[0020] [Table 2]

[0021] 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

[0022] [Example 1] (Preparation of sesame protein solution) The study was carried out using sesame protein as an allergen protein. The sesame protein solution was prepared by grinding sesame in a mill and then defatting it with acetone, referring to "Testing methods for foods containing allergens (Reference) (March 26, 2014, Consumer Affairs Agency)". The protein concentration in the sesame protein solution was measured using a 2-D QuantKit (GE Healthcare Life Sciences, 80-6483-56).

[0023] [Measurement of allergen proteins using sandwich ELISA] The types of surfactants used in the study are shown in Table 3 below.

[0024] [Table 3]

[0025] (Preparation of extraction solution) Three types of extraction solutions (36 types in total) were prepared for each of the 12 surfactants by adding each of the 12 surfactants to a final concentration of 1.0%, 5.0%, or 10.0% 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), 0.1% sodium thiosulfate (Wako Pure Chemical Industries, 197-03605), and 0.09% sodium azide (Wako Pure Chemical Industries, 195-11092).

[0026] (Immobilization of antibodies) Anti-sesame mouse monoclonal antibody (NITE P-02042) was adjusted to 5 μg / mL in PBS, and 100 μL of the solution was added to each well of a microplate (Nunc-Immuno Module plate F8 NAL, 468667) and reacted at 37°C for 1 hour and 30 minutes. After the reaction, each well was washed five times with 300 μL of PBS containing 0.05% TWEEN (registered trademark) 20 (hereinafter also referred to as "PBST"). All subsequent washings were performed using the same procedure.

[0027] (blocking) 200 μL of PBS containing 1% bovine serum albumin (Sigma-Aldrich, A3059) was added to each well, and the wells were incubated at 37° C. for 1 hour and then washed.

[0028] (Sample reaction) Sesame protein solution was added to each of the 36 types of extraction solutions so that the sesame protein was 62.5ppb, 250ppb, and 1000ppb to prepare crude measurement solutions, and then the crude measurement solutions were diluted 20-fold with PBST containing 0.5% fish gelatin (Sigma-Aldrich, 7765) to adjust the sesame 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 solution without sesame protein was used as a negative control.

[0029] (Enzyme-labeled antibody reaction) 100 μL of horseradish peroxidase-labeled anti-sesame 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.

[0030] (Color development and stop) 100 μL of 3,3',5,5' tetramethylbenzidine solution (Promega, G7431) was added to each well and reacted for 10 minutes at room temperature in the dark. Then, 100 μL of 1N hydrochloric acid (Wako Pure Chemical Industries, 080-01066) was added to each well to stop the reaction, and the absorbance was measured at a main wavelength of 450 nm and a secondary wavelength of 620 nm using a microplate reader.

[0031] (Judgment of results) A graph was created using the absorbance values ​​for sesame protein concentrations of 3.125 ppb, 12.5 ppb, and 50 ppb for each of the 12 types of surfactants. When the surfactant concentration was 1.0%, 5.0%, or 10.0%, and the absorbance at 50 ppb was 0.5 or higher, it was determined that the surfactant was measurable.

[0032] (result) The results are shown in Figures 1 to 12 (see "x20 measurement solution" on the right side of each figure). From these results, it was confirmed that, except for surfactant 1, surfactants 2 to 12 had an absorbance of 0.5 or more at 50 ppb when the surfactant concentration was 1.0%, 5.0%, or 10.0%, and could be measured satisfactorily. Surfactant 1 (sodium dodecyl sulfate) could be measured at 1%, but the reaction was low at 5% and it could not be measured at 10%. The other surfactants 2 to 12 could be measured from 1% to 10%.

[0033] [Example 2] (Measurement under conditions where the measurement solution contains a high concentration of surfactant) The reactivity was confirmed when the extraction solutions containing 1.0%, 5.0% or 10.0% surfactant in Example 1 were measured without diluting 20 times with a buffer solution (see Table 1). A sesame protein solution was added to the extraction solution at 50 ppb to prepare a high-concentration surfactant-measurement solution, and measurements were performed by sandwich ELISA in the same manner as in Example 1.

[0034] (Judgment of results) When the surfactant concentration is 1.0% and the absorbance at 50 ppb is 0.5 or more, the surfactant at that concentration is determined to be measurable.

[0035] (result) The results are shown in Table 4 and Figures 1 to 12 (see "x1 measurement solution" on the left side of each figure). From these results, it was confirmed that, except for surfactant 1 and surfactant 5, when the surfactant concentration was 1.0%, the absorbance at 50 ppb was 0.5 or more, and good measurement was possible.

[0036] [Table 4]

[0037] [Example 3] (Evaluation of the effect of food components on measurements) Among the surfactants that could be measured satisfactorily in Example 2 above, surfactant 2, surfactant 3, and surfactant 4, which showed high absorbance at 1.0%, were examined for their ability to be measured in the presence of ham, a processed food, taking into consideration the actual food inspection situation. The surfactant concentration in the extraction solution used for the measurement was 1.0%.

[0038] In a 50 mL tube, 1 g of ham and 19 mL of the extraction solution were mixed to prepare a food (ham)-containing extraction solution. To the food-containing extraction solution, 0.05 μg, 0.25 μg, 0.50 μg, 1.00 μg, 5.00 μg, or 10.00 μg of sesame protein was added, heated in boiling water for 10 minutes, and then cooled to room temperature. The solution was centrifuged (room temperature, 3,000 rpm, 20 minutes) and filtered to prepare a food (ham) specimen measurement solution, which was measured by sandwich ELISA in the same manner as in Example 1. At the same time, the extraction solution to which the sesame protein solution was added was measured, and the sesame protein concentration in the ham specimen measurement solution was calculated from the obtained standard curve. The recovery rate was calculated by dividing the amount of sesame protein obtained by the measured value, with the amount of added sesame protein being 100%. If the amount of sesame protein contained in the ham specimen measurement solution exceeded the measurement range, it was diluted with the extraction solution so as to fall within the range before measurement.

[0039] (result) The results are shown in Table 5. As is clear from Table 5, it was confirmed that for all of surfactant 2 (sodium laureth-5 carboxylate), surfactant 3 (lauryl glucoside), and surfactant 4 (PEG-3 coconut fatty acid amide MEA sodium sulfate), good recovery rates of 59.0% to 175.5% were obtained when the addition amount was in the range of 0.05 ppm to 10.00 ppm.

[0040] [Table 5]

[0041] [Example 4] (Preparation of almond protein solution) The study was carried out using almond protein as an allergen protein. The almond protein solution was prepared by grinding almonds in a mill and then defatting them with acetone, referring to "Testing methods for foods containing allergens (Reference) (March 26, 2014, Consumer Affairs Agency)". The protein concentration in the almond protein solution was measured using a 2-D QuantKit (GE Healthcare Life Sciences, 80-6483-56).

[0042] (Measurement of allergen proteins using sandwich ELISA) The procedures were carried out in accordance with Examples 1 and 2, except that anti-almond monoclonal antibodies (NITE P-03345 and NITE P-03346) were used.

[0043] (result) The results are shown in Figures 13 to 24. From the results of "x1 measurement solution" on the left side of Figures 13 to 24, it was confirmed that, except for surfactant 1, when the surfactant concentration was 1.0%, the absorbance at 50 ppb was 0.5 or more, and good measurement was possible.

[0044] (summary) The outline of the notified method and the new ELISA method of the present invention are shown in Figure 25. According to the present invention, it was confirmed that by selecting three particularly preferred types of surfactants (sodium laureth-5 carboxylate, lauryl glucoside, and sodium PEG-3 coconut fatty acid amide MEA sulfate) that can be measured at a surfactant concentration of 1 to 10% and can be measured with high sensitivity without dilution, the selected surfactants can be quantified without being affected by food components.

Claims

1. A method for extracting allergens in food for use in a method for detecting allergens in food using an ELISA method, characterized in that the food is mixed with an extraction liquid containing one or more of the following surfactants, and the allergens in the food are extracted. (Surfactant) ・Sodium laureth-5 carboxylate ・Lauryl glucoside ・PEG-3 coconut fatty acid amide MEA sodium sulfate ・Sodium oleate ・Potassium coconut fatty acid ・2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine ・PEG-8 glyceryl isostearate ・Sodium cocoyl glycine ・Polyoxyethylene oleyl ether ・Sodium lauraminopropionate

2. 2. The method for extracting allergens in food according to claim 1, wherein the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate.

3. A method for measuring allergens in food by sandwich ELISA, which comprises mixing 1 g of food with 19 mL of an extract of the allergens in the food containing 1% of the following surfactant, heating and extracting in boiling water for 10 minutes, cooling, and then filtering the solution, without dilution, to use as the measurement solution. (Surfactant) ・Sodium laureth-5 carboxylate ・Lauryl glucoside ・PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium N-dodecanoyl sarcosinate ・Potassium coconut fatty acid ・2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine ・PEG-8 glyceryl isostearate ・Sodium cocoyl glycine ・Polyoxyethylene oleyl ether ・Sodium lauraminopropionate

4. 4. The method for measuring allergens in food according to claim 3, wherein the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate.

5. An allergen measurement kit for measuring allergens in food by ELISA, comprising an extract containing the following surfactant, and a monoclonal antibody that specifically recognizes the allergen to be measured. (Surfactant) ・Sodium laureth-5 carboxylate ・Lauryl glucoside ・PEG-3 coconut fatty acid amide MEA sodium sulfate ・Potassium coconut fatty acid ・2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine ・PEG-8 glyceryl isostearate ・Sodium cocoyl glycine ・Polyoxyethylene oleyl ether ・Sodium lauraminopropionate

6. 6. The allergen measuring kit according to claim 5, wherein the surfactant is sodium laureth-5 carboxylate, lauryl glucoside or sodium PEG-3 coconut fatty acid amide MEA sulfate.

7. A method for screening surfactants for use in a method for extracting allergens in food, comprising mixing 1 g of food with 19 mL of an extract of the allergens in the food containing 1% surfactant, heating and extracting in boiling water for 10 minutes, cooling and then filtering the resulting solution without dilution as a measurement solution, and selecting a surfactant that shows an absorbance value of 0.5 or more at wavelengths of 450-620 nm for 50 ppb of allergens in a sandwich ELISA method.

8. The following surfactant for allergen measurement, obtained by the screening method according to claim 7: (Surfactant) ・Sodium laureth-5 carboxylate ・Lauryl glucoside ・PEG-3 coconut fatty acid amide MEA sodium sulfate Sodium N-dodecanoyl sarcosinate ・Potassium coconut fatty acid ・2-Alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine ・PEG-8 glyceryl isostearate ・Sodium cocoyl glycine ・Polyoxyethylene oleyl ether ・Sodium lauraminopropionate

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