JP2004333171A - Quantitative measurement method for target element included in food - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To detect a target element included in food accurately and quickly. <P>SOLUTION: Food or processed food and particles (preferably latex particles) on which antibodies or fragments of antibody which selectively react with a target element are connected are made contact with each other in a liquid. A degree of agglutination cased by an antigen-antibody reaction is used as an index to measure the target element quantitatively. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００３５】
〔試験例２〕
ウシラクトフェリンを含有する乳製品（ヨーグルト（森永乳業社製）、ラクトフェリン錠菓（森永乳業社製））のウシラクトフェリン濃度をＥＬＩＳＡ法及び本発明の定量方法により測定した。なお、ヨーグルトのウシラクトフェリン含有量は５０．０ｍｇ／１００ｇであり、ラクトフェリン錠菓のウシラクトフェリン含有量は２０．０ｇ／１００ｇである。
【００３６】
ＥＬＩＳＡ法及び本発明の定量方法は試験例１と同様に実施した。但し、ＥＬＩＳＡ法においては、ヨーグルトを３１，２５０倍及び６２，５００倍に希釈し、ラクトフェリン錠菓を１．２５×１０^７倍及び２．５×１０^７倍に希釈した。また、本発明の方法においては、ヨーグルトを５００倍、１０００倍及び２０００倍に希釈し、ラクトフェリン錠菓を２×１０^５倍、４×１０^５倍及び８×１０^５倍に希釈した。
【００３７】
ＥＬＩＳＡ法及び本発明の方法による測定は、１日あたり併行して６回行った。これと同時に吸光度とウシラクトフェリン濃度との相関・回帰分析を行い（検量線の作成）、吸光度の測定値から希釈倍率を乗じ、サンプル中のウシラクトフェリン濃度を算出した。結果を表２に示す。表２中の数値は、サンプル１００ｇあたりのウシラクトフェリン量（ｍｇ）の平均値であり、括弧内の数値は、不偏標準偏差値である。
【００３８】
【表２】

【００３９】
試験例１及び２に示される結果から、本発明の定量方法によれば、たとえ食品中の標的成分の含有量が微量であっても、ＥＬＩＳＡ法よりも迅速かつ精確に、標的成分を定量できることが判明した。
【００４０】
【発明の効果】
本発明によれば、たとえ食品中の標的成分の含有量が微量であっても、標的成分を迅速かつ精確に定量できる方法が提供される。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for quantifying a target component contained in food, particularly to a quantification method using a latex immunoagglutination method.
[0002]
[Prior art]
Foods may contain useful substances or allergenic substances, and it may be necessary to quantify specific components contained in the foods.
Conventionally, an ELISA method (enzyme-linked immunosorbent assay) has been widely used for quantification of a specific component contained in food.
However, although the ELISA method has excellent quantitative sensitivity, it has a problem that measurement requires a long time. On the other hand, as a food analysis technique using a latex immunoagglutination method, a method for detecting a specific bacterium in a food and a reagent for detection (Patent Document 1), a method for judging a spoilage bacterium in a milky carbonated beverage and a judgment reagent (Patent Document 2) ) Are known, but these are all methods for detecting bacteria contained in foods, that is, methods for qualitatively detecting bacteria, and methods capable of rapidly and accurately quantifying specific components contained in foods. Absent.
[0003]
[Patent Document 1]
JP-A-9-133684 [Patent Document 2]
Japanese Patent Application Laid-Open No. 2002-58426
[Problems to be solved by the invention]
An object of the present invention is to provide a method for quickly and accurately quantifying a specific component contained in a food.
[0005]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides the following quantification method.
(1) A method for quantifying a target component contained in a food, wherein the food or a processed product thereof is brought into contact with a fine particle to which an antibody or a fragment thereof specifically reacting with the target component is bound in a liquid, The above-described quantification method, wherein the target component is quantified using the degree of aggregation generated by the antigen-antibody reaction as an index.
(2) The method according to (1), wherein the fine particles are latex particles.
(3) The quantification method according to (1) or (2), wherein the degree of the aggregation is optically detected.
(4) The quantification method according to any one of (1) to (3), wherein the target component is a useful substance or an allergy-causing substance.
(5) The quantification method according to any one of (1) to (4), wherein only the F (ab ′) ₂ fragment of the antibody is used as the antibody or a fragment thereof to be bound to the fine particles.
(6) The method according to any one of (1) to (5), wherein the food or the processed product is treated with a stabilizer before the food or the processed product is brought into contact with the fine particles. Quantitation method.
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, "food" includes any food and drink, and specific examples of food include milk and dairy products such as milk, processed milk, butter, cheese, condensed milk, milk powder, yogurt; soft drinks, carbonated drinks , Nutritional drinks, fruit drinks, lactic acid drinks, etc .; ice cream, ice sorbet, shaved ice, etc .; candy, candy, gum, chocolate, snacks, biscuits, jellies, jams, creams, baked goods, etc .; buckwheat , Udon, Harusame, Gyoza Peel, Shumai Peel, Chinese Noodles, Instant Noodles and Other Noodles; Fish and Livestock Processed Foods such as Kamaboko, Ham, Sausage; Salad Oil, Tempura Oil, Margarine, Mayonnaise, Shortening, Whipped Cream, Dressing Fats and oils and processed foods; sauces, seasonings such as sauces, and the like.
[0007]
In the present invention, the “target component” means a component to be quantified, and its type is not particularly limited as long as a specifically reactive antibody can be produced. Specific examples of the target component include a protein, a peptide, a saccharide (including a saccharide portion of a glycoprotein) or a mixture thereof, and specifically, lactoferrin, lactalbumin, lactoperoxidase, immunoglobulin, lysozyme, and the like. Useful substances: Allergenic substances such as milk (casein, β-lactoglobulin), egg (ovalbumin), wheat (gliadin), buckwheat, and peanuts. Further, the content of the target component in the food is not particularly limited, but the content of the target component in the food is usually 0.00001 to 50% by mass, preferably 0.0001 to 20% by mass. The quantification method of the present invention enables quick and accurate quantification of a target component even if the content of the target component in the food is minute.
[0008]
When the target component contained in the food is in a state capable of being dispersed in a liquid (for example, when the food is a liquid or the food is dissolvable in a liquid), the food directly reacts specifically with the target component. Antibody or a fragment thereof can be brought into contact with a liquid in a liquid, but when the target component contained in the food is not in a dispersible state in the liquid, the food is subjected to an appropriate treatment to obtain the target component. Is dispersible in a liquid. Examples of the treatment applied to food include crushing and the like.
[0009]
The food or its processed product is preferably treated with a stabilizing agent before it is brought into contact with microparticles to which an antibody or a fragment thereof specifically reacting with the target component is bound in a liquid. The food or its processed product contains various components such as protein, fat, carbohydrate and ash, and various matrices are in a non-uniform state, but by treating the food or its processed product with a stabilizer. , The presence of various matrices can be homogenized, and after such homogenization is performed, by contacting the antibody or a fragment thereof that specifically reacts with the target component with fine particles to which the target component is bound, quantitative sensitivity of the target component is obtained. Can be improved.
[0010]
Specific examples of the stabilizer include serum albumin; L-amino acids such as glycine, cysteine and glutamic acid; monosaccharides such as glucose, mannose, galactose and fructose; sugar alcohols such as mannitol, inositol and xylitol; sucrose; maltose; Saccharides such as disaccharides such as lactose, polysaccharides such as dextran, hydroxypropyl starch, chondroitin sulfate, and hyaluronic acid and derivatives thereof; celluloses such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose sodium Derivatives; polyethylene glycol (preferably having a molecular weight of 6000) and the like. The treatment with the stabilizer can be performed by mixing the food or the processed product thereof with the stabilizer-containing liquid. It is preferable to use a buffer containing 1.2 to 2.4% NaCl as the solvent of the stabilizer-containing liquid, and it is preferable that the amount of the stabilizer added is 0.2 to 5% by mass.
[0011]
In the present invention, "antibody" includes both monoclonal antibodies and polyclonal antibodies, and "antibody fragments" include any fragments as long as they can react with a target component contained in food. Specific examples of antibody fragments include Fab fragments, Fab 'fragments, and F (ab') ₂ fragments. Further, “specifically reacts with the target component” means that the component reacts with the target component but does not react with other components contained in the food.
[0012]
An antibody can be obtained, for example, as follows using a target component as an antigen for immunization.
In preparing a polyclonal antibody, mammals such as rats, mice, guinea pigs, rabbits, sheep, horses, and cows are first immunized using an antigen for immunization. In immunization, in order to induce antibody production, it is preferable to carry out immunization a plurality of times after emulsifying with an immune aid such as Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FIA). The antibody titer against the target component is measured, and blood is collected after the antibody titer has increased to obtain an antiserum.
[0013]
In preparing a monoclonal antibody, a mammal is immunized with an immunizing antigen in the same manner as in the case of the polyclonal antibody, and then antibody-producing cells are collected. Examples of the antibody-producing cells include spleen cells, lymph node cells, thymocytes, peripheral blood cells, and the like, and spleen cells are generally used. Next, in order to obtain a hybridoma, cell fusion between the antibody-producing cell and the myeloma cell is performed. After the cell fusion treatment, the cells are cultured using a selection medium, and the desired hybridoma is selected. Next, it is screened whether the target antibody is present in the culture supernatant of the grown hybridoma. Next, the hybridoma is cloned by a limiting dilution method, a soft agar method, a fibrin gel method, a fluorescence excitation cell sorter method or the like, and finally a hybridoma producing a monoclonal antibody is obtained. As a method for collecting a monoclonal antibody from the obtained hybridoma, a usual cell culture method or the like can be used. In addition, after transplanting the hybridoma into the abdominal cavity of a mouse or the like, ascites may be collected, and a monoclonal antibody may be obtained from the ascites.
[0014]
When purification of polyclonal or monoclonal antibodies is required, it is possible to appropriately select a method such as salting out with ammonium sulfate, gel chromatography, ion exchange chromatography, affinity chromatography or the like, or to use a combination thereof. it can.
[0015]
It is preferable to use only the F (ab ') ₂ fragment as the antibody or its fragment to be bound to the microparticles. It is better to use microparticles to which only the F (ab ') ₂ fragment is bonded than to use microparticles to which a mixture of Fab fragments, Fab' fragments, F (ab ') ₂ fragments, and Fc fragments are bonded. In addition, the sensitivity of quantifying the target component can be improved. The F (ab ') ₂ fragment can be obtained by treating the antibody with pepsin and then purifying it using affinity chromatography or the like. The isoelectric point of F (ab ') ₂ is preferably from 4.0 to 6.0.
[0016]
The “fine particles” are not particularly limited in the material as long as they are insoluble in a liquid used when the target component and the fine particles are brought into contact. Examples of the material of the fine particles include latex, bentonite, and collodion. , Kaolin, sheep erythrocytes and the like. The latex includes various synthetic resin latexes synthesized by emulsion polymerization or a polymerization method using no emulsifier. Specific examples of the latex include polystyrene, styrene-methacrylic acid copolymer, and styrene-glycidyl (meth) acrylate copolymer. Examples thereof include a polymer, a styrene-styrene sulfonate copolymer, a methacrylic acid polymer, an acrylonitrile butadiene styrene copolymer, a vinyl chloride-acrylate copolymer, and a polyvinyl acetate acrylate. As the latex, a homopolymer and a copolymer containing styrene as a constitutional unit are preferable from the viewpoint that particles having a uniform particle size can be synthesized with good reproducibility.
[0017]
The particle size of the fine particles is not particularly limited as long as it can be dispersed in a liquid used when the target component and the fine particles are brought into contact with each other. It is preferably 0.5 μm, and more preferably 0.1 to 0.3 μm.
[0018]
As a method for binding an antibody or a fragment thereof to microparticles, a physical adsorption method using a hydrophobic bond or a functional group (for example, a carboxyl group, an amino group, a hydroxyl group, a tosyl group, or a thiol group) possessed by the microparticles or the antibody is used. Covalent bonding and the like can be mentioned. When latex particles are used, the antibody or a fragment thereof can be easily bound to the latex particles without special treatment. The antibody or fragment thereof is preferably bound to the microparticles in a sufficient amount. When latex particles are used, for example, a liquid containing latex particles and the antibody or fragment thereof is added at 20 to 25 ° C. for 0.5 to 6 hours. By stirring, a sufficient amount of the antibody or a fragment thereof can be bound (sensitized) to the latex particles.
[0019]
After binding the antibody or fragment thereof to the microparticles, it is preferable to block unreacted sites on the surface of the microparticles with bovine serum albumin or the like. As a result, non-specific binding can be prevented, and the stability of the fine particles in the reaction solution can be improved.
[0020]
As the liquid used when the food or the processed product thereof is brought into contact with fine particles to which an antibody or a fragment thereof specifically reacting with the target component is bound, the target component, the antibody or a fragment thereof can be stably present. There is no particular limitation as long as the buffer is, for example, a buffer such as a phosphate buffer, a glycine buffer, a Tris buffer, a borate buffer, and a triethanolamine buffer. The buffer may contain any components as long as they do not interfere with the antigen-antibody reaction between the target component and the antibody or fragment thereof.
[0021]
Conditions for contacting a food or a processed product thereof with microparticles to which an antibody or a fragment thereof specifically reacting with a target component is attached may cause an antigen-antibody reaction between the target component and the antibody or a fragment thereof. The reaction temperature is usually 0 to 40 ° C., preferably 20 to 37 ° C., and the reaction time is usually 0.5 to 5 minutes, preferably 0.5 to 2.5 minutes. It is.
[0022]
Aggregates of antigens and antibodies generated by the antigen-antibody reaction are very small, and it is difficult to detect aggregation when the amount of antigen is small.However, in the present invention, the antigen-antibody reaction is carried out in the form of aggregation of fine particles. Since it appears, aggregation can be easily detected.
[0023]
The method for detecting aggregation is not particularly limited, but optical detection is preferred. Examples of the optical detection method include a method of measuring the absorbance (transmitted light) or scattered light of a liquid. The absorbance (transmitted light) or scattered light can be measured using a commercially available optical instrument. The wavelength used for the measurement varies depending on the type of food, target component, fine particles, etc., but is usually 300 to 1000 nm, preferably 550 to 900 nm.
[0024]
In the present invention, the target component can be quantified by using the degree of aggregation produced by the antigen-antibody reaction as an index. That is, the target component can be quantified from the degree of aggregation based on the correlation (calibration curve) between the degree of aggregation and the concentration of the target component. The “degree of aggregation” includes the amount of aggregation and the rate of aggregation, and according to the optical detection method, the degree of aggregation can be measured as the turbidity of the liquid or the rate of change in turbidity.
[0025]
【Example】
[Example 1]
50 mg of bovine lactoferrin (purity 99%, manufactured by Mirai) is added to 100 g of prepared milk powder (containing no lactoferrin, manufactured by Meiji Dairies) and 100 mL of ultra-high temperature heat treated (UHT) pasteurized milk (containing no lactoferrin, manufactured by Morinaga Dairies) Then, 100 mg of bovine lactoferrin was added to 100 g of yogurt (containing no lactoferrin, manufactured by Morinaga Milk Industry Co., Ltd.). In addition, the purity of lactoferrin was measured by HPLC (Shodex C4P-504D; 4.6 mmID × 150 mmL).
The bovine lactoferrin concentrations of the above prepared milk powder, milk and yogurt were measured by the ELISA method and the quantitative method of the present invention.
[0026]
(1) Preparation of test solution for immunoassay [ELISA method]
Dilutions were performed using PBS containing 0.05% Tween and 1% gelatin. Milk powder and milk were diluted 31,250-fold and 62,500-fold, and yogurt was diluted 62,500-fold and 125,000-fold.
[0027]
[Quantitative method of the present invention]
Dilutions were made using a 1.2% NaCl solution containing 2% bovine serum albumin (BSA). The prepared milk powder and the milk were diluted 500 times, 1000 times and 2000 times, and the yogurt was diluted 1000 times, 2000 times and 4000 times.
[0028]
(2) Immunoassay for bovine lactoferrin [ELISA method]
The affinity-purified goat anti-bovine lactoferrin antibody (Bethyl A10-126A-2) was diluted with 0.1 mol / L sodium bicarbonate (pH 9.6) to 0.5 μg / mL, and 50 μL was added to each well of Nunc-Immunoplate Maxisorp F96. Added. After incubation at room temperature for 2 hours, the antibody solution was removed, and each well was washed four times with PBS containing 0.05% Tween. Then, 200 μL of PBS containing 1% gelatin (Biorad 170-6537) was added to each well and incubated for 1 hour at room temperature. Then, the PBS was removed and each well was washed four times with PBS containing 0.05% Tween.
[0029]
Test solution (50 μL) was added to each well and incubated for 1.5 hours at room temperature. After incubation, the test solution was removed and each well was washed four times with PBS containing 0.05% Tween. Horseradish peroxidase-conjugated goat anti-bovine lactoferrin antibody (Bethyl A10-126P-4) was diluted 2000-fold with PBS containing 0.05% Tween and 1.0% gelatin, and 50 μL was added to each well. After incubation at room temperature for 1 hour, the antibody solution was removed, and each well was washed with PBS containing 0.05% Tween. Immediately before use, an O-phenylenediamine (OPD) tablet (manufactured by Sigma) was dissolved in 20 mL of distilled water, and 100 μL of the solution was added to each well. After incubation for 15 minutes, 20 μL of 8 mol / L H ₂ SO ₄ was added to each well. The absorbance at 492 nm of each well was measured using a microplate reader.
[0030]
[Quantitative method of the present invention]
Rabbit polyclonal IgG was purified from rabbit antiserum by performing ammonium sulfate purification followed by anion exchange chromatography. Rabbit polyclonal IgG was digested with pepsin to obtain F (ab ') ₂ fragment, which was then purified by affinity chromatography to remove undigested IgG and Fc fragment. As a result of measuring the degree of purification by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), no contamination of undigested IgG and Fc fragment was detected. The isoelectric point of the F (ab ') ₂ fragment was 4.0 to 4.6.
[0031]
Polystyrene latex particles having a diameter of 0.1 to 0.3 μm and a specific gravity of 1.002 were used. The latex particles were slowly mixed with the F (ab ') ₂ fragment with gentle shaking. The solution was incubated at 25 ° C. for 4 hours with gentle stirring in a thermostat. After incubation, the solution was centrifuged and the supernatant was removed. In order to avoid non-specific binding and to improve the stability of the latex particles coated with F (ab ') ₂ fragments in the reaction buffer, unreacted binding sites on the latex particle surface are converted to bovine serum albumin (BSA). Blocked by.
[0032]
The test solution (15 μL) was added to 200 μL of Tris buffer (reagent 1) containing 1% polyethylene glycol 6000 (stabilizer) and 1.2% NaCl and stirred. Six minutes later, 30 μL of a uniformly mixed latex particle solution (reagent 2) was added. The rate of change in absorbance at 800 nm of the reaction mixture was measured for 30 to 150 seconds after the addition of Reagent 2.
[0033]
The measurement by the ELISA method and the method of the present invention was performed 6 times per day in parallel, and this was performed for 3 days. At the same time, a correlation / regression analysis between the absorbance and the bovine lactoferrin concentration was performed (preparation of a calibration curve), and the measured absorbance value was multiplied by the dilution factor to calculate the bovine lactoferrin concentration in the sample. Table 1 shows the results. The numerical values in Table 1 represent the amount (mg) of bovine lactoferrin per 100 g or 100 mL of the sample.
[0034]
[Table 1]

[0035]
[Test Example 2]
The concentration of bovine lactoferrin in dairy products containing bovine lactoferrin (yogurt (manufactured by Morinaga Dairy Co., Ltd.) and lactoferrin tablet confectionery (manufactured by Morinaga Dairy Co., Ltd.)) was measured by the ELISA method and the quantitative method of the present invention. In addition, the bovine lactoferrin content of yogurt is 50.0 mg / 100 g, and the bovine lactoferrin content of lactoferrin tablet confections is 20.0 g / 100 g.
[0036]
The ELISA method and the quantification method of the present invention were performed in the same manner as in Test Example 1. However, in the ELISA method, yogurt was diluted 31,250-fold and 62,500-fold, and lactoferrin tablet was diluted 1.25 × 10 ⁷ and 2.5 × 10 ^7- fold. In the method of the present invention, yogurt was diluted 500 times, 1000 times and 2000 times, and lactoferrin tablet was diluted 2 × 10 ⁵ times, 4 × 10 ⁵ times and 8 × 10 ⁵ times.
[0037]
The measurement by the ELISA method and the method of the present invention was performed 6 times per day in parallel. At the same time, a correlation / regression analysis between the absorbance and the bovine lactoferrin concentration was performed (preparation of a calibration curve), and the measured absorbance value was multiplied by the dilution factor to calculate the bovine lactoferrin concentration in the sample. Table 2 shows the results. The numerical values in Table 2 are average values of the amount (mg) of bovine lactoferrin per 100 g of the sample, and the numerical values in parentheses are unbiased standard deviation values.
[0038]
[Table 2]

[0039]
From the results shown in Test Examples 1 and 2, according to the quantification method of the present invention, even if the content of the target component in the food is very small, the target component can be quantified more quickly and more accurately than the ELISA method. There was found.
[0040]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, even if the content of the target component in a foodstuff is very small, the method of quantifying a target component quickly and accurately is provided.

Claims (6)

A method for quantifying a target component contained in a food, wherein the food or a processed product thereof is brought into contact with a fine particle to which an antibody or a fragment thereof specifically reacting with the target component is bound in a liquid, and the antigen-antibody reaction is performed. The method for quantifying the target component, wherein the target component is quantified using the degree of agglutination caused by the reaction as an index. The method according to claim 1, wherein the fine particles are latex particles. The quantification method according to claim 1, wherein the degree of the aggregation is optically detected. The method according to any one of claims 1 to 3, wherein the target component is a useful substance or an allergy-causing substance. The method according to any one of claims 1 to 4, wherein only the F (ab ') ₂ fragment of the antibody is used as the antibody or a fragment thereof to be bound to the fine particles. The method according to any one of claims 1 to 5, wherein the food or the processed product is treated with a stabilizer before the food or the processed product is brought into contact with the fine particles.