JP2019189557A - Extract of allergen suitable for inspection method using monoclonal antibody - Google Patents

Abstract

To provide an extract for a food product allergen detection method using a monoclonal antibody, capable of extracting allergen protein from a sample such as a food product (including processed food product subjected to cooking), seed, raw material powder, capable of detecting allergen without occurrence of false positive in a food product allergen detection method using the monoclonal antibody.SOLUTION: The extract comprises: 0.05-10 (w/v)% of zwitter surfactant; 0.05-1.5 (v/v)% of polyoxyethylene octylphenyl ether (CAS registration number 9002-93-1:TritonX-100 (registered trademark)); 0.05-10 (v/v)% of octylphenyl polyethylene glycol (CAS registration number 9036-19-5:IGEPAL CA-630(registered trademark)) or 0.05-0.9 (v/v)% of polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5:Tween20 (registered trademark)), and comprises no reductant, or the extract comprises 0.005-0.09 (w/v)% of anionic surfactant and 0.05-10 (v/v)% of nonionic surfactant as surfactant.SELECTED DRAWING: None

Description

  The present invention relates to an allergen extract suitable for a test method using a monoclonal antibody.

In recent years, there has been an increase in people with food allergies that cause allergic symptoms due to specific foods. Food manufacturers need to perform allergen tests and provide consumers with information about allergens in food.
Specified raw materials are 7 ingredients including allergens (shrimp, crab, wheat, buckwheat, egg, milk, peanuts). In order to prevent the occurrence of health hazards for people with allergies, it is legally applied to food. Raw material labeling is mandatory.
Inspection of specific raw materials in processed foods is to be conducted in accordance with the notice of the Consumer Affairs Agency. Since there is a duty to display when the remaining amount of allergen protein in the food is about 10 ppm or more, the allergen test is first performed by a screening test using a quantitative test method (ELISA method), and if positive, a qualitative test method (Western blot method) (Or PCR method). Regarding the ELISA method, it is stipulated to use designated inspection kits commercially available from several companies.
However, since the ELISA method is complicated and requires several hours for measurement, the routine self-inspection uses the same antigen-antibody reaction as the ELISA method, and can be easily and quickly determined for allergen testing. Often, immunochromatography is used. This immunochromatography also has a plurality of commercially available kits as in the ELISA method.
Immunochromatography is prepared by combining either or both of a polyclonal antibody and a monoclonal antibody. The allergen extract attached to each kit has a composition suitable for each immunochromatography.
Conventionally, 2-mercaptoethanol was used as a reducing agent in an allergen protein extract to increase extraction efficiency. However, in July 2008, 2-mercaptoethanol was designated as a toxic substance and became subject to regulation, making it difficult to use 2-mercaptoethanol in food factories, thus enabling highly efficient inspection in food factories. Therefore, the development of the extract has been demanded.
As an extract containing no 2-mercaptoethanol for extracting an allergen from food, for example, in Patent Document 1, a reducing agent (as sulfite, sodium sulfite, sodium bisulfite, potassium sulfite, ammonium sulfite and iron sulfite is selected. And at least one kind of solubilizing aid and sodium dodecyl sulfate (SDS) are provided. Patent Document 2 provides an extract containing SDS as an anionic surfactant and thiosulfate or SDS and Tween 20 as a nonionic surfactant. Further, Patent Document 3 provides an extract containing SDS, which is thioglycerol as a reducing agent and urea or alkyl sulfate as a solubilizer.
However, when the inventor of the present application conducted a test method using a homemade monoclonal antibody with an extract containing no 2-mercaptoethanol, it cannot be said that all of the allergen extraction efficiency is sufficient, and a false positive reaction. As a result, the occurrence or undetectable.

JP2013-33062A Table 2010/095469 Table 2013/179663

  The present invention is an extract for a method for detecting food allergens using a monoclonal antibody, and can extract allergen proteins from specimens such as food (including cooked processed foods), seeds, and raw material powders. An object of the present invention is to provide an extract capable of detecting an allergen without producing false positives when performing a test method using a monoclonal antibody.

  The present inventors have used as surfactants 0.05 to 10 (w / v)% zwittersurfactant, 0.05 to 1.5 (v / v)% polyoxyethylene octylphenyl ether ( CAS registration number 9002-93-1: Triton X-100 (registered trademark), 0.05 to 10 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (registration) Trademark))) or 0.05-0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20 (registered trademark)) only, and no reducing agent 0.005 to 0.09 (w / v)% anionic surfactant and 0.05 to 10 (v / v)% nonionic surfactant as an extract or surfactant Extract allergen protein from samples of food (including cooked processed foods), seeds, raw material powders, etc., with an extract for detecting food allergens using monoclonal antibodies, It has been found that food allergen detection methods using monoclonal antibodies can detect allergens without showing false positives.

That is, the present invention is as follows.
[1] 0.05 to 10 (w / v)% zwitterionic surfactant, 0.05 to 1.5 (v / v)% polyoxyethylene octylphenyl ether (CAS registration number) as the surfactant 9002-93-1: Triton X-100 (registered trademark)), 0.05 to 10 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (registered trademark)) Or an extract containing only 0.05 to 0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20 (registered trademark)) and no reducing agent. An extract for a food allergen detection method using a monoclonal antibody.
[2] Extract containing 0.005-0.09 (w / v)% anionic surfactant and 0.05-10 (v / v)% nonionic surfactant as a surfactant An extract for a food allergen detection method using a monoclonal antibody.
[3] The extract according to [2], further including a reducing agent.
[4] A method for extracting a food allergen in a sample, wherein the extract according to any one of [1] to [3] is used in a method for extracting a food allergen from a sample.
[5] Food allergen detection, wherein a food allergen extracted from a specimen using the extract according to any one of [1] to [3] is detected by a measurement method using a monoclonal antibody Law.
[6] A food allergen detection kit comprising a monoclonal antibody against food allergen and the extract according to any one of [1] to [3].

  By using the extract of the present invention, allergen proteins are efficiently extracted from specimens such as food (including cooked processed foods), seeds and raw powders, and food allergen detection methods using self-prepared monoclonal antibodies , Allergens can be detected without showing false positives.

The extract of the present invention is an extract for a food allergen detection method using a monoclonal antibody. In the present invention, specimens that are targets of food allergen detection methods using monoclonal antibodies are foods containing food allergen proteins such as eggs, wheat, milk, shrimp, buckwheat, and peanuts (cooked and processed foods). Food), seeds, raw powder, etc. For example, in the case of wheat, processed foods made using flour such as flour, bread, confectionery, white sauce, etc., soba, soba seeds, buckwheat, buckwheat processed into noodles, buckwheat, etc. In the case of peanuts such as processed foods produced using powder, butter peanuts, peanut oil, peanut butter and processed foods using them are included. In addition, when a food containing a protein that becomes a food allergen is used in the same production line, food raw materials and processed foods that may contain a protein that becomes a food allergen are also included.
In the present invention, a food allergen detection method using a monoclonal antibody is a well-known immunological measurement method, and an immunochromatography method, an ELISA method, Western blotting or the like is used, preferably an immunochromatography.
Here, the monoclonal antibody is a monoclonal antibody against a food allergen protein, and an antibody actually produced for a specific food allergen protein according to a conventional method or a commercially available antibody may be used. Examples thereof include casein and β-lactoglobulin, which are major allergens of milk, ovomucoid and ovalbumin, which are major allergens of eggs, and Flag e1, Fage 2, which are major allergens of buckwheat, and monoclonal antibodies that recognize globulin and albumin. For example, as a monoclonal antibody that recognizes the buckwheat allergen protein Flag e2, as of April 30, 2014 (notification number: 2013-0606), the National Institute of Technology and Evaluation, National Institute of Microbiology (Address: Kazusakami, Kisarazu City, Chiba Prefecture) Examples include antibodies produced by NITE AP-01791 and NITE AP-01792 deposited and accepted on foot 2-5-8).

  The first aspect of the extract of the present invention comprises 0.05 to 10 (w / v)% zwitterionic surfactant and 0.05 to 1.5 (v / v)% poly as a surfactant. Oxyethylene octyl phenyl ether (CAS registration number 9002-93-1: Triton X-100 (registered trademark)), 0.05 to 10 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (registered trademark)) or 0.05-0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20 (registered trademark)) only Does not contain a reducing agent.

  In the present invention, the surfactant is added to the extract in order to solubilize a protein that is hardly soluble in water or a protein that is difficult to extract (for example, heat-denatured) and extract it efficiently from the processed food.

The amphoteric surfactants include 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO), decyldimethyl (3-sulfopropyl) ammonium hydroxide inner salt (Zwittergent 3-10), dodecyldimethyl (3-sulfopropyl) ammonium hydroxide inner salt (Zwittergent 3-12), and the like are preferable. Is CHAPS or CHAPSO. When the extract contains only the zwitterionic surfactant as the surfactant, the concentration of the zwitterionic surfactant is preferably 0.1 to 7 (w / v)%, more preferably 0.5 to 5 (w). / V)%.
When only Triton X-100 is included as a surfactant in the first aspect of the extract of the present invention, the concentration of Triton X-100 in the extract is preferably 0.1 to 1.5 (v / v)%, more Preferably it is 0.1-1 (v / v)%.
When only IGEPAL CA-630 is included as a surfactant in the first aspect of the extract of the present invention, the concentration of IGEPAL CA-630 in the extract is preferably 0.05 to 7 (v / v)%, more Preferably it is 0.5-7 (v / v)%.
When only Tween 20 is included as a surfactant in the first aspect of the extract of the present invention, the concentration of Tween 20 in the extract is preferably 0.05 to 0.5 (v / v)%, more preferably 0. 07-0.5 (v / v)%.

The first aspect of the extract of the present invention may further contain a preservative. The preservative is not essential for the extraction of food allergens, but is preferably added to the extract to prevent microorganisms from entering, growing and growing in the buffer and minimizing spoilage.
Any preservative can be used without particular limitation as long as it is usually used in foods and cosmetics. Specific examples include sodium benzoate, calcium propionate, sodium propionate and Procrine 300 (Sigma Aldrich), and Procrine 300 is preferable. The concentration of the preservative in the extract is preferably 0.01 to 0.05 (w / v)%, more preferably 0.015 to 0.035 (w / v)%.

The first aspect of the extract of the present invention may further contain a chelating agent. The chelating agent is preferably added to the extract because it has the property of capturing metal ions that promote oxidation.
The chelating agent can be used without particular limitation as long as it is a component that is usually blended for the purpose of maintaining the quality of food and cosmetics, such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid. (DTPA) and the like are mentioned, and EDTA is preferred. The concentration of the chelating agent in the extract is preferably 0.1 to 0.7 mM, more preferably 0.5 mM.

  In the first aspect of the extract of the present invention, preferably, the above components can be contained in a buffer solution generally used in experiments such as ELISA, Western blot, and immunohistochemical staining. Examples of such a buffer include Tris-HCl buffer, Tris-maleate buffer, Tris-buffered saline, phosphate buffer, phosphate buffered saline PBS (pH 7.4), and the like. Use PBS.

Moreover, the 2nd aspect of the extract of this invention is 0.005-0.09 (w / v)% anionic surfactant and 0.05-10 (v / v)% as surfactant. Nonionic surfactants.
Examples of the anionic surfactant include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium cholate and the like, preferably SDS. Examples of nonionic surfactants include IGEPAL CA-630, Triton X-100, Tween 20, Bridge 35 (Brij 35), n-octyl-β-D-glucopyranoside, and the like. When an anionic surfactant and a nonionic surfactant are included as surfactants in the extract, the nonionic surfactant is preferably IGEPAL CA-630, Triton X-100 or Tween 20, more preferably Triton X -100.
In the second aspect of the extract of the present invention, the concentration of the anionic surfactant is preferably 0.01 to 0.09 (w / v)%, more preferably 0.04 to 0.07 (w / V)%. When an anionic surfactant and a nonionic surfactant are included as surfactants in the extract, the concentration of the nonionic surfactant is preferably 0.1 to 7 (v / v)%, more preferably Is 0.3-7 (v / v)%.

The second aspect of the extract of the present invention may further contain a reducing agent. When the food allergen detection target is processed food, it is often manufactured under harsh conditions such as heating and pressurization, which may cause a problem that the allergen protein in the specific raw material to be tested is insoluble and difficult to extract. Therefore, it is preferable to add a reducing agent to the extract to prevent deterioration in extraction efficiency.
As a reducing agent, in consideration of safety and storage stability, specifically, α-tocopherol, β-tocopherol, γ-tocopherol, σ-tocopherol, BHA (butylhydroxyanisole) already used in foods and cosmetics. , BHT (dibutylhydroxytoluene), L-ascorbate and isoascorbate, and preferably at least one of sodium L-ascorbate and sodium isoascorbate. In the present invention, the reducing agent does not include 2-mercaptoethanol. The concentration of the reducing agent in the extract is preferably 0.005 to 7 (w / v)%, more preferably 0.005 to 1.5 (w / v)%.

The second aspect of the extract of the present invention may further contain a preservative. The preservative is not essential for the extraction of food allergens, but is preferably added to the extract to prevent microorganisms from entering, growing and growing in the buffer and minimizing spoilage.
Any preservative can be used without particular limitation as long as it is usually used in foods and cosmetics. Specific examples include sodium benzoate, calcium propionate, sodium propionate and Procrine 300 (Sigma Aldrich), and Procrine 300 is preferable. The concentration of the preservative in the extract is preferably 0.01 to 0.05 (w / v)%, more preferably 0.015 to 0.035 (w / v)%.

The second aspect of the extract of the present invention may further contain a chelating agent. The chelating agent is preferably added to the extract because it has the property of capturing metal ions that promote oxidation.
The chelating agent can be used without particular limitation as long as it is a component that is usually blended for the purpose of maintaining the quality of foods and cosmetics, and examples thereof include EDTA, NTA, and DTPA, and EDTA is preferred. . The concentration of the chelating agent in the extract is preferably 0.1 to 0.7 mM, more preferably 0.5 mM.

  In the second aspect of the extract of the present invention, preferably, the above components can be contained in a buffer solution generally used in experiments such as ELISA, Western blot, and immunohistochemical staining. Examples of such a buffer include Tris-HCl buffer, Tris-maleate buffer, Tris-buffered saline, phosphate buffer, and phosphate buffered saline PBS (pH 7.4). Use PBS.

The method for extracting a food allergen in a sample of the present invention is characterized by using the above extract in a method for extracting a food allergen from a sample. The method for extracting a food allergen in a sample of the present invention can be carried out according to a conventional method except that the above-mentioned extract is used in the method for extracting a food allergen from a sample, and conventionally used grinding, emulsification, stirring, shaking and shaking. An appropriate method such as boiling or boiling can be used alone or in combination.
For example, it can be carried out by adding an extract to a specimen, stirring at room temperature using a vortex mixer, and then centrifuging and collecting the supernatant.

The food allergen detection method of the present invention is characterized in that the food allergen extracted using the above extract is detected by a measurement method using a monoclonal antibody. The food allergen detection method of the present invention can be performed according to a conventional method except that the food allergen extracted using the above extract is detected and used by a measurement method using a monoclonal antibody.
In the food allergen detection method of the present invention, a measurement method using a monoclonal antibody is a well-known immunological measurement method, and an immunochromatography method, an ELISA method, Western blotting, or the like is used, preferably an immunochromatography.

  The food allergen detection kit of the present invention comprises a monoclonal antibody against a food allergen and the above extract. One or more monoclonal antibodies may be included in the food allergen detection kit. In the food allergen detection kit of the present invention, the monoclonal antibody against the food allergen is preferably fixed to a solid layer as a support. Included in form. In addition, the food allergen detection kit of the present invention may contain a standard product of allergen protein, a detection reagent, a dilution buffer, and the like.

  EXAMPLES Examples will be shown below for specifically explaining the present invention, but the present invention is not limited to the following examples.

<Production example (production of immunochromatography)>
1. Monoclonal antibody (MAb)
The MAb used recognizes the buckwheat allergen protein Fage 2. The hybridoma producing MAb is a patent microbiology deposit center of the National Institute of Technology and Evaluation (Adress: 2-5-8 Kazusa Kama feet, Kisarazu City, Chiba Prefecture) as of April 30, 2014 (notification number: 2013-0606) NITE AP-01791 and NITE AP-01792 deposited and accepted by (Japanese Patent Laid-Open No. 2015-178461)
2. Preparation of antibody-immobilized membrane MAb (NITE AP-01792) prepared by setting a membrane dedicated for immunochromatography (Merck Millipore) on a mass application manufacturing machine (Nippon Engineering) and adjusting to 1 mg / ml with PBS. The solution was applied linearly and dried at 50 ° C. for 30 minutes.
3. Preparation of gold colloid-labeled antibody-immobilized conjugate pad 9 ml of gold colloid solution (manufactured by BBI), 1 ml of MAb (NITE AP-01791) solution prepared to 1 mg / ml with PBS and 1 ml of 50 mM potassium phosphate buffer Mixed. Add 0.55 ml of 1% PEG and 1.1 ml of 10% BSA, centrifuge, adjust to OD520 = 1-2 with 1% BSA / Tris (pH 8.2) solution, and conjugate pad (Merck Millipore) And then freeze-dried.
4. Assemble immunochromatography Affixing membrane, conjugate pad for immobilizing colloidal gold-labeled antibody, sample pad (Merck Millipore), absorption pad (Merck Millipore) are pasted in this order on the backing sheet (Nippon Engineering). It was. This sheet was cut into a width of 5 mm with a compact cutter (manufactured by Nipple Engineering Co., Ltd.) to obtain an immunochromatography.

<Test Example 1 (Examination of surfactant)>
1. Preparation of extract liquid Each surfactant is 5 to 0.001 (w / v)% for anionic surfactant, zwitterionic surfactant and chaotropic reagent, and for nonionic surfactant. The extract was dissolved in 0.5 mM EDTA / PBS so as to be 5 to 0.001 (v / v)%.
2. Extraction method Add 20 mg of buckwheat primary standard powder to 980 μL of each extract, mix for 1.5 minutes at room temperature using a vortex mixer, and then centrifuge (3,000 × g, 20 minutes, 4 ° C.) The supernatant was collected. This solution was diluted 200 times with each extract to obtain an extract solution.
The soba primary standard powder was obtained by mixing equal amounts of soba from Ibaraki Prefecture and China (northern China) and then pulverizing and passing through a 14-mesh sieve (aperture 1.18 mm).
3. Determination method Each extraction solution was diluted 10-fold with a developing solution (phosphate buffer solution) to obtain a measurement solution. 100 μL of the measurement solution was added dropwise to the immunochromatography and developed at room temperature.
As a negative control, each extract was diluted 10-fold with a developing solution, and 100 μL was dropped.
After 10 minutes, the color density of the reaction line was measured with an immunochromatographic reader (manufactured by NIPPON ENGINEERING).
Concentrations that showed positive without producing false positives were taken as the compatible range.
The judgment was according to the following table.

０.１〜５（ｗ／ｖ）％では偽陽性を生じることなく陽性を示し良好であった。 4. Results (1) Extract containing the zwitterionic surfactant CHAPS

In 0.1-5 (w / v)%, it showed the positive without producing a false positive and was good.

０.１〜５（ｗ／ｖ）％では偽陽性を生じることなく陽性を示し良好であった。 (2) Extract containing the amphoteric surfactant CHAPSO

In 0.1-5 (w / v)%, it showed the positive without producing a false positive and was good.

０.１〜１（ｖ／ｖ）％では偽陽性を生じることなく陽性を示し良好であった。 (3) Extract containing nonionic surfactant Triton X-100

In 0.1-1 (v / v)%, it showed a positive without producing a false positive and was good.

０.０１〜５（ｖ／ｖ）％では偽陽性を生じることなく陽性を示し良好であった。０.０１（ｖ／ｖ）％では測定値が低かった。 (4) Extract containing nonionic surfactant IGEPAL CA-630

0.01 to 5 (v / v)% was good without showing a false positive and showing good results. The measured value was low at 0.01 (v / v)%.

０.１（ｖ／ｖ）％では偽陽性を生じることなく陽性を示し良好であった。 (5) Extract containing nonionic surfactant Tween 20

0.1 (v / v)% was positive and good without causing false positives.

０.０１〜０.０５（ｗ／ｖ）％では偽陽性を生じることなく陽性を示したが、測定値は低かった。 (6) Extract containing an anionic surfactant SDS

0.01-0.05 (w / v)% showed positive without causing false positive, but the measured value was low.

０.００１〜０.０１（ｗ／ｖ）％では偽陽性を生じることなく陽性を示したが、測定値は低かった。 (7) Extract containing urea as a chaotropic reagent

0.001 to 0.01 (w / v)% showed a positive result without producing a false positive, but the measured value was low.

<Test Example 2 (Study on combinations of surfactants)>
The case where an anionic surfactant and a nonionic surfactant were combined as a surfactant was examined.
As surfactant, 0.05% (w / v) anionic surfactant and 0.005 to 5 (v / v)% for nonionic surfactant were added to 0.5 mM EDTA / PBS. Except that it was dissolved and used as an extract, an extract was prepared according to the method of Test Example 1, extracted, and judged. The results are shown below.

０.０５（ｗ／ｖ）％ＳＤＳと０.１〜１（ｖ／ｖ）％ＴｒｉｔｏｎＸ-１００を組み合わせた場合、ＳＤＳ単独又はＴｒｉｔｏｎＸ-１００単独の場合と比較して、全般的に測定値が高くなり０.１〜１（ｖ／ｖ）％の範囲で偽陽性を生じることなく陽性を示し良好であった。 (1) Extract containing an anionic surfactant SDS and a nonionic surfactant TritonX-100

When 0.05 (w / v)% SDS and 0.1 to 1 (v / v)% Triton X-100 are combined, the measured value is generally higher than that of SDS alone or Triton X-100 alone. It became high and showed a positive without producing a false positive in the range of 0.1 to 1 (v / v)%, and was good.

０.０５（ｗ／ｖ）％ＳＤＳと０.１〜５（ｖ／ｖ）％ＩＧＥＰＡＬ ＣＡ−６３０を組み合わせた場合、ＳＤＳ単独又はＩＧＥＰＡＬ ＣＡ−６３０単独の場合と比較して、全般的に測定値が高くなり偽陽性を生じることなく陽性を示し良好であった。 (2) Extract containing anionic surfactant SDS and nonionic surfactant IGEPAL CA-630

When 0.05 (w / v)% SDS and 0.1 to 5 (v / v)% IGEPAL CA-630 are combined, it is measured in general compared to SDS alone or IGEPAL CA-630 alone. The value was high and it was positive and positive without producing false positives.

０.０５（ｗ／ｖ）％ＳＤＳと０.１〜１（ｖ／ｖ）％ Ｔｗｅｅｎ２０を組み合わせた場合、Ｔｗｅｅｎ２０単独の場合と比較して、全般的に測定値が高くなった。０.０５（ｗ／ｖ）％ＳＤＳと０.１〜０.４（ｖ／ｖ）％ Ｔｗｅｅｎ２０では偽陽性を生じることなく陽性を示し良好であった。 (3) Extract containing an anionic surfactant SDS and a nonionic surfactant Tween 20

When 0.05 (w / v)% SDS and 0.1 to 1 (v / v)% Tween 20 were combined, the measured values were generally higher than when Tween 20 was used alone. 0.05 (w / v)% SDS and 0.1 to 0.4 (v / v)% Tween 20 were positive without producing false positives and were good.

<Test Example 3 (Study on the effect of adding a reducing agent)>
1. Reagents examined (1) Sodium L-ascorbate (2) Sodium isoascorbate

2. Preparation of Extract 0.05 (w / v)% SDS, 0.6 (v / v)% Triton X-100, 0.5 mM EDTA / PBS Effective in Test Example 1 as Solvent for Reducing Agent (1) and (2) were dissolved so as to be 1 (w / v)%.

3. Extraction method and determination method The same procedure as in Test Example 1 was performed.

Ｌ−アスコルビン酸ナトリウムとイソアスコルビン酸ナトリウムのいずれの還元剤を添加しても偽陽性を生じず陽性を示した。還元剤を添加することで添加しないものと比較して高い測定値となった。 4. Results

Even if any reducing agent of sodium L-ascorbate and sodium isoascorbate was added, a false positive was not generated and the result was positive. By adding a reducing agent, the measured value was higher than that not added.

<Test Example 3 (Examination of Reducing Agent Concentration)>
The extraction efficiency of sodium L-ascorbate was measured using buckwheat primary standard powder or an actual sample. A commercial baked confectionery “Sobabouro” (Heiwa Seika), a processed food containing soba, was used as the actual sample.
1. Preparation of Extract 0.05- (w / v)% SDS, 0.6 (v / v)% TritonX-100 so that sodium L-ascorbate is 5 to 0.01 (w / v)% , 0.5 mM EDTA / PBS.
2. Extraction Method and Judgment Method A test using buckwheat primary standard powder was performed in the same manner as in Test Example 1.
The test using the soba bowl was performed as follows.
19 ml of each extract was added to 1 g of buckwheat and mixed with a vortex mixer (room temperature, 1.5 minutes), then centrifuged (3,000 × g, 20 minutes, 4 ° C.), and the supernatant was recovered. . The recovered liquid was diluted 100 times with each extract to obtain an extract solution.

いずれの場合も、５〜０.０１（ｗ／ｖ）％で偽陽性を生じることなく陽性を示した。 3. Results

In any case, 5 to 0.01 (w / v)% showed a positive without causing a false positive.

<Test Example 4 (Examination of preservative)>
1. Reagents examined (1) Procrine 300 (Sigma Aldrich)
(2) Sodium benzoate (3) Calcium propionate (4) Sodium propionate

2. Preparation of Extract 0.05 (w / v)% SDS, 0.6 (v / v)% Triton X-100, 0.01 (w / w) Effective in Test Example 3 as a Preservative Solvent v)% Sodium L-ascorbate, 0.5 mM EDTA / PBS was used, and the preservative was dissolved to 0.02 (w / v)%.
3. Extraction method and determination method The same procedure as in Test Example 1 was performed.

いずれの試薬でも偽陽性を生じることなく陽性を示した。 4. Results

Both reagents showed positive without producing false positives.

<Test Example 5 (Detection of allergen from model processed food)>
A model processed food containing buckwheat primary standard powder was prepared and detected by immunochromatography.
1. Preparation of model processed food (a) Retort food To 100g of meat sauce (Ohmy), add soba primary standard powder equivalent to 10mg in terms of total protein (170mg as the weight of buckwheat primary standard powder) or 1mg equivalent ( Each of which was added as a negative control) was put in a retort bag and sealed. This bag was subjected to retort treatment (121 ° C., 15 minutes) with a hot water hot water type retort sterilizer (manufactured by Nisaka Seisakusho).
(B) Baked confectionery A buckwheat primary standard powder was added to 100 g of a commercially available cookie mix (Ohmai Co., Ltd.) in the same manner as (a) and cooked by the method described in the product (heat treatment was 170 ° C., 15 minutes).
(C) Steamed confectionery A buckwheat primary standard powder was added to 100 g of commercially available steamed bread mix (Ohmai Co., Ltd.) in the same manner as (a) and cooked by the method described in the product (heat treatment was 100 ° C., 15 minutes).
(D) Deep-fried confectionery A buckwheat primary standard powder was added to 100 g of a commercially available hot cake mix (Ohmai Co., Ltd.) in the same manner as (a) and cooked by the method described in the product (heat treatment was 180 ° C., 8 minutes).
2. Extract 0.05 (w / v)% SDS, 0.6 (v / v)% Triton X-100, 0.01 (w / v)% L-sodium ascorbate, 0.5 mM EDTA, 0.5 02 (w / v)% Procrine 300, 0.5 mM EDTA / PBS
3. Extraction method and measurement method
Add 19 ml of extract to 1 g of retorted or cooked model processed food, mix for 1.5 minutes at room temperature using a vortex mixer, then centrifuge (3,000 × g, 20 minutes, 4 ° C.) The supernatant was collected. This solution was diluted 10 times with a developing solution to obtain a measurement solution.
The measurement method was the same as in Test Example 1.

そばのアレルゲンタンパク質は高温、高圧処理した加工食品からも検出可能であった。 4. Results

Soba allergen protein was also detectable in processed food processed at high temperature and pressure.

Claims (6)

  As the surfactant, 0.05 to 10 (w / v)% zwitterionic surfactant, 0.05 to 1.5 (v / v)% polyoxyethylene octylphenyl ether (CAS registration number 9002-93) -1: Triton X-100 (registered trademark)), 0.05 to 10 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (registered trademark)) or 0. An extract containing only 05-0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20 (registered trademark)) and no reducing agent, Extract for food allergen detection method using monoclonal antibody.   An extract containing 0.005 to 0.09 (w / v)% anionic surfactant and 0.05 to 10 (v / v)% nonionic surfactant as a surfactant, Extract for food allergen detection method using monoclonal antibody.   The extract according to claim 2, further comprising a reducing agent.   A method for extracting a food allergen in a sample, wherein the extract of any one of claims 1 to 3 is used in a method for extracting a food allergen from a sample.   A food allergen detection method, comprising: detecting a food allergen extracted from a sample using the extract according to any one of claims 1 to 3 by a measurement method using a monoclonal antibody.   A kit for detecting a food allergen, comprising a monoclonal antibody against a food allergen and the extract according to any one of claims 1 to 3.