JP6944679B2 - Method for quantifying antigens in food - Google Patents

Description

The present invention relates to a method for quantifying an antigen in a food, and more particularly to a method for measuring a food sample by an immunological method after performing a specific pretreatment.

Eggs, milk, wheat, soybeans, buckwheat, peanuts and the like are well known as food materials that cause food allergies. In particular, seven items (eggs, milk, wheat, buckwheat, peanuts, shrimp and crab) that cause serious clinical symptoms such as itchy skin, diarrhea, abdominal pain and anaphylactic shock are required to be labeled as specified raw materials by the Food Sanitation Law. ing. The other 20 items (soybeans, abalone, squid, salmon roe, orange, kiwifruit, beef, walnuts, salmon, mackerel, chicken, pork, matsutake mushrooms, thighs, yams, apples, gelatin, bananas, sesame, and cashew nuts) It is recommended as an allergy labeling item as it conforms to specific raw materials. In the present specification, the above-mentioned specified raw materials and those equivalent to the above-mentioned specified raw materials are referred to as specified raw materials and the like.

Food allergen inspection of foods containing specific raw materials and the like is basically carried out in accordance with "Guidelines for Evaluation of Improvement Method of Inspection Method of Foods Containing Allergens" (Non-Patent Document 1). Patent Document 1 discloses an immunochromatography method and a detection kit for measuring soybean allergen and sesame allergen. In this method, a gold colloid-labeled antibody in which a gold colloid is bound to a monoclonal antibody of a food allergen, a development support in which a monoclonal antibody that recognizes an epitope different from the gold colloid-labeled antibody is immobilized, and an allergen extracted from a test sample are used. After developing the developing solution on the developing support using the developing solution, soybean allergen is detected by the presence or absence of accumulation of colloidal gold. Although monoclonal antibodies can suppress non-specific reactions, when many types of antigens such as soybean allergens are present, it is necessary to prepare monoclonal antibodies corresponding to them. Further, when it is desired to quantify an antigen for which a monoclonal antibody is unknown, a great deal of labor and cost are required for obtaining the monoclonal antibody. Therefore, it is not easy to widely apply a technique such as Patent Document 1 to food allergens.

In Non-Patent Document 2, 7S globulin in various soybeans and their processed foods is measured by direct ELISA using a mixed antibody against three subunits of 7S globulin (also called "β-conglycinin") which is a kind of soybean allergen. bottom. Direct ELISA is said to be poor in quantitativeness and specificity.

Sandwich ELISA is an immunological method that is superior in specificity and quantification as compared with direct ELISA. In Patent Documents 2 and 3, for detecting food allergens (egg white albumin, milk β-lactoglobulin / casein, wheat gliadin, peanut Arah2 component, soybean 7S globulin, soybean 13S globulin, etc.) derived from the above-mentioned specific raw materials and the like, respectively. Examples of the use of commercially available sandwich ELISA kits have been reported. These kits use polyclonal antibodies for both immobilized and labeled antibodies.

The above-mentioned commercially available sandwich ELISA kit has been developed for the purpose of highly sensitively detecting trace amounts of food allergens intentionally or unintentionally contained in processed foods, and the range of immunological measurement methods is extremely narrow. For example, the quantification range of a commercially available sandwich ELISA kit for measuring soy protein (7S globulin) is approximately 1 to 20 μg / g. When trying to measure processed soybean foods (frozen tofu and soymilk) with a 7S globulin concentration on the order of mg / g using this sandwich ELISA kit, the sample extract is divided into several stages so that the measured values are within the calibration curve of the measurement kit. Also needed to be diluted. The increase in the number of times the sample extract was diluted was one of the factors that increased the measurement error.

Antigens such as food allergens have multiple epitopes. When the polyclonal antibody detects these epitopes, the antigen is cross-linked in a chain reaction via the polyclonal antibody, and as a result, the measurement signal tends to be elevated. In particular, when food allergens such as soybean allergens and soybean allergens are the main storage proteins of specific raw materials, the antigen concentration is high when trying to quantify the antigen concentration of foods containing them (for example, soymilk and soybeans). In addition, the variation in measured values becomes even greater.

JP-A-2016-21967 (Method for detecting allergens by lateral flow test) Japanese Patent Application Laid-Open No. 2013-33062 (Food component extraction method, food inspection method, and food inspection kit) Re-table 2013/179663 (Food ingredient extract and extraction method)

Notification of Deputy Director of Consumer Affairs Agency, Food Consumption Table No. 36 dated March 26, 2014 "Guidelines for Evaluation of Improvement Methods for Inspection Methods for Foods Containing Allergens" Moriyama T., et al. (2005). A Novel Enzyme-Linked Immunosorbent Assay for Quantification of Soybean β-Conglycinin, a Major Soybean Storage Protein, in Soybean and Soybean Food Products. J. Nutr. Sci. Vitaminol., 51, 34-39.

Specific methods for quantifying antigens when food contains a large amount of antigens have not been sufficiently established so far. An object of the present invention is to easily and accurately detect an antigen such as a food allergen contained in a large amount in a food material or a processed product thereof, even by using a commercially available ELISA kit prepared for highly sensitive detection of a trace protein. It is to provide a method of quantification. Another object of the present invention is to provide a method for quantifying with high accuracy even when a polyclonal antibody that recognizes a polyvalent antigen such as a food allergen is used.

The present inventors have attempted to perform accurate quantification with good reproducibility by devising the pretreatment of the sample when measuring a food sample having a high antigen concentration with a commercially available ELISA kit for protein detection. As a result, it was surprisingly found that the above problems can be solved according to the following invention. That is, the present invention is a method for quantifying antigens in foods, and the following steps:
It comprises a step of mixing a sample containing the food with a powder diluent to obtain a diluent mixture, and a step of measuring the amount of antigen in the diluent mixture by an immunological method.
Provided is a method for quantifying an antigen in a food product, characterized in that the bulk density of the powder diluent is 0.40 g / mL or more.

The oil content of the sample is preferably 20.0% by mass or less.

The quantification method preferably further includes a step of degreasing the sample so that the oil content is 20.0% by mass or less before the step of obtaining the diluent mixture.

The degreasing is carried out using at least one of the group consisting of, for example, alcohol, acetone, ether, hexane and chloroform.

The quantification method may include a step of drying the sample before the step of mixing with the powder diluent.

It is preferable that the powder diluent does not substantially contain the antigen to be measured for the food.

In the measurement step by the immunological method, it is preferable that the powder diluent does not react with the antibody of the antigen.

The powder diluent is preferably at least one selected from the group consisting of rice flour, wheat flour, aluminum oxide, silicon dioxide and calcium carbonate.

The immunological method is, for example, ELISA.

The immunological method may use a polyclonal antibody.

The food samples include, for example, soybeans, eggs, milk, wheat, buckwheat, peanuts, shrimp, crabs, abalone, squid, how much, oranges, kiwifruit, beef, walnuts, salmon, mackerel, chicken, pork, matsutake, thighs, etc. Includes chicken, apples, gelatin, bananas, sesame, and cashew nuts, and at least one of these processed foods. The food sample preferably contains soybean or processed soybean food.

The antigen is, for example, a food allergen.

The food allergen specifically comprises 7S globulin.

When a food having a high antigen concentration is measured by an immunological method, it is possible to suppress the variation in the measured values by performing a pretreatment of mixing the food sample with a powder diluent having specific physical characteristics according to the present invention. It becomes.

Existing kits for food allergen testing (eg, ELISA kits) are suitable for measuring trace amounts of allergens in food samples. In order to measure, for example, 7S globulin, which is a major soybean storage protein, with such a kit, it was necessary to increase the number of dilutions with the extract in order to dilute to an appropriate concentration of ELISA. This led to variations in measurement. In the present invention, when quantifying an antigen by an immunological method, the amount of antigen can be easily and accurately quantified by mixing a food sample with a diluent powder that is not involved in the antigen-antibody reaction as a pretreatment. .. INDUSTRIAL APPLICABILITY The present invention has established a method for easily and accurately quantifying an antigen, particularly for a food sample containing a large amount of food allergen, even if an existing trace measurement kit is used.

Existing ELISA kits often use polyclonal antibodies. When food allergens were detected with a conventional ELISA kit, the measurement signal was increased by non-specific reactions and cross-linking reactions based on polyclonal antibodies, and it was easy to vary. As shown in Examples described later, the variation in the measurement signal was remarkable in the material rich in food allergens. In the method of the present invention, it is possible to reduce the variation in the measurement signal even when the antigen is measured with an ELISA kit using a polyclonal antibody.

Antigens in foods may be proteins with bioactivity. For example, it has been clarified by animal experiments and human clinical trials that soybean 7S globulin, which is known as a soybean allergen, has physiological functions such as a neutral fat lowering effect. Soybean 7S globulin is expected to be used as a material for functional foods. The ability to easily and accurately quantify 7S globulin in foods according to the present invention is also useful for research and development and production of useful proteins such as 7S globulin.

It is a figure which shows the correlation of the 7S globulin concentration between soybean refined 7S globulin and soybean total protein in the measurement test of ELISA. A clear proportional relationship was observed in the absorbance between the purified soybean 7S globulin and the total soybean protein, and 0.23 as a correction coefficient when calculating the 7S globulin concentration of the soybean sample using the calibration curve of the total soybean protein. Got The relationship between the bulk density of the powder diluent (X-axis) and the coefficient of variation of absorbance during ELISA analysis (Y-axis) is shown. From FIG. 2, in order to suppress fluctuations in immunological measurement of antigens in food samples, the bulk density of the powder diluent needs to be 0.4 g / mL or more, preferably 0.5 g / mL or more. be.

The method for quantifying an antigen in a food according to the present invention is as follows:
It comprises a step of mixing a sample containing the food with a powder diluent to obtain a diluent mixture, and a step of measuring the amount of antigen in the diluent mixture by an immunological method.
The powder diluent is characterized in that the bulk density is 0.40 g / mL or more.

The antigen is not particularly limited as long as it has antigenicity to animals including humans. The antigen is a food allergen particularly contained in specific raw materials such as soybean, milk, wheat, soybean, egg, shrimp / crab, sesame, peanut / cashew nut and the like. A particularly suitable target food allergen for the method of the invention is a soybean allergen such as 7S globulin. 7S globulin comprises a trimer structure of α', α and β subunits having a molecular weight of about 75, 70 and 50 kDa, respectively, and has a molecular weight of 150 to 200 kDa. The three subunits of 7S globulin are highly antigenic as Class I-related antigens, and because 7S globulin is the major storage protein of soybeans (accounting for about 25-30% of total protein in seeds), soybeans. It is of great significance to measure the amount of 7S globulin in food with high accuracy. In addition, since soybean 7S globulin has physiological functions such as a neutral fat lowering effect, it is of great significance to measure the amount with high accuracy.

Foods for which the method of the present invention is suitable are foods having a high antigen concentration that exceeds the measurement range in a normal food protein test kit. For example, soymilk, soybean flour and frozen tofu usually have 7S globulin concentrations of about 10 mg / g or more, 80 mg / g or more, and 140 mg / g, respectively.

Examples of foods having a high antigen concentration are foods and processed foods containing at least one of the above-mentioned specific raw materials as a main material. Foods containing soybeans as the main ingredient and processed foods include frozen tofu, tofu, roasted tofu, thick-fried tofu, cancer-like, fried, yuba, okara, tofu steak, tofu hamburger, soybean sausage, soybean ham, soybean meat, soybean. Solid soybean processed foods such as flour, yellow flour, tofu powder, boiled beans, tofu cookies, soybean senbei, soybean granola; liquid soybean processed foods such as soymilk, prepared soymilk, soymilk beverages; natto, miso, soybean oil, soybean tempe, etc. Soybean fermented foods can be mentioned. Foods containing milk as the main ingredient include dairy products such as milk, powdered milk, condensed milk, cream, pudding, ice cream, and lactic acid drinks; fermented milk such as yogurt, cheese, and butter; and casein-containing foods such as ham and sausage. Can be mentioned. Foods containing wheat as the main ingredient include bread, udon, macaroni, spaghetti, Chinese noodles, okonomiyaki, takoyaki, dumplings, spring rolls and other powders; fried foods such as tonkatsu and tempura; baked goods such as cakes, etc. Be done. Foods containing buckwheat as a main ingredient include buckwheat, buckwheat flour crepe, buckwheat bun, buckwheat tea, buckwheat shochu and the like. Examples of foods containing eggs as a main ingredient include egg white-containing foods such as mayonnaise, pudding, ice cream, pastry, ham, and wieners. Examples of foods containing shrimp and crab as the main material include fresh and cooked shrimp and crab foods, soups made by boiling shrimp and crab, and shrimp crackers.

If the oil content of the food sample is high, it may affect the homogeneity and the dilution time when mixed with the powder diluent, and it is preferable that the oil content of the food sample is low. The oil content of the food sample may be 35% by mass or less, preferably 20% by mass or less, more preferably 10% by mass or less, and particularly preferably 6% by mass or less, although it depends on the type of food. be. If the oil content of the food sample is more than 35% by mass, problems such as uneven distribution of the powder diluent when the food sample and the powder diluent are mixed and a time required for uniform mixing may occur. There is no particular lower limit on the oil content of food samples.

The method for reducing the oil content of the food sample (hereinafter, may be referred to as degreasing) is not particularly limited. For example, the food sample or a mixture of diluents thereof may be immersed in an alcohol such as ethanol or methanol, or a volatile organic solvent such as acetone, ether, hexane or chloroform, mixed and stirred as appropriate, and then the organic solvent may be removed. Further, the oil content may be reduced by means such as absorbing the oil content with a filter paper or the like, or mechanically squeezing the oil content with an oil squeezing machine or the like. For liquid foods, the sample may be centrifuged to separate the oil content. A food sample containing oil may be mixed with a powder diluent and then degreased to further homogenize.

The form of the food sample used in the method of the present invention may be any solid or liquid material such as a dried product, a hydrated material, and a suspended product, but a dried product is preferable. Therefore, when the target of the quantification method of the present invention is a hydrous, suspension or liquid (for example, soymilk), the step of drying the sample is included before the step of mixing with the powder diluent. Is preferable. The drying step may be carried out by an appropriate drying means, for example, heat drying, sun drying, air drying, freeze drying, vacuum drying, spray drying and the like.

The food sample is usually pulverized and homogenized using an appropriate pulverizing / mixing means. Examples of such means include homogenizers, blenders, mill mixers, millers, food cutters, bead mills, wholesalers, sieves and the like. The average particle size of the food sample after pulverization may be usually 350 μm or less, preferably 200 μm or less, and particularly preferably 150 μm or less. In order to obtain a food sample having a desired average particle size, the ground product may be sieved using a sieve having a mesh size of, for example, 125 μm.

The food sample is mixed with a powder diluent. The bulk density of this powder diluent needs to be 0.4 g / mL or more. If the bulk density is less than 0.4 g / mL, problems such as uneven distribution of the powder diluent occur when the powder diluent is mixed with the food sample. As a result, there is a large variation in the measurement of immunological methods. The bulk density of the powder diluent is preferably 0.5 g / mL or more, more preferably 0.8 g / mL or more, and particularly preferably 1.0 g / mL or more.

The powder diluent preferably does not contain an antigen to be measured in a food sample or an antigen similar thereto. Further, it is preferable that the powder diluent does not react with the antibody of the antigen in the measurement step by the immunological method.

Examples of the powder diluent used in the method of the present invention are preferably calcium carbonate, silicon dioxide, rice flour, or aluminum oxide, more preferably rice flour or aluminum oxide, and particularly preferably rice flour. The powder diluent may be used alone or in combination of two or more of these powder diluents.

The dilution ratio of the food sample with the powder diluent is usually less than 20 mg / g, preferably 15 mg / g or less, and more preferably 12 mg / g or less in terms of the antigen concentration of the diluent mixture. For example, in the case of processed soybean foods such as frozen tofu, soybean flour or soymilk, the ratio of the powder diluent (dry matter equivalent) is usually 9 to 99 parts by weight, preferably 19 parts by weight with respect to 1 part by weight of the food sample. ~ 99 parts by weight.

The mixing method is not particularly limited. For example, plastic, iron, or ceramic balls (for example, 1 to 6 mmΦ) are appropriately placed in a container filled with a diluent mixture and shaken by a manual or mechanical mixing means.

Next, a step of measuring the amount of antigen in the diluent mixture by an immunological method will be described. First, the target protein (antigen) is obtained from the above diluent mixture by using a known protein extract, for example, an anionic surfactant such as sodium dodecyl sulfate (SDS), polyoxyethylene (20) sorbitan monolaurate (Tween 20), or the like. Extract with nonionic surfactant. Extraction may be performed with the extract supplied with a commercially available ELISA kit.

An immunoassay is a biochemical test that uses the reaction of an antigen to an antibody to measure the level of a substance contained in a sample. Examples of the immunological methods include ELISA (Enzyme-Linked ImmunoSorbent Assay) method (including direct adsorption method, sandwich method, and competitive method), radioimmunoassay method, laterochromatography method, Western blotting method, aggregation method, and the like. Examples include a turbidity method and a turbidity measurement method. The immunological measurement method is preferably an ELISA method, particularly preferably a sandwich ELISA method.

The above immunological method is based on a conventional method. For example, in sandwich ELISA, a target antigen is bound (primary reaction) to an antibody immobilized on a plate to obtain a complex I composed of an immobilized antibody / antigen, and then an enzyme is added to the antigen on the complex. The labeled polyclonal antibody is bound (secondary reaction) to obtain a complex II consisting of a immobilized antibody / antigen / enzyme-labeled antibody, and finally, the enzyme bound to the complex II on the plate and the enzyme substrate solution are combined. It is reacted (enzymatic reaction) to develop color. The amount of antigen in a food sample is determined by measuring the absorbance of the color-developing solution with a plate reader and applying the obtained absorbance value to a standard curve of a specific protein concentration.

In the above immunological measurement method, a commercially available immunological measurement kit can be used without limitation. For example, commercially available ELISA kits for detecting food proteins include Morinaga FASPEK Eliza II (manufactured by Morinaga Seigaku Kenkyusho Co., Ltd.) and FASTKIT Eliza ver. III (manufactured by Nippon Ham Co., Ltd.) can be mentioned.

The antibody used in the immunological method may be either a polyclonal antibody or a monoclonal antibody. Although the polyclonal antibody can be produced more easily and cheaply than the monoclonal antibody, the antigen measurement value tends to vary due to the higher reactivity and cross-reactivity than the monoclonal antibody. However, in the method of the present invention, even if a polyclonal antibody is used, the variation in the measured value of the antigen becomes small. Therefore, the method of the present invention is particularly advantageous when using polyclonal antibodies. The polyclonal antibody or monoclonal antibody used for the above immunological method can be obtained based on a conventional method. As these antibodies, those attached to a commercially available ELISA kit for protein detection or the like can be used.

The present invention will be described in more detail by presenting examples according to the present invention. However, the present invention is not limited to the following examples.

[Preparation example]
1. 1. Preparation of food samples Various processed soybean foods (frozen tofu manufactured by Misuzu Corporation, soymilk manufactured by Kikkoman Co., Ltd., soybean flour manufactured by Mitake Food Industry Co., Ltd., and natto manufactured by Yukiguni Maitake Co., Ltd.) were prepared as food samples. Soymilk and natto were prepared into dried products by freeze-drying.

The above-mentioned various food samples were appropriately homogenized by crushing / mixing means, and then immersed in ether for mixing / stirring to transfer the oil content in the food sample to ether. Then, the food sample was allowed to stand on the filter paper to remove the ether in which the oil was dissolved. The filter paper-like food sample thus degreased was dried and then sieved with a mesh opening of 125 μm (JIS Z 8801) to collect the food under the sieve. Table 1 shows the oil content before and after degreasing of food samples.

2. Preparation of powder reagent As a powder reagent used for diluting the above food samples, methyl cellulose (product name: Metrose Foods, manufactured by Shin-Etsu Chemical Industries, Ltd.), rice flour (commercially available), wheat flour (weak flour, commercial product) , Silicon dioxide (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.), aluminum oxide (special grade reagent, manufactured by Wako Pure Chemical Industries, Ltd.), and calcium carbonate (special grade reagent, manufactured by Yoneyama Yakuhin Kogyo Co., Ltd.) were prepared. The bulk density of each diluent is shown in Table 2.

3. 3. Immunological measurement test and determination of correction coefficient for antigen quantification As an immunological measurement method, a commercially available sandwich ELISA kit for soybean protein detection (product name: Morinaga FASPEK Eliza II Soybean, manufactured by Morinaga Seigaku Kenkyusho Co., Ltd.) is adopted. bottom. This ELISA kit uses an anti-7S globulin polyclonal antibody as an immobilized antibody for antigen 7S globulin and an enzyme-labeled antibody.

Soybeans according to the method of Nagano et al. (Nagano T., et al., "Dynamic viscoelastic study on the gelation of 7S globulin from soybeans", J. Agric. Food Chem., 1992, 40 (6), pp 941-944). Soybean purified 7S globulin was obtained by purifying the protein.

The total soybean protein attached to the ELISA kit and the purified 7S globulin were measured using the ELISA kit, respectively. The ELISA test was performed according to the instruction manual included with the ELISA kit. The correlation of 7S globulin concentration between purified soybean 7S globulin and total soybean protein based on the result of ELISA measurement is shown in FIG. As shown in FIG. 1, a clear proportional relationship was observed in absorbance between purified soybean 7S globulin and total soybean protein. From the correlation equation of FIG. 1, 0.23 was obtained as a correction coefficient when calculating the 7S globulin concentration of processed soybean food using the calibration curve of total soybean protein.

[Examples 1 to 5 and Comparative Examples 1 to 2] Selection of diluent for processed soybean food and quantitative test of 7S globulin According to the present invention, a food having a high antigen concentration such as processed soybean food is mixed with a powder diluent. Then, by subjecting the obtained diluent mixture to an immunological measurement method, it is possible to accurately measure the concentration of the antigen in the food. Therefore, the powder of processed soybean food was diluted with a powder diluent and a quantitative test of 7S globulin, which is an antigen in the diluted mixture, was carried out according to the following basic procedure.

The powder diluent was evaluated as follows.
(1) Evaluation of Antigenicity of Diluent Using the above ELISA kit, an ELISA measurement test of the diluent alone shown in Table 2 was performed. If the absorbance is 0.100 or less, it is judged that the soybean allergen is not contained, that is, the diluent does not adversely affect the antigen quantification test. The absorbance values of the ELISA test are shown in Table 2.

(2) Evaluation of mixing operability of diluent The operability of mixing each powder diluent into a food sample (defatted frozen tofu powder, bulk density 0.38 g / mL) is visually checked according to the following criteria:
× Difficult to mix food sample and powder diluent △ Powder diluent is biased in the mixture of food sample and powder diluent 〇 Because the mixture of food sample and powder diluent is homogenized evaluated. The evaluation results are shown in Table 2.

(3) ELISA analysis A food sample (defatted frozen tofu powder) was diluted with the powder diluent shown in Table 1, and then sandwich ELISA was performed using the above ELISA kit in order to quantify 7S globulin. The ELISA test was carried out according to the following procedure according to the instruction manual attached to the ELISA kit except that a diluent was used.
1) 9.5 g of various powder diluents shown in Table 1 and 0.5 g (19: 1) of a food sample (powder of defatted frozen tofu, average particle size 150 μm) were accurately weighed and placed in a 50 mL tube.
2) 12 g of a toy bullet (6 mmΦ, made of plastic) was put into the above tube and the lid was closed.
3) The tube was shaken by hand for 5 minutes after closing the lid.
4) Exactly 0.5 g of the homogenized mixture in the tube was weighed into another 50 mL tube and 19.5 mL of the sample extract included in the kit was added. As a blank, extraction with only the diluent was also performed (that is, 19.9 mL of the sample extract was added to 0.1 g of the diluent).
5) A parafilm was wrapped around the lid, and shaking was performed at a temperature of 25 ° C. at 100 rpm overnight.
6) The tube was centrifuged (3000 g, 20 minutes).
7) The intermediate layer of the sample (upper layer: oil, intermediate layer: sample solution, lower layer: precipitate) was diluted 20-fold with the sample diluent I attached to the kit (that is, 950 μL of the diluent was added to 50 μL of the sample solution. bottom).
8) A food sample (frozen tofu powder) was diluted with the sample diluent II attached to the kit to prepare a measurement solution. The food sample was diluted 40 million times.
9) The standard solution (total soybean protein) included in the kit was prepared.
10) 100 μL each of the sample solution or the standard solution was dispensed into the wells of the ELISA.
11) The reaction was allowed to stand at room temperature for 1 hour.
12) The solution in the well was completely removed and washed 6 times with a washing solution (300 μL / well).
13) 100 μL of each enzyme-labeled antibody solution was dispensed.
14) The reaction was allowed to stand at room temperature for 30 minutes.
15) The solution in the well was completely removed and washed 6 times with a washing solution (300 μL / well).
16) 100 μL of each enzyme substrate solution (TMB solution) was dispensed.
17) The reaction was allowed to stand at room temperature for 20 minutes.
18) 100 μL of each of the reaction terminators was dispensed.
19) Using a plate reader (product name: Wallac ARVO, manufactured by PerkinElmer Japan Co., Ltd.), the absorbance at a main wavelength of 450 nm and a sub-wavelength of 620 nm was measured. As Comparative Example 1, the same procedure as the above operation was performed except that the diluent was not used in 1).

により求めた。また、補正係数Ｋを０．２３とした。 The 7S globulin concentration per 1 g of frozen tofu is calculated by the following formula:

Obtained by. The correction coefficient K was set to 0.23.

により算出した。結果を表２に示す。 The above ELISA test was performed three times to determine the average value and standard deviation of the 7S globulin concentration. The results are shown in Table 2. Furthermore, using these values, the coefficient of variation of the 7S globulin concentration is calculated by the following formula:

Calculated by The results are shown in Table 2.

When wheat flour, silicon dioxide, rice flour and aluminum oxide were analyzed by ELISA, the absorbance was extremely low and no color was visually developed (Table 2). Even with methyl cellulose and calcium carbonate, the absorbance was low and almost no color was developed. From this result, it was confirmed that none of the diluents contained soybean allergen, that is, it did not adversely affect the antigen quantification test of food samples.

As shown in Table 2, the operability of mixing the powder diluent with the defatted food sample was inferior to that of the methyl cellulose of Comparative Example 1 and the wheat flour of Example 1, and the calcium carbonate, silicon dioxide, rice flour and oxidation of Examples 2 to 5 were inferior. It was excellent with aluminum.

In Comparative Example 1 in which the food sample was subjected to ELISA analysis without using a powder diluent, the measured value of 7S globulin concentration was as low as 29.3 mg / g, and the coefficient of variation indicating the stability of 7S globulin detection was as high as 42.2%. .. In Comparative Example 2 in which methyl cellulose was used as the powder diluent, the 7S globulin concentration was as high as 126.8 mg / g, but the coefficient of variation was as high as 17.7%. On the other hand, in Examples 1 to 5 in which calcium carbonate, silicon dioxide, rice flour or aluminum oxide was used as the powder diluent, the coefficient of variation was improved to 15.3% to 5.6% while showing a higher 7S globulin concentration. Was done.

The above results suggest that the bulk density of the diluent affects the operability of mixing the powder diluent with the powdered food sample and the fluctuation of the immunological measurement value of the antigen in the food sample. That is, when a powder diluent having a bulk density of less than 0.4 g / mL is used as in Comparative Example 2, the powder diluent is difficult to mix with the food sample, the food sample is not diluted evenly, and the ELISA measurement is performed. The variation becomes large. When a powder diluent having a bulk density slightly exceeding 0.4 g / mL is used as in Example 1, the mixing operability is poor, but the variation in the ELISA measurement values is improved. Further, when a powder diluent having a bulk density of 0.5 g / mL or more is used as in Examples 2 to 5, the mixing operability is improved and the variation of the ELISA measured value is also improved. In particular, in Examples 4 and 5 having a bulk density of 1.0 g / mL or more, the variation in the ELISA measurement value is remarkably improved.

FIG. 2 shows the relationship between the bulk density (X-axis) and the coefficient of variation (Y-axis) of the powder diluent. As can be seen from FIG. 2, the bulk density of the powder diluent needs to be 0.4 g / mL or more, preferably 0.5 g, in order to keep the fluctuation of immunological measurement of the antigen in the food sample low. / ML, more preferably 0.8 g / mL or more, and particularly preferably 1.0 g / mL or more. Further, examples of the diluent capable of providing such bulk density are preferably calcium carbonate, silicon dioxide, rice flour, or aluminum oxide, more preferably rice flour or aluminum oxide, and particularly preferably rice flour.

[Example 6] Test for changing the dilution ratio with a powder diluent In Example 4, the same operation as in Example 4 except that the compounding ratio of the powder diluent to the food sample was changed as shown in Table 3. And ELISA analysis was performed. The results are shown in Table 3 together with Example 4.

As shown in Table 3, it was confirmed that the 7S globulin concentration of the food sample can be measured with high accuracy even if the compounding ratio of the powder diluent is increased.

[Examples 7 to 8] Evaluation test of degreasing of food sample A test was conducted to investigate the influence of the presence or absence of degreasing on the food sample on the operability of mixing the powder diluent with the food sample and the stability of the ELISA measurement. Specifically, the same operation was performed except that the degreased frozen tofu was changed to frozen tofu in Example 4, and the mixing operability of the powder diluent (rice flour) and the measurement stability during the ELISA analysis were evaluated. Since the 7S globulin concentration fluctuates between lots of food, Example 4 was retested using a degreased product of frozen tofu whose raw material lot and production date were different from those of Example 4. The results are also shown in Table 4 as Example 7.

As shown in Table 4, it was reconfirmed that the mixing operability of the powder diluent was good and the variation in the ELISA measurement values was small in Example 7 using the defatted frozen tofu having a different lot from that of Example 4. On the other hand, in Example 8 in which the sample was not degreased, the mixing operability of the powder diluent was deteriorated, and the variation in the ELISA measured values was also large. From this, it can be said that the food sample to be diluted with the powder diluent is preferably degreased.

[Examples 9 to 11] Food sample change test The defatted frozen tofu of Example 4 was changed to defatted soybean flour (Example 9), defatted soybean milk powder (Example 10), and defatted powder natto (Example 11). When the food sample was natto in step 8) of the ELISA test, the same operation as in Example 4 was performed except that the food sample was diluted 20 million times, and the mixing operability of the powder diluent and the measurement stability during the ELISA analysis were performed. Was evaluated. The results are shown in Table 5 together with Example 4.

As shown in Table 5, the concentration of the antigen (7S globulin) could be measured with high accuracy for processed soybean foods other than frozen tofu. According to the present invention, when a food sample having a high antigen concentration is measured by an immunological method, it is possible to suppress the variation in the measured values by performing a pretreatment for diluting the food sample with a powder diluent.

Claims (8)

A method for quantifying 7S globulin in foods having a high concentration of 7S globulin, wherein the following steps:
Wherein the step of measuring to obtain a diluent mixture of the foods are mixed with the powder diluent, and the amount of 7S globulin of the diluent mixture in ELISA,
The powder diluent is selected from those bulk density Ri der least 0.40 g / mL, which does not substantially contain a 7S globulin, and do not react with the 7S globulin antibody in the step of measuring by the ELISA , A method for quantifying 7S globulin in the food. The method for quantifying 7S globulin in a food according to claim 1, wherein the oil content of the food is 20.0% by mass or less. The method for quantifying 7S globulin in a food according to claim 2, further comprising a step of degreasing the food so that the oil content is 20.0% by mass or less before the step of obtaining the diluent mixture. The method for quantifying 7S globulin in a food product according to claim 3, wherein the degreasing is performed using at least one of the group consisting of alcohol, acetone, ether, hexane and chloroform. The method for quantifying 7S globulin in a food according to any one of claims 1 to 4 , which comprises a step of drying the food before the step of mixing with the powder diluent. The method for quantifying 7S globulin in a food according to any one of claims 1 to 5 , wherein the powder diluent contains at least one selected from the group consisting of rice flour, wheat flour, aluminum oxide, silicon dioxide and calcium carbonate. The method for quantifying 7S globulin in a food according to any one of claims 1 to 6 , wherein the ELISA uses a polyclonal antibody. The food article comprises a soybean or processed soybean foods, 7S globulin quantification methods in food according to claim 1.

Images