JP7101035B2 - Allergen extract suitable for testing methods using monoclonal antibodies - Google Patents

Description

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

In recent years, an increasing number of people have food allergies that cause allergic symptoms due to specific foods. Food manufacturers are required to perform allergen testing and provide consumers with information about allergens in food.
Specified raw materials are 7 items of foodstuffs containing allergens (shrimp, crab, wheat, buckwheat, egg, milk, peanuts), and to prevent the occurrence of health hazards for people with allergies, to foods by law. Ingredient labeling is obligatory.
Inspection of specific raw materials in processed foods is to be carried out in accordance with the notification of the Consumer Affairs Agency. Since labeling is obligatory when the residual amount of allergen protein in food is about 10 ppm or more, a screening test by quantitative test method (ELISA method) is first performed in the allergen test, and if it is positive, a qualitative test method (Western blotting method) is performed. Or perform a definitive test by 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 takes several hours for measurement, the same antigen-antibody reaction as the ELISA method is used for daily self-inspection, and it is for allergen inspection that can be determined easily and in a short time. Immunochromatography is often used. Similar to the ELISA method, there are a plurality of commercially available kits for this immunochromatography.
The immunochromatography is made by combining either one of polyclonal antibodies or monoclonal antibodies or both. The allergen extract attached to each kit has a composition suitable for each immunochromatography.
Conventionally, 2-mercaptoethanol has been used as a reducing agent in the extract of allergen protein in order to improve the extraction efficiency. However, in July 2008, 2-mercaptoethanol was designated as a poison and subject to regulation, making it difficult to use 2-mercaptoethanol in food factories, enabling highly efficient inspections in food factories. Therefore, the development of an extract is required.
As an extract containing no 2-mercaptoethanol for extracting allergens from food, for example, in Patent Document 1, a reducing agent (sodium sulfite, sodium hydrogen sulfite, potassium sulfite, ammonium sulfite and iron sulfite is selected as the sulfite). And at least one) and an extract to which sodium dodecyl sulfate (SDS) is added as a solubilizing agent is provided. Further, Patent Document 2 provides an extract containing SDS of an anionic surfactant and thiosulfate or SDS and Tween 20 of a nonionic surfactant. Further, Patent Document 3 provides an extract containing thioglycerol as a reducing agent and urea or an alkyl sulfate SDS as a solubilizer.
However, when the inventor of the present application conducted a test method using a homemade monoclonal antibody with these 2-mercaptoethanol-free extracts, the extraction efficiency of allergens was not sufficient in any of them, and a false positive reaction was carried out. The result was the occurrence or undetectability of.

JP 2013-33062 Re-table 2010/0954669 Re-table 2013/179663

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

As surfactants, we have 0.05-10 (w / v)% bidirectional surfactants and 0.05-1.5 (v / v)% polyoxyethylene octylphenyl ethers ( CAS registration number 9002-93-1: TritonX-100®, 0.05-10 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (registration) Includes only 0.05-0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20®) and no reducing agent. Extract or extract containing 0.005 to 0.09 (w / v)% anionic surfactant and 0.05 to 10 (v / v)% nonionic surfactant as the surfactant Therefore, allergen proteins are efficiently extracted from samples such as foods (including cooked processed foods), seeds, and raw material powders by an extract for a food allergen detection method using a monoclonal antibody, and a monoclonal antibody is obtained. In the food allergen detection method used, it was found that the allergen can be detected without showing a false positive.

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

A food allergen detection method using a self-prepared monoclonal antibody by efficiently extracting allergen proteins from samples such as foods (including cooked processed foods), seeds, and raw material powders by using the extract of the present invention. In, the allergen 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, the sample to be the target of the food allergen detection method using a monoclonal antibody is a food containing a protein that becomes a food allergen such as egg, wheat, milk, shrimp, crab, buckwheat, and peanut (cooked processing). Includes food), seeds, raw material powder, etc. For example, in the case of wheat, processed foods made from wheat flour, breads, confectionery, white sauce, etc., and in the case of soba, peanuts, peanuts, noodle-processed soba, soba boro, etc. In the case of peanuts, such as processed foods produced using flour, butter peanuts, peanut oil, peanut butter and processed foods using them are included. In addition, when foods containing proteins that become food allergens are used on the same production line, food raw materials and processed foods that may be contaminated with proteins that become food allergens are also included.
In the present invention, the 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, and is preferably an immunochromatography.
Here, the monoclonal antibody is a monoclonal antibody against a food allergen protein, and an antibody actually prepared by a conventional method against a specific food allergen protein 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, Fag e1 and Fag e2, 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 Fage2, on April 30, 2014 (notification number: 2013-0606), the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Address: Kazusakamatari, Kisarazu City, Chiba Prefecture) Examples include antibodies produced by NITE AP-01791 and NITE AP-01792 deposited and accepted in Feet 2-5-8).

The first aspect of the extract of the present invention is, as a surfactant, 0.05 to 10 (w / v)% bidirectional surfactant and 0.05 to 1.5 (v / v)% poly. Oxyethylene octylphenyl ether (CAS registration number 9002-93-1: TritonX-100®), 0.05-10 (v / v)% octylphenylpolyethylene glycol (CAS registration number 9036-19-5:): IGEPAL CA-630®) or 0.05-0.9 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween 20®) only. , Does not contain reducing agents.

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

As a bidirectional surfactant, 3-[(3-colamidepropyl) dimethylammonio] propanesulfonate (CHASP), 3-[(3-colamidepropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate. (CHASPO), decyldimethyl (3-sulfopropyl) ammonium hydroxide intramolecular salt (Zwittergent3-10), dodecyldimethyl (3-sulfopropyl) ammonium hydroxide intramolecular salt (Zwittergent3-12) and the like are preferable. Is CHAPS or CHASPO. When the extract contains only the twin surfactant as the surfactant, the concentration of the twin surfactant is preferably 0.1 to 7 (w / v)%, more preferably 0.5 to 5 (w). / V)%.
When only Triton X-100 is contained 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. It is preferably 0.1 to 1 (v / v)%.
When only IGEPAL CA-630 is contained 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. It is preferably 0.5 to 7 (v / v)%.
When only Tween 20 is contained 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. It is 07 to 0.5 (v / v)%.

The first aspect of the extract of the present invention may further contain a preservative. Preservatives are not essential for the extraction of food allergens, but are preferably added to the extract to prevent the invasion, growth and proliferation of microorganisms into the buffer and to minimize spoilage.
The preservative can be used without particular limitation as long as it is usually used for foods and cosmetics. Specific examples thereof include sodium benzoate, calcium propionate, sodium propionate and Proclin 300 (Sigma-Aldrich), with Proclin 300 being preferred. 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 foods and cosmetics, and can be used without particular limitation, such as ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and diethylenetriaminepentaacetic acid. (DTPA) and the like can be mentioned, but EDTA is preferable. 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, the above-mentioned components can be preferably contained in a buffer solution generally used in experiments such as ELISA method, Western blotting method and immune tissue staining method. Examples of such a buffer include Tris hydrochloride buffer, Trismaleic acid buffer, Tris buffered saline, phosphate buffer, PBS (pH 7.4) which is phosphate buffered saline, and the like, which are preferable. Uses PBS.

The second aspect of the extract of the present invention is, as a surfactant, 0.005 to 0.09 (w / v)% of anionic surfactant and 0.05 to 10 (v / v)%. Contains nonionic surfactants.
Examples of the anionic surfactant include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate, sodium cholic acid and the like, and SDS is preferable. Examples of the nonionic surfactant include IGEPAL CA-630, TritonX-100, Tween20, Bridge 35 (Brij35), n-octyl-β-D-glucopyranoside and the like. When the extract contains an anionic surfactant and a nonionic surfactant as the surfactant, 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 the extract contains an anionic surfactant and a nonionic surfactant as the surfactant, the concentration of the nonionic surfactant is preferably 0.1 to 7 (v / v)%, more preferably. Is 0.3 to 7 (v / v)%.

The second aspect of the extract of the present invention may further contain a reducing agent. When the target for detecting food allergens is processed foods, many of them are manufactured under harsh conditions such as heating and pressurization, and there may be a problem that the allergen protein in the specific raw material to be inspected is insoluble and difficult to be extracted. Therefore, it is preferable to add a reducing agent to the extract in order to prevent deterioration of the extraction efficiency.
As the reducing agent, considering safety and storage stability, specifically, α-tocopherol, β-tocopherol, γ-tocopherol, σ-tocopherol, BHA (butylhydroxyanisole) already used in foods and cosmetics. , BHT (dibutylhydroxytoluene), L-ascorbic acid salt and isoascorbic acid salt, but preferably at least one of L-sodium ascorbic acid and sodium isoascorbic acid. In the present invention, the reducing agent does not contain 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. Preservatives are not essential for the extraction of food allergens, but are preferably added to the extract to prevent the invasion, growth and proliferation of microorganisms into the buffer and to minimize spoilage.
The preservative can be used without particular limitation as long as it is usually used for foods and cosmetics. Specific examples thereof include sodium benzoate, calcium propionate, sodium propionate and Proclin 300 (Sigma-Aldrich), with Proclin 300 being preferred. 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 an ingredient that is usually blended for the purpose of maintaining the quality of foods and cosmetics, and examples thereof include EDTA, NTA, and DTPA, but EDTA is preferable. .. The concentration of the chelating agent in the extract is preferably 0.1 to 0.7 mM, more preferably 0.5 mM.

A second aspect of the extract of the present invention can preferably contain the above components in a buffer commonly used in experiments such as ELISA, Western blotting, immune tissue staining and the like. Examples of such a buffer include Tris hydrochloride buffer, Trismaleic acid buffer, Tris buffered saline, phosphate buffer, PBS (pH 7.4) which is phosphate buffered saline, and the like, which are preferable. Uses PBS.

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

The kit for detecting a food allergen of the present invention is characterized by containing a monoclonal antibody against the food allergen and the above-mentioned extract. The monoclonal antibody included in the food allergen detection kit may be one or more. In the food allergen detection kit of the present invention, the monoclonal antibody against the food allergen is preferably immobilized on a solid layer that is a support, for example, immunochromatography on which the antibody is immobilized, a plate on which the antibody is immobilized, or the like. Included in form. In addition, the food allergen detection kit of the present invention can include standard products of allergen proteins, detection reagents, dilution buffers and the like.

Hereinafter, examples will be shown for the purpose of specifically explaining the present invention, but the present invention is not limited to the following examples.

<Manufacturing example (manufacturing of an immunochromatography)>
1. Monoclonal antibody (MAb)
The MAb used recognizes the buckwheat allergen protein Fag e2. The hybrid dome that produces MAb was released on April 30, 2014 (Notification No .: 2013-0606), National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture). NITE AP-01791 and NITE AP-01792 deposited and accepted in Japan. (Japanese Patent Laid-Open No. 2015-178461)
2. Preparation of antibody-immobilized membrane MAb (NITE AP-01792) prepared by setting a membrane for immunochromatography (manufactured by Merck Millipore) in a mass coating manufacturing machine (manufactured by Nippon Engineering) and preparing it at 1 mg / ml with PBS. The solution was applied linearly and dried at 50 ° C. for 30 minutes.
3. Preparation of colloidal gold-labeled antibody-immobilized conjugate pad 1 ml of colloidal gold 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, prepare with 1% BSA / Tris (pH 8.2) solution to OD520 = 1-2, and conjugate pad (Merck Millipore). After being applied to (manufactured by), it was freeze-dried.
4. Attach the antibody-fixed membrane, gold colloid-labeled antibody-fixed conjugate pad, sample pad (Merck Millipore), and absorption pad (Merck Millipore) to the backing sheet (manufactured by Nippon Engineering) in order. rice field. This sheet was cut into a width of 5 mm with a compact cutter (manufactured by Nippon Engineering Co., Ltd.) to obtain an immunochromatography.

<Test Example 1 (Examination of Surfactant)>
1. Preparation of extract The respective surfactants are 5 to 0.001 (w / v)% for anionic surfactants, twin surfactants and chaotropic reagents, and 5 to 0.001 (w / v)% for nonionic surfactants. It was dissolved in 0.5 mM EDTA / PBS so as to be 5 to 0.001 (v / v)% to prepare an extract.
2. Extraction method Add 20 mg of primary standard powder of buckwheat to 980 μL of each extract, mix at room temperature for 1.5 minutes using a vortex mixer, and then centrifuge (3,000 × g, 20 minutes, 4 ° C). The supernatant was collected. This solution was diluted 200-fold with each extract to obtain an extract.
The primary standard powder of buckwheat is obtained by mixing equal amounts of buckwheat from Ibaraki prefecture and China (northern China), crushing the powder, and passing through a 14-mesh sieve (opening 1.18 mm).
3. Judgment method Each extraction solution was diluted 10-fold with a developing solution (phosphate buffer solution) to prepare a measurement solution. 100 μL of the measurement solution was dropped onto an immunochromatography and developed at room temperature.
For the negative control, each extract was diluted 10-fold with a developing solution and 100 μL was added dropwise.
After 10 minutes, the color density of the reaction line was measured with an immunochromatographic reader (manufactured by Nippon Flour Mills).
Concentrations that showed positives without causing false positives were defined as the applicable range.
The judgment was according to the table below.

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 zwitterionic surfactant CHAPS

At 0.1 to 5 (w / v)%, positive results were shown without causing false positives, which 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 zwitterionic surfactant CHASPO

At 0.1 to 5 (w / v)%, positive results were shown without causing false positives, which 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

At 0.1 to 1 (v / v)%, positive results were shown without causing false positives, which 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

At 0.01 to 5 (v / v)%, positive results were shown without causing false positives, which was good. 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 Tween20

At 0.1 (v / v)%, it showed positive without causing false positives and was good.

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 anionic surfactant SDS

From 0.01 to 0.05 (w / v)%, it showed positive without causing false positives, 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, which is a chaotropic reagent

At 0.001 to 0.01 (w / v)%, positives were shown without causing false positives, but the measured values were low.

<Test Example 2 (Examination of combination of surfactants)>
The case where an anionic surfactant and a nonionic surfactant were combined as a surfactant was investigated.
0.5 mM EDTA / PBS so that the amount of anionic surfactant as a surfactant is 0.05 (w / v)% and that of nonionic surfactant is 0.001 to 5 (v / v)%. An extract was prepared according to the method of Test Example 1 except that it was dissolved and used as an extract, and extraction was performed to make a judgment. 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 anionic surfactant SDS and nonionic surfactant Triton X-100

When 0.05 (w / v)% SDS and 0.1 to 1 (v / v)% Triton X-100 are combined, the measured values are generally higher than those of SDS alone or Triton X-100 alone. It increased and showed positive in the range of 0.1 to 1 (v / v)% without causing false positives, which 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

Overall measurements when 0.05 (w / v)% SDS and 0.1-5 (v / v)% IGEPAL CA-630 are combined, compared to SDS alone or IGEPAL CA-630 alone. The value was high and positive was shown without causing false positives, which was good.

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 anionic surfactant SDS and nonionic surfactant Tween20

When 0.05 (w / v)% SDS and 0.1 to 1 (v / v)% Tween 20 were combined, the measured values were generally higher than those of Tween 20 alone. 0.05 (w / v)% SDS and 0.1 to 0.4 (v / v)% Tween 20 showed positive results without causing false positives and were good.

<Test Example 3 (Examination of the effect of adding a reducing agent)>
1. Reagents examined (1) Sodium L-ascorbic acid (2) Sodium isoascorbate

2. Preparation of extract solution 0.05 (w / v)% SDS, 0.6 (v / v)% TritonX-100, 0.5 mM EDTA / PBS, which was effective as a solvent for the reducing agent in Test Example 1. (1) and (2) were dissolved so as to be 1 (w / v)%.

3. Extraction method and judgment method The same procedure as in Test Example 1 was carried out.

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. Result

No false positives were generated and positives were obtained even when any reducing agent of sodium L-ascorbic acid or sodium isoascorbic acid was added. With the addition of the reducing agent, the measured value was higher than that without the addition.

<Test Example 3 (Examination of Reducing Agent Concentration)>
The extraction efficiency of sodium L-ascorbic acid was measured using buckwheat primary standard powder or an actual sample. As the actual sample, a commercially available baked confectionery "Soba Bolo" (Heiwa Seika), which is a processed food containing buckwheat, was used.
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 The test using the buckwheat primary standard powder was carried out in the same manner as in Test Example 1.
The test using buckwheat bolo was performed as follows.
19 ml of each extract was added to 1 g of buckwheat bolo, mixed with a vortex mixer (room temperature, 1.5 minutes), and then centrifuged (3,000 × g, 20 minutes, 4 ° C.) to collect the supernatant. .. The recovered solution was diluted 100-fold with each extract to obtain an extract.

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. Result

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

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

2. Preparation of extract Solution 0.05 (w / v)% SDS, 0.6 (v / v)% TritonX-100, 0.01 (w /), which was effective as a solvent for preservatives in Test Example 3. v)% Sodium L-ascorbate, 0.5 mM EDTA / PBS was used to dissolve the preservative to 0.02 (w / v)%.
3. Extraction method and judgment method The same procedure as in Test Example 1 was carried out.

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. Result

All reagents showed positives without producing false positives.

<Test Example 5 (Detection of allergens from model processed foods)>
A model processed food containing the primary standard powder of buckwheat was prepared, and the detection by immunochromatography was confirmed.
1. Preparation of model processed food (a) Retort pouch food To 100 g of minced meat sauce (Ohmai), add 10 mg or 1 mg of the primary standard powder of buckwheat in terms of total protein amount (170 mg of the primary standard powder of buckwheat) ( (The one without addition was used as a negative control), and each was placed in a retort pouch and sealed. This bag was retort-treated (121 ° C., 15 minutes) with a hot water storage type retort sterilizer (manufactured by Hisaka Works).
(B) Baked confectionery To 100 g of a commercially available cookie mix (Ohmai), the primary standard powder of buckwheat was added in the same manner as in (a), and the product was cooked by the method described in the product (heat treatment at 170 ° C. for 15 minutes).
(C) Steamed confectionery To 100 g of a commercially available steamed bread mix (Ohmai), the primary standard powder of buckwheat was added in the same manner as in (a) and cooked by the method described in the product (heat treatment at 100 ° C. for 15 minutes).
(D) Deep-fried confectionery To 100 g of a commercially available pancake mix (Ohmai), the primary standard powder of buckwheat was added in the same manner as in (a), and cooked by the method described in the product (heat treatment at 180 ° C. for 8 minutes).
2. Extract 0.05 (w / v)% SDS, 0.6 (v / v)% TritonX-100, 0.01 (w / v)% Sodium L-ascorbate, 0.5 mM EDTA, 0. 02 (w / v)% Procrine 300, 0.5 mM EDTA / PBS
3. Extraction method and measurement method
Add 19 ml of the extract to 1 g of the retorted or cooked model processed food, mix at room temperature for 1.5 minutes using a vortex mixer, and then centrifuge (3,000 xg, 20 minutes, 4 ° C). The supernatant was collected. This solution was diluted 10-fold 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. Result

The buckwheat allergen protein was also detectable in processed foods treated at high temperature and high pressure.

Claims (6)

An extract consisting of a surfactant and 0.5 mM EDTA / PBS for a buckwheat allergen detection method using a monoclonal antibody, wherein the surfactant is 0.1 to 5 (w / v)%. CHAPS or CHASPO bidirectional surfactant, 0.1-1 (v / v)% polyoxyethylene octylphenyl ether (CAS registration number 9002-93-1: Triton X-100®), 0. 01-5 ( v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630®) or 0.1 (v / v)% polyoxyethylene sorbitan monolaur The extract containing only the rate (CAS registration number 9005-64-5: Tween 20®) and no reducing agent. An extract consisting of a surfactant and 0.5 mM EDTA / PBS for a buckwheat allergen detection method using a monoclonal antibody, which is an anionic surfactant and a nonionic surfactant as surfactants. The anionic surfactant is 0.05 (w / v)% SDS and the nonionic surfactant is 0.1-1 (v / v)% polyoxyethylene octylphenyl ether. (CAS registration number 9002-93-1: TritonX-100®), 0.1-5 (v / v)% octylphenyl polyethylene glycol (CAS registration number 9036-19-5: IGEPAL CA-630 (CAS registration number 9036-19-5: IGEPAL CA-630) A monoclonal antibody which is 0.1 to 0.4 (v / v)% polyoxyethylene sorbitan monolaurate (CAS registration number 9005-64-5: Tween20®) was used. Extract for buckwheat allergen detection method. The extract according to claim 2, further comprising a reducing agent. A method for extracting a buckwheat allergen in a sample, which comprises using the extract according to any one of claims 1 to 3 in a method for extracting a buckwheat allergen from a sample. A method for detecting a buckwheat allergen, which comprises detecting a buckwheat 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 buckwheat allergen, which comprises a monoclonal antibody against buckwheat allergen and the extract according to any one of claims 1 to 3.