JP2016211967A - Allergen detection method using immuno-chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an allergen detection method using immuno-chromatography, and an allergen detection kit for immuno-chromatography, with which a soybean allergen and a sesame allergen are extracted from a test sample without using 2-mercaptoethanol, and further with which the allergens are rapidly and accurately detected by suppressing nonspecific reactions accompanying disintegration of colloidal gold conjugated to antibodies.SOLUTION: In an allergen detection method using immuno-chromatography, there are used: a colloidal gold-labeled antibody in which colloidal gold is conjugated to a monoclonal antibody for recognizing both denatured and native allergens; a developing support medium to which a monoclonal antibody for recognizing an epitope different from that recognized by the colloidal gold-labeled antibody is fixed; a measuring sample of allergens extracted from a test sample by using an anionic surfactant such as SDS, and thiosulfate and a nonionic surfactant; and a developing solution containing FBS. After developing the developing solution on a developing support medium in the allergen detection method using immuno-chromatography, soybean 7S globulin and sesame 11S globulin are detected according to the presence/absence of colloidal gold deposition.SELECTED DRAWING: None

Description

  The present invention extracts a major allergen of soybeans and sesame from a test sample such as a food containing the major allergens of soybean and sesame using an anionic surfactant, thiosulfate, and a nonionic surfactant. These allergens can be efficiently extracted with anionic surfactants, thiosulfates, and nonionic surfactants in any state of denaturation / denaturation, and accompanied by the collapse of colloidal gold bound to antibodies. The present invention relates to a method for detecting an allergen by an immunochromatography method capable of suppressing a non-specific reaction and detecting it quickly and an allergen detection kit for an immunochromatography which can be used therefor.

  Due to various factors such as a decrease in the natural environment, exhaust gas from cars and factories, housing conditions, and changes in food, one in three people is now said to have some allergic disease. In particular, food allergies are harmful immune reactions caused by the intake of allergens (hereinafter referred to as food allergens) contained in foods, causing dermatitis, asthma, gastrointestinal disorders, anaphylactic shock, etc. The increasing number of patients with food allergies has caused serious problems in medicine and the food industry. These harms can be fatal and require action. To that end, the need to provide information to consumers through the labeling is increasing, and the FAO / WHO Joint Food Standards Committee has confirmed that for foods containing eight kinds of raw materials known as allergic substances. Agreed to indicate that it is included, and decided to consider a labeling method suitable for each country's system in the member countries (June 1999). In Japan, a labeling method was established for 24 food items that had a history of causing severe allergic symptoms in consideration of the degree and frequency of past health hazards (effective from April 2002). Later, it was reviewed based on a survey of food allergy patients, and “banana” was added in 2004 and “sesame” and “cashew nuts” were added in 2013 as recommended display items. In addition, “Shrimp” and “Crab”, which were recommended display items, became display-required items in 2008, and as of May 2015, a display method is defined for a total of 27 food items. As foods causing allergies, eggs, milk, meat, fish, crustaceans, mollusks, cereals, beans, nuts, fruits, vegetables, brewer's yeast, gelatin and the like are known.

  In order to detect the above-mentioned allergen quickly and easily, as an immunoassay for detecting a target substance comprising a specific antigen or antibody using a specific reaction by an antigen-antibody, the target substance in a sample is An agglutination method that binds an antibody or antigen sensitized to microparticles by an immune reaction and measures the aggregation state of the microparticles resulting from the binding is a simple immunoassay method, and is generally used because it can be visually judged. It is the method that has been.

  In addition, a radioimmunoassay method or enzyme immunity method in which an antibody or antigen labeled with a labeling substance composed of a radioisotope, an enzyme or a fluorescent substance is bound to an analyte in a sample by an immune reaction, and the bound labeling substance is measured. Measurement methods or fluorescence immunoassays are also employed. In these immunoassays, competitive reaction and sandwich reaction are widely used. Of these, immunochromatography is known as a so-called sandwich-type reaction measurement method (see, for example, Patent Document 1). In addition to high specificity resulting from an antigen-antibody reaction, various features characterized by simplicity and rapidity. Allergen detection kits are sold.

  Samples applied to such immunochromatography include biological samples and extracts from foods, but depending on the type of sample, a so-called non-specific reaction that shows a light color at the capture site despite the absence of the specimen. May cause a decrease in accuracy in inspection. Therefore, the buffer contains a polymer having a phosphorylcholine group at a concentration of 0.005 to 0.3 w / v%, and the number average molecular weight of the polymer is 40,000 or more. There has been proposed a developing solvent (see, for example, Patent Document 2) that prevents non-specific aggregation and non-specific reaction during measurement and thus enables measurement with high accuracy.

  Further, in the above immunoassay method, when the protein is denatured by heating or pressurization, the measurement result is sometimes lowered or cannot be measured. In the sandwich ELISA method listed in Food Safety No. 012001, which is notified by the Ministry of Health, Labor and Welfare as a method for inspecting specific raw materials, a denaturant and a reducing agent are used to sufficiently extract each allergen from a heated test sample. A method of extraction using an extraction solution using (2-mercaptoethanol) is employed. The sample preparation method of SDS-polyacrylamide electrophoresis used for protein analysis, which has been conventionally used, is applied. In order to increase the extraction efficiency of each allergen from a test sample, a denaturant and a reducing agent are used. The agent was considered essential. Moreover, when these were applied to the immunochromatography method, since many non-specific reactions were observed, an accurate test could not be performed, which caused a problem when measuring denatured proteins.

  Applicant can extract allergens quickly and accurately by suppressing non-specific reactions associated with colloidal collapse of gold colloids, even when extracted using an extraction solution containing a denaturant and a reducing agent and adapted to immunochromatography. The immunochromatography method which can be performed is proposed (for example, refer patent document 3). In this method, since allergens were sufficiently extracted from a heated test sample and further tested by a simple immunochromatography method, accuracy and simplicity could be dramatically improved. However, since the reducing agent used (2-mercaptoethanol) has a peculiar odor and 2-mercaptoethanol has been designated as a toxic substance on July 1, 2008, it can be used easily in a food manufacturing factory. Has become difficult. Therefore, there has been a demand for a safer and more efficient extraction method and an immunochromatography method in which a non-specific reaction is not observed when these are applied to the immunochromatography method and an accurate test can be performed.

JP-A-5-010950 JP 2003-344406 A JP 2007-278773 A

  An object of the present invention is to extract soy allergen and sesame allergen from a test sample such as food containing main allergens of soybean and sesame without using 2-mercaptoethanol, and further, colloidal gold bound to the antibody. An object of the present invention is to provide a method for detecting an allergen by an immunochromatographic method capable of detecting an allergen quickly and accurately, suppressing a non-specific reaction associated with the decay, and a kit for detecting an allergen for immunochromatography that can be used in such a method.

  The present inventors provide a gold colloid-labeled antibody obtained by binding a gold colloid to a monoclonal antibody against a denatured and native allergen and a monoclonal antibody against a denatured and native allergen that recognizes a different epitope from the gold colloid-labeled antibody. Using an anionic surfactant and a thiosulfate, or an anionic surfactant and a nonionic surfactant from a test sample such as a food carrier containing allergens and a development support fixed in position In an immunochromatography method in which allergen is detected by the presence or absence of accumulation of colloidal gold, the fetal bovine serum (FBS) is developed by using a developing solution containing the extracted denatured and undenatured allergen measurement samples and then developing on a development support. An allergen by immunochromatography characterized by using a developing solution containing at least 10% by weight A detection method (International Publication WO2010 / 095469 pamphlet) has already been developed, αs1 casein as a major component of milk allergen, β-lactoglobulin as a major component of whey allergen, ovalbumin and ovomucoid as egg white allergen, wheat allergen As a major component of the protein, excellent detection ability has been confirmed for gliadin, a buckwheat major protein having molecular weights of 24 kDa and 76 kDa, and a denatured and undenatured allergen selected from Arah1, a major protein of peanut. The present inventors continued further investigation and developed an excellent monoclonal antibody against soybean 7S globulin or sesame 11S globulin after more than one year of trial and error. In addition, by extracting from a test sample using an extract containing an anionic surfactant, thiosulfate, and a nonionic surfactant, the main allergens of soybeans and sesame are extracted using the antibody. The inventors have found that detection can be performed quickly and accurately, and the present invention has been completed.

  That is, the present invention includes (1) a colloidal gold-labeled antibody in which a colloidal gold is bound to a monoclonal antibody that recognizes both denatured and undenatured allergens, and recognizes both denatured and undenatured allergens and is different from the gold colloid-labeled antibody. A development support in which a monoclonal antibody that recognizes an epitope is fixed at a predetermined position, an allergen measurement sample extracted from a test sample using an extract, and fetal bovine serum (FBS) at least 10% by weight An immunochromatographic method for detecting an allergen based on the presence or absence of accumulation of colloidal gold at a predetermined position after a mixed solution of a colloidal gold labeled antibody, a measurement sample and a developing solution is developed on a developing support. The allergen is soybean 7S globulin or sesame 11S globulin, and the extract is anionic A method for detecting allergens by immunochromatography, characterized in that it contains an ionic surfactant, a thiosulfate, and a nonionic surfactant, and (2) at least 30% by weight of fetal bovine serum (FBS) (1) or (1), wherein the allergen detection method by immunochromatography according to (1) above, wherein (3) sodium dodecyl sulfate is used as an anionic surfactant. 2) The allergen detection method by the immunochromatography method according to any one of (1) to (3) above, wherein the allergen detection method by the immunochromatography method or (4) Tween 20 is used as a nonionic surfactant. And (5) a hybridoma (NITE AP-02) as a monoclonal antibody against soybean 7S globulin. 039) produced by PDSY1 and hybridoma (NITE AP-02040) produced by PDSY2, the method for detecting an allergen by immunochromatography according to any one of (1) to (4), Any one of (1) to (4) above, wherein PDSE1 produced by a hybridoma (NITE AP-02041) and PDSE2 produced by a hybridoma (NITE AP-02042) are used as monoclonal antibodies against sesame 11S globulin. The present invention relates to a method for detecting allergens by immunochromatography.

  The present invention also provides (7) a colloidal gold-labeled antibody in which a colloidal gold is bound to a monoclonal antibody that recognizes both denatured and undenatured allergens, an epitope that recognizes both denatured and undenatured allergens and is different from the colloidal gold-labeled antibody. Extraction containing a development support on which a monoclonal antibody recognizing is fixed at a predetermined position and an anionic surfactant, thiosulfate, and a nonionic surfactant for extracting allergen from a test sample And an allergen detection kit for immunochromatography, characterized in that the allergen is soybean 7S globulin or sesame 11S globulin, and a developing solution containing fetal bovine serum (FBS) or fetal bovine serum (FBS), 8) The said (7) description characterized by containing sodium dodecyl sulfate as an anionic surfactant. (9) The immunochromatographic allergen detection kit according to (7) or (8) above, which contains Tween 20 as a nonionic surfactant, and (10) soybean. The monoclonal antibody against 7S globulin is PDSY1 produced by a hybridoma (NITE AP-02039) and PDSY2 produced by a hybridoma (NITE AP-02040), according to any one of the above (7) to (9) The detection kit for allergens for immunochromatography and (11) monoclonal antibody against sesame 11S globulin is PDSE1 produced by hybridoma (NITE AP-02041) and PDSE2 produced by hybridoma (NITE AP-02042). The immunochromatographic allergen detection kit according to any one of (7) to (9) above, and (12) PDSY1 produced by a hybridoma (NITE AP-02039) and a hybridoma (NITE AP-02040) produced A combination of monoclonal antibodies against soybean 7S globulin characterized by containing PDSY2 and (13) PDSE1 produced by hybridoma (NITE AP-02041) and PDSE2 produced by hybridoma (NITE AP-02042) The present invention relates to a combination of monoclonal antibodies against sesame 11S globulin.

  According to the method for detecting an allergen by the immunochromatography method of the present invention, when an extract containing an anionic surfactant, thiosulfate, and a nonionic surfactant is used, the colloid of gold bound to the antibody is disrupted. Non-specific reaction can be suppressed and soybeans and sesame can be detected quickly and accurately.

It is a graph which shows the specificity with respect to the antigen of a solid phase of five types of antibodies which are positive with respect to soybean 7S globulin. It is a graph which shows the specificity with respect to the antigen of a solid phase of six types of antibodies which are positive with respect to sesame 11S globulin.

  As the method for detecting allergens by the immunochromatography method of the present invention, a gold colloid-labeled antibody in which gold colloid is bound to a monoclonal antibody that recognizes both denatured and undenatured allergens, and both denatured and undenatured allergens are recognized. A development support on which a monoclonal antibody recognizing an epitope different from a colloid-labeled antibody is fixed at a predetermined position, an allergen measurement sample extracted from a test sample using an extract, and at least 10 fetal bovine serum (FBS) Using a developing solution containing 5% by weight, a mixture of a colloidal gold labeled antibody, a measurement sample and a developing solution is developed on a developing support, and then the allergen is determined depending on whether or not gold colloid is accumulated at the predetermined position. And the allergen is soybean 7S globulin or sesame 11S. The detection method is not particularly limited as long as it is a detection method characterized in that it is lobulin and the extract contains an anionic surfactant, a thiosulfate, and a nonionic surfactant. Samples can include test samples such as foods that may contain allergens. Examples of test samples such as foods include various foods and raw materials used to produce the foods. Residue remaining in the apparatus used to produce the food, residue such as precipitates, cleaning liquid for cleaning the apparatus, rinsing liquid used for removing the cleaning liquid, wrapping paper packaging the food, Samples that may have secondary presence of allergens present in food, such as packaging containers, can be included.

  The immunochromatographic allergen detection kit of the present invention includes a colloidal gold-labeled antibody in which a colloidal gold is bound to a monoclonal antibody that recognizes both denatured and undenatured allergens, and both the denatured and undenatured allergens. A development support in which a monoclonal antibody that recognizes an epitope different from the labeled antibody is fixed in place, an anionic surfactant, thiosulfate, and nonionic surfactant for extracting allergens from a test sample A detection kit comprising an extract containing an agent and a developing solution containing fetal bovine serum (FBS) or fetal bovine serum (FBS), wherein the allergen is soybean 7S globulin or sesame 11S globulin. Although there is no particular limitation, it is practical even when stored at room temperature for more than one year from the date of manufacture. Those having Eur accuracy and stability is desired.

  Examples of fetal bovine serum (FBS) concentration in the developing solution include 10 to 50% by weight, preferably 20 to 40% by weight, more preferably 25 to 35% by weight, and less than 10% by weight. It is not preferable because a specific reaction is likely to occur. In the developing solution, in addition to fetal bovine serum (FBS), various additives such as other surfactants, preservatives, and inorganic salts are suspended, emulsified or dissolved in the buffer solution as necessary. Can be prepared. The buffer solution preferably has a pH of 4 to 10, particularly preferably pH 6 to 8. For example, a phosphate buffer solution (PBS) or a Tris buffer solution (TBS) can be suitably exemplified.

  A method for producing a colloidal gold-labeled antibody in which colloidal gold is bound to the above monoclonal antibody is not particularly limited, including a conventionally known method. For example, a 2 mM boronic acid solution is added to a colloidal gold solution prepared to pH 9.0 with 0.2 M potassium carbonate solution. An example is a method in which a solution in which a monoclonal antibody is dissolved in an acid buffer (pH 9.0) is added and reacted at room temperature for 30 minutes, and then a 10% BSA solution is added, further reacted for 15 minutes, and centrifuged. The colloidal gold labeled antibody carrier can be produced by applying the colloidal gold labeled antibody produced above to, for example, a glass wool conjugate pad and drying.

  The development support is, for example, a buffer solution containing a monoclonal antibody that recognizes both a modified and non-denatured allergen that recognizes an epitope different from that of a colloidal gold-labeled antibody, for example, linearly applied to a nitrocellulose membrane and dried. It can be produced by a blocking treatment.

  As an anionic surfactant in the extract used when preparing the measurement sample of allergen extracted from the test sample using the extract, higher alcohol sulfate, alkylnaphthalene sulfonate, alkylbenzene sulfonate Salt, alkyl phosphate ester salt, and the like. Specific examples thereof include sodium dodecyl sulfate (SDS). Examples of the thiosulfate include sodium thiosulfate, potassium thiosulfate, and ammonium thiosulfate. Specifically, sodium thiosulfate can be preferably exemplified. Nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene fatty acid amide, polyoxyethylene alkylamine, sorbitan fatty acid ester, etc. Specifically, polyoxyethylene (20) sorbitan monolaurate (Tween 20) can be preferably exemplified. The concentration of the anionic surfactant is 0.1 to 2.0%, preferably 0.25% to 1%, and the concentration of thiosulfate is 0.01 to 5.0%, preferably 0.8. The concentration of the nonionic surfactant is from 0.01% to 1.0%, preferably from 0.1% to 0.5%, and the anionic interface in these concentration ranges. Use of an extract containing an activator, a thiosulfate, and a nonionic surfactant is preferable in that the extraction efficiency is high and nonspecific reactions can be suppressed.

  An example of the sample carrier that can carry the measurement sample is a glass wool sample pad. The sample carrier, the colloidal gold-labeled antibody support, the development support, and preferably the other end of the development support are connected to the other end of the development support, such as an absorbent pad, for immunochromatography. It can be a test piece. Then, when the measurement sample is spotted on a sample carrier and immersed in a developing solution containing fetal bovine serum, the allergen in the measurement sample moves due to capillary action or the like and binds to the colloidal gold labeled antibody, and this antigen-antibody complex is The antigen-antibody complex is captured at a predetermined position where a monoclonal antibody that recognizes both a denatured and native allergen that recognizes an epitope different from that of a colloidal gold-labeled antibody is immobilized by moving on the development support by capillary action. Thus, the allergen can be detected based on the presence or absence of a colored line that appears at a predetermined position due to the accumulation of colloidal gold.

  The monoclonal antibody that recognizes both the modified and native allergens specifically recognizes the modified and native allergens selected from 7S globulin as a major soybean allergen and 11S globulin as a major sesame allergen 2 A variety of monoclonal antibodies can be preferably exemplified.

  More preferably, as the anti-soybean 7S globulin monoclonal antibody prepared by the present inventors, the anti-soybean 7S globulin monoclonal antibody PDSY1 produced by the hybridoma (NITE AP-02039) or the hybridoma (NITE AP-02040) is produced. Anti-soybean 7S globulin monoclonal antibody PDSY2 can be mentioned. As anti-sesame 11S globulin monoclonal antibody, anti-sesame 11S globulin monoclonal antibody PDSE1 produced by hybridoma (NITE AP-02041) and hybridoma (NITE AP-02042) are produced. Mention may be made of the anti-sesame 11S globulin monoclonal antibody PDSE2. As a combination of two monoclonal antibodies against soybean 7S globulin, PDSY1 produced by hybridoma (NITE AP-02039) and PDSY2 produced by hybridoma (NITE AP-02040) can be preferably mentioned. As a combination of two kinds of monoclonal antibodies against the above, PDSE1 produced by a hybridoma (NITE AP-02041) and PDSE2 produced by a hybridoma (NITE AP-02042) can be preferably mentioned. The hybridoma (NITE AP-02039), the hybridoma (NITE AP-02040), the hybridoma (NITE AP-02041), and the hybridoma (NITE AP-02042) were issued on May 7, 2015 as an independent administrative agency product evaluation technology infrastructure ( NITE) Patent Microbiology Deposit Center (NPMD) (address: 2-5-8, 122, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture).

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

1. Establishment of anti-soybean 7S globulin monoclonal antibody (1) Preparation of soybean 7S globulin Soybeans were pulverized with a miller, degreased with acetone, air-dried overnight at room temperature, and acetone-removed soybean powder was obtained. This defatted soybean powder was purified using Prep Cell 960 (BioRad). The 7S globulin fraction was dialyzed and lyophilized. Using these freeze-dried powders, a 0.1% 7S globulin solution is prepared with physiological saline, and dispensed in an amount of 500 μl each in a 1 ml Eppendorf tube to prepare an antigen solution, which is stored frozen at −40 ° C. until immunization. did.

(2) Immunization Five 6-week-old BALB / c mice (manufactured by CLEA Japan, Inc.) were used as test animals. For the first immunization, an emulsion prepared by adding an equal amount of complete Freund's adjuvant (Difco) to an Eppendorf tube containing 500 μl of a 0.1% 7S globulin solution and stirring with a vortex mixer was used. This emulsion was injected intraperitoneally at 150 μl per tail. Further, booster immunization was performed twice at intervals of 3 weeks. For immunization, an emulsion prepared by adding an equal volume of incomplete Freund's adjuvant (Difco) to an Eppendorf tube containing 500 μl of a 0.1% 7S globulin solution and stirring with a vortex mixer was used. This emulsion was injected intraperitoneally at 150 μl per tail.

(3) Measurement of antibody titer in blood One week after the first or booster injection of 7S globulin, blood was collected from the tail vein of each BALB / c mouse. The collected blood was allowed to stand at room temperature for 2 hours and then centrifuged to obtain serum. Ten-fold dilutions of these sera were prepared, and the anti-7S globulin antibody titer in mouse blood was examined by non-competitive ELISA. As the secondary antibody, alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (manufactured by Jackson ImmunoReserch Laboratories Inc.) was used.

(4) Production of hybridoma Hybridoma was produced according to the method of Keller and Milstein (1975). That is, 100 μl of a 0.1% 7S globulin solution was injected from the tail vein into mice whose antibody titers were sufficiently increased. Four days after intravenous injection, the spleen was aseptically removed from the mouse. The spleen was minced, washed with RPMI 1640, and passed through a sterile nylon mesh (Cell Strainer, 70 mm, Becton Dickinson) to obtain a spleen cell suspension. Spleen cells were collected by centrifugation at 1000 rpm × 10 minutes, resuspended in RPMI 1640 and counted. This spleen cell suspension and mouse myeloma cell (P3X63Ag8.653) suspension were mixed so that the cell number was 10: 1, and centrifuged again at 1000 rpm × 10 minutes to obtain a pellet. To this pellet, 45% polyethylene glycol having an average molecular weight of 3350 was dropped to perform cell fusion. RPMI1640 was added to the cell solution for dilution, and then pelleted by centrifugation. To this pellet, hybridoma medium (RPMI 1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / ml penicillin, 100 mg / ml streptomycin) in 100 μM hypoxanthine, 0.4 μM aminopterin, Add HAT selection medium containing 16 μM thymidine and dispense into a 24-well cell culture plate (Becton Dickinson) at 5 × 10 ⁶ cells / well and culture at 37 ° C. with 5% CO _2. did.

(5) Cloning by limiting dilution The culture supernatant of each well of the cell culture plate was used as a primary antibody for ELISA to examine the presence of a hybridoma producing an anti-7S globulin antibody. Hybridomas in wells that were positive for 7S globulin by ELISA were transferred to a 96-well cell culture plate (Becton Dickinson) at 0.9 cells / well and cloned by limiting dilution. . As feeder cells, 4-week-old BALB / c mouse thymocytes were added to each well of a 96-well cell culture plate at 5 × 10 ⁶ cells / well. RPMI1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin was used for the culture of the cloned hybridoma.

(6) Antibody Screening Monoclonal antibody screening is performed using native soybean crude protein (hereinafter “N-SOY”) containing soybean 7S globulin and urea-treated soybean crude protein (hereinafter “D-SOY”) containing soybean 7S globulin. The clones having different specificities were obtained by examining the difference in reactivity to the two types of proteins. D-SOY weighs 1 mg of defatted soybean powder, adds 100 μl of 5% EDTA, 6.0 g of urea, 0.2 ml of 2-mercaptoethanol, 1 ml of 50 mM Tris-HCl buffer (pH 8.6), 1.5 ml of distilled water, After covering with an aluminum foil, the film was heated and modified in an oil bath at 100 ° C. for 1 hour. The reactivity of the purified antibody to N-SOY and D-SOY was examined by direct ELISA, respectively, and five types of antibodies positive for N-SOY and D-SOY were selected.

(7) Ascites Collection and MAb Purification According to Jones et al. (1990), BALB / c mice were first injected with 0.2 ml of incomplete Freund's adjuvant intraperitoneally. One week later, each hybridoma producing the selected antibody was inoculated with 5 × 10 ⁶ cells per mouse. After ascites retention, ascites was collected with a syringe. The collected ascites was purified by Protein G column (manufactured by GE Healthcare) to obtain five types of purified monoclonal antibodies (MAbs) having specificity for modified and unmodified soybean 7S globulin. , PDSY1, and PDSY2.

(8) Characteristics of anti-soybean 7S globulin MAb Specificity of five kinds of purified antibodies showing positive for soybean 7S globulin was confirmed. The reactivity with respect to the native soybean crude protein (N-SOY) containing soybean 7S globulin in the solid phase or the urea-treated soybean crude protein (D-SOY) containing soybean 7S globulin is shown in FIG.

(result)
As is clear from FIG. 1, it was confirmed that all of the five types of antibodies, A, B, C, PDSY1, and PDSY2, recognize native soybean 7S globulin and modified soybean 7S globulin. The intensity of specificity for globulin and modified soybean 7S globulin was different for each antibody.

(9) Examination of antibody combinations In order to test the above-mentioned five kinds of purified antibodies for sandwich ELISA, each of them was labeled with HRP. According to the instruction manual of Peroxidase Labeling Kit-SH (manufactured by Dojindo Laboratories, Inc.), an HRP-labeled monoclonal antibody was obtained. MAb combinations were selected from the viewpoint of detection sensitivity in sandwich ELISA, using 5 types of MAbs that showed a positive reaction with solid phase antigen as solid phase or HRP labeled antibodies, respectively. As a measurement object, PBS containing 0.5% SDS, 0.1% sodium thiosulfate and 0.2% Tween 20 added to defatted soybean powder and heated in boiling water for 10 minutes (hereinafter referred to as “S-SOY”) Were prepared and appropriately diluted. Table 1 below shows the results of reactivity (absorbance value at 1 ppm of S-SOY) by sandwich ELISA of five types of MAb combinations having different reactivities.

(result)
As is clear from Table 1, the plate-immobilized antibody PDSY1 (NITE AP-02039) and the HRP-labeled antibody PDSY2 (NITE AP-02040) were selected as the most sensitive combinations that can detect S-SOY.

2. Detection of soybean allergen extracted with anionic surfactant, thiosulfate, and nonionic surfactant by immunochromatography (1) Preparation of gold colloid-labeled antibody 1 mg / ml with 2 mM borate buffer (pH 9.0) A MAb solution of PDSY2 (NITE AP-02040) was prepared so as to be ml. Add 500 μl of MAb solution to 5 ml of colloidal gold solution (manufactured by Sigma) adjusted to pH 9.0 with 0.2 M potassium carbonate solution in advance, react at room temperature for 30 minutes, add 635 μl of 10% BSA solution, and react for another 15 minutes I let you. Centrifugation was performed, and a 1% BSA solution was prepared so that OD ₅₂₅ = 1.0.

(2) Production of antibody-immobilized membrane A MAb solution of PDSY1 (NITE AP-02039) was prepared so as to be 4 mg / ml with PBS, and applied linearly to a nitrocellulose membrane and dried. Thereafter, the mixture was blocked with TBS containing 1% skim milk at 37 ° C. for 1 hour, washed with TBS and dried.

(3) Assembling the immunochromatographic strip In addition to the antibody-immobilized membrane, a glass wool sample pad for the test solution spot and a glass wool absorption pad for absorbing the test solution are prepared separately. The absorbent pads were pasted in this order to form an immunochromatographic strip. The following model meat products were used as detection samples.

(4) Preparation of soybean protein Soybean powder was pulverized with miller and degreased with acetone to obtain soybean protein.

(5) Model meat product Ham and hamburg were selected as model meat products for testing, and model ham and model hamburger containing soy protein at various concentrations were prepared according to the formulations shown in Tables 2 and 3, respectively. Additives were weighed according to each formulation, mixed in a food processor, and filled into a PVC tube.

(6) Heating temperature and time Model ham was heated at 75 ° C. for 30 minutes, and model hamburger was heated with boiling water for 10 minutes. After heating, the sample made uniform by a food processor was used as a sample for detection.

(7) Sample Pretreatment Weigh 1 g of the sample for detection, and add 19 ml of PBS containing 0.5% SDS, 0.1% sodium thiosulfate, and 0.2% Tween 20 as an extract, and stir. The sample was heated in boiling water for 10 minutes, cooled and centrifuged, and the supernatant was used as a measurement sample.

(8) Confirmation of detection by immunochromatography 20 μl of the prepared gold colloid-labeled antibody, 30 μl of fetal bovine serum (FBS) as a developing solution, and 50 μl of a measurement sample were added to an immunochromatographic strip to confirm the detection.

(result)
Next, the detection results are shown in Table 4. Judgment was expressed as ++ and + in order from the strongest line, and negative was set as −.

  As shown in Table 4, all the measurement samples were determined to be positive up to 2 ppm, and there was no non-specific reaction at 0 ppm. An accurate immunochromatography kit can be constructed.

  Based on the above results, a colloidal gold-labeled antibody carrier in which a colloidal gold antibody bound to a monoclonal antibody against soybean 7S allergen is supported, and a monoclonal antibody against soybean 7S allergen that recognizes an epitope different from the colloidal gold-labeled antibody. A fixed support, a buffer containing an anionic surfactant, thiosulfate, and a nonionic surfactant for extracting allergen from a test sample, and an extracted soybean 7S allergen For detecting soybean 7S allergen by preparing a detection kit comprising a sample carrier capable of supporting a measurement sample of the above and a fetal bovine serum (FBS) or a developing solution containing fetal bovine serum (FBS) A highly accurate immunochromatography kit could be constructed. Further, it has been confirmed that such an immunochromatography kit can maintain the accuracy that can withstand practicality even when stored at room temperature for one year or more from the date of manufacture.

1. Establishment of anti-sesame 11S globulin monoclonal antibody (1) Preparation of sesame 11S globulin Sesame seeds were pulverized with a miller, degreased with acetone, then air-dried overnight at room temperature, and acetone-removed sesame powder was obtained. The defatted sesame powder was purified using a prep cell 960 (manufactured by BioRad). The 11S globulin fraction was dialyzed and lyophilized. Using these lyophilized powders, a 0.1% 11S globulin solution is prepared with physiological saline, and 500 μl is dispensed into a 1 ml Eppendorf tube to make an antigen solution, which is stored frozen at −40 ° C. until immunization. did.

(2) Immunity
As test animals, five 6-week-old BALB / c mice (manufactured by CLEA Japan, Inc.) were used. For the first immunization, an emulsion prepared by adding an equal volume of complete Freund's adjuvant (Difco) to an Eppendorf tube containing 500 μl of a 0.1% 11S globulin solution and stirring with a vortex mixer was used. This emulsion was injected intraperitoneally at 150 μl per tail. Further, booster immunization was performed twice at intervals of 3 weeks. For immunization, an incomplete Freund's adjuvant (Difco) was added to an Eppendorf tube containing 500 μl of a 0.1% 11S globulin solution, and an emulsion prepared by stirring with a vortex mixer was used. This emulsion was injected intraperitoneally at 150 μl per tail.

(3) Measurement of antibody titer in blood Blood was collected from the tail vein of each BALB / c mouse one week after the injection of 11S globulin in the first or booster immunization. The collected blood was allowed to stand at room temperature for 2 hours and then centrifuged to obtain serum. Ten-fold dilutions of these sera were prepared, and the anti-11S globulin antibody titer in mouse blood was examined by non-competitive ELISA. As the secondary antibody, alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (manufactured by Jackson ImmunoReserch Laboratories Inc.) was used.

(4) Production of hybridoma Hybridoma was produced according to the method of Keller and Milstein (1975). That is, 100 μl of 0.1% 11S globulin solution was injected from the tail vein into mice with sufficiently increased antibody titers. Four days after intravenous injection, the spleen was aseptically removed from the mouse. The spleen was minced, washed with RPMI 1640, and passed through a sterile nylon mesh (Cell Strainer, 70 mm, Becton Dickinson) to obtain a spleen cell suspension. Spleen cells were collected by centrifugation at 1000 rpm × 10 minutes, resuspended in RPMI 1640 and counted. This spleen cell suspension and mouse myeloma cell (P3X63Ag8.653) suspension were mixed so that the cell number was 10: 1, and centrifuged again at 1000 rpm × 10 minutes to obtain a pellet. To this pellet, 45% polyethylene glycol having an average molecular weight of 3350 was dropped to perform cell fusion. RPMI1640 was added to the cell solution for dilution, and then pelleted by centrifugation. To this pellet, hybridoma medium (RPMI 1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / ml penicillin, 100 mg / ml streptomycin) in 100 μM hypoxanthine, 0.4 μM aminopterin, Add HAT selection medium containing 16 μM thymidine and dispense into a 24-well cell culture plate (Becton Dickinson) at 5 × 10 ⁶ cells / well and culture at 37 ° C. with 5% CO _2. did.

(5) Cloning by limiting dilution The culture supernatant of each well of the cell culture plate was used as a primary antibody for ELISA, and the presence of a hybridoma producing an anti-sesame 11S globulin antibody was examined. The well hybridoma positive for sesame 11S globulin by ELISA was transferred to a 96-well cell culture plate (Becton Dickinson) at 0.9 cells / well and cloned by limiting dilution. It was. As feeder cells, 4-week-old BALB / c mouse thymocytes were added to each well of a 96-well cell culture plate at 5 × 10 ⁶ cells / well. RPMI1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin was used for the culture of the cloned hybridoma.

(6) Antibody Screening Monoclonal antibody screening is performed using native sesame crude protein containing sesame 11S globulin (hereinafter referred to as “N-SSM”) and urea-treated sesame crude protein containing sesame 11S globulin (hereinafter referred to as “D-SSM”). The clones having different specificities were obtained by examining the difference in reactivity to the two types of proteins. D-SSM weighed 1 mg of defatted sesame powder, added 5% EDTA 100 μl, urea 6.0 g, 2-mercaptoethanol 0.2 ml, 50 mM Tris-HCl buffer (pH 8.6) 1 ml, distilled water 1.5 ml, After covering with an aluminum foil, the film was heated and modified in an oil bath at 100 ° C. for 1 hour. The reactivity of the purified antibody to N-SSM and D-SSM was examined by direct ELISA, and 6 types of antibodies positive for N-SSM and D-SSM were selected.

(7) Ascites Collection and MAb Purification According to Jones et al. (1990), BALB / c mice were first injected with 0.2 ml of incomplete Freund's adjuvant intraperitoneally. One week later, each hybridoma producing the selected antibody was inoculated at 5 × 10 ⁶ cells per mouse. After ascites retention, ascites was collected with a syringe. The collected ascites was purified with a Protein G column (manufactured by GE Healthcare) to obtain 6 types of purified monoclonal antibodies (MAbs) having specificity for denatured and native 11S globulin, and D, E, F, They were called PDSE1 and PDSE2.

(8) Characteristics of anti-sesame 11S globulin MAb Specificity of 6 types of purified antibodies that were positive for sesame 11S globulin was confirmed. The reactivity of the solid sesame crude protein (N-SSM) containing sesame 11S globulin or urea-treated soybean crude protein (D-SSM) containing sesame 11S globulin is shown in FIG.

(result)
As is clear from FIG. 2, it was confirmed that all of the six antibodies, D, E, F, PDSE1, and PDSE2, recognize native sesame 11S globulin and modified sesame 11S globulin. The intensity of specificity for globulin and modified sesame 11S globulin was different for each antibody.

(9) Examination of antibody combinations In order to test the above-mentioned 6 kinds of purified antibodies for sandwich ELISA, each of them was labeled with HRP. According to the instruction manual of Peroxidase Labeling Kit-SH (manufactured by Dojindo Laboratories, Inc.), an HRP-labeled monoclonal antibody was obtained. Each MAb that showed a positive reaction against the antigen on the solid phase was used as a solid phase or an HRP-labeled antibody, and a combination of MAbs was selected from the viewpoint of detection sensitivity in sandwich ELISA. As a measurement object, PBS containing 0.5% SDS, 0.1% sodium thiosulfate and 0.2% Tween 20 added to defatted sesame powder and heated in boiling water for 10 minutes (hereinafter referred to as “S-SSM”) Were prepared and appropriately diluted. Table 5 below shows the results of reactivity (absorbance value at 1 ppm of S-SSM) by sandwich ELISA of 6 kinds of MAb combinations having different reactivity.

(result)
As is clear from Table 5, S-SSM was detected in any of the antibody combinations, and the ability of antigen capture by sandwich ELISA was confirmed. However, the most sensitive combination capable of detecting S-SSM. As a combination, plate-immobilized antibody PDSE1 (NITE AP-02041) and HRP-labeled antibody PDSE2 (NITE AP-02042) were selected.

2. Detection of sesame allergens extracted with anionic surfactant, thiosulfate, and nonionic surfactant by immunochromatography (1) Preparation of gold colloid-labeled antibody 1 mg / mL with 2 mM borate buffer (pH 9.0) A MAb solution of PDSE2 (NITE AP-02042) was prepared to be ml. Add 500 μl of MAb solution to 5 ml of colloidal gold solution (manufactured by Sigma) adjusted to pH 9.0 with 0.2 M potassium carbonate solution in advance, react at room temperature for 30 minutes, add 635 μl of 10% BSA solution, and react for another 15 minutes. I let you. Centrifugation was performed, and a 1% BSA solution was prepared so that OD525 = 1.0.

(2) Production of antibody-immobilized membrane A MAb solution of PDSE1 (NITE AP-02041) was prepared to 4 mg / ml with PBS, applied linearly to a nitrocellulose membrane and dried. Thereafter, the mixture was blocked with TBS containing 1% skim milk at 37 ° C. for 1 hour, washed with TBS and dried.

(3) Assembling the immunochromatographic strip In addition to the antibody-immobilized membrane, a glass wool sample pad for the test solution spot and a glass wool absorption pad for absorbing the test solution are prepared separately. The absorbent pads were pasted in this order to form an immunochromatographic strip. The following model meat products were used as detection samples.

(4) Preparation of sesame protein Sesame powder was pulverized with a miller and degreased with acetone as sesame protein.

(5) Model meat product Ham and hamburg were selected as model meat products for testing, and model ham and model hamburger containing sesame protein at various concentrations were prepared according to the formulations shown in Tables 6 and 7, respectively. Additives were weighed according to each formulation, mixed in a food processor, and filled into a PVC tube.

(6) Heating temperature and time Model ham was heated at 75 ° C. for 30 minutes, and model hamburger was heated with boiling water for 10 minutes. After heating, the sample made uniform by a food processor was used as a sample for detection.

(7) Sample Pretreatment Weigh 1 g of the sample for detection, and add 19 ml of PBS containing 0.5% SDS, 0.1% sodium thiosulfate, and 0.2% Tween 20 as an extract, and stir. The sample was heated in boiling water for 10 minutes, cooled and centrifuged, and the supernatant was used as a measurement sample.

(8) Confirmation of detection by immunochromatography 20 μl of the prepared gold colloid-labeled antibody, 30 μl of fetal bovine serum (FBS) as a developing solution, and 50 μl of a measurement sample were added to an immunochromatographic strip to confirm the detection.

(result)
Next, Table 8 shows the detection results. Judgment was expressed as ++ and + in order from the strongest line, and negative was set as −.

  As shown in Table 8, all the measurement samples were determined to be positive up to 2 ppm, and there was no non-specific reaction at 0 ppm.

  Based on the above results, a colloidal gold labeled antibody carrier in which a colloidal gold antibody bound to a monoclonal antibody against sesame 11S allergen is supported, and a monoclonal antibody against sesame 11S allergen that recognizes a different epitope from the colloidal gold labeled antibody. A fixed support, a buffer containing an anionic surfactant, thiosulfate, and a nonionic surfactant for extracting allergens from a test sample, and an extracted 11S allergen Accuracy for detecting sesame 11S allergen by preparing a detection kit comprising a sample carrier capable of carrying a measurement sample and fetal bovine serum (FBS) or a developing solution containing fetal bovine serum (FBS) A good immunochromatography kit could be constructed. Further, it has been confirmed that such an immunochromatography kit can maintain the accuracy that can withstand practicality even when stored at room temperature for one year or more from the date of manufacture.

  Food allergens such as soybean allergen and sesame allergen can be detected quickly and accurately, the allergen detection method by the immunochromatography method of the present invention, and the immunochromatographic allergen detection kit of the present invention which can be used for the food, Useful in industry.

Claims (13)

Gold colloid-labeled antibody in which colloidal gold is bound to a monoclonal antibody that recognizes both denatured and native allergens, and a monoclonal antibody that recognizes both denatured and undenatured allergens and recognizes an epitope different from the gold colloid-labeled antibody are predetermined. Using a development support fixed at a position, an allergen measurement sample extracted from the test sample using the extraction liquid, and a development liquid containing at least 10% by weight of fetal bovine serum (FBS), In an immunochromatography method in which an allergen is detected based on the presence or absence of accumulation of colloidal gold at the predetermined position after a mixed solution of a colloid-labeled antibody, a measurement sample and a developing solution is developed on a developing support, the allergen is soybean 7S globulin. Or sesame 11S globulin, wherein the extract is an anionic surfactant, thiosulfate And method for detecting allergens by immunochromatography which comprises a nonionic surfactant. 2. The method for detecting an allergen by immunochromatography according to claim 1, wherein a developing solution containing at least 30% by weight of fetal bovine serum (FBS) is used. The method for detecting an allergen by immunochromatography according to claim 1 or 2, wherein sodium dodecyl sulfate is used as the anionic surfactant. The method for detecting an allergen by the immunochromatography method according to any one of claims 1 to 3, wherein Tween 20 is used as the nonionic surfactant. The immunochromatography method according to any one of claims 1 to 4, wherein PDSY1 produced by a hybridoma (NITE AP-02039) and PDSY2 produced by a hybridoma (NITE AP-02040) are used as monoclonal antibodies against soybean 7S globulin. Method for detecting allergens. The immunochromatography method according to any one of claims 1 to 4, wherein PDSE1 produced by a hybridoma (NITE AP-02041) and PDSE2 produced by a hybridoma (NITE AP-02042) are used as monoclonal antibodies against sesame 11S globulin. Method for detecting allergens. A colloidal gold-labeled antibody in which colloidal gold is bound to a monoclonal antibody that recognizes both denatured and native allergens, and a monoclonal antibody that recognizes both denatured and native allergens and recognizes an epitope different from the gold colloid-labeled antibody A deployment support fixed in position, an extract containing an anionic surfactant, thiosulfate, and a nonionic surfactant for extracting allergens from a test sample, fetal bovine serum (FBS) Or a developing solution containing fetal bovine serum (FBS), and the allergen is soybean 7S globulin or sesame 11S globulin. The kit for detecting an allergen for immunochromatography according to claim 7, comprising sodium dodecyl sulfate as an anionic surfactant. The kit for detecting an allergen for immunochromatography according to claim 7 or 8, comprising Tween 20 as a nonionic surfactant. The monoclonal antibody against soybean 7S globulin is PDSY1 produced by a hybridoma (NITE AP-02039) and PDSY2 produced by a hybridoma (NITE AP-02040), for immunochromatography according to any one of claims 7 to 9 Allergen detection kit. The monoclonal antibody against sesame 11S globulin is PDSE1 produced by a hybridoma (NITE AP-02041) and PDSE2 produced by a hybridoma (NITE AP-02042), for immunochromatography according to any one of claims 7 to 9 Allergen detection kit. A combination of monoclonal antibodies against soybean 7S globulin, comprising PDSY1 produced by a hybridoma (NITE AP-02039) and PDSY2 produced by a hybridoma (NITE AP-02040). A combination of monoclonal antibodies against sesame 11S globulin, comprising PDSE1 produced by a hybridoma (NITE AP-02041) and PDSE2 produced by a hybridoma (NITE AP-02042).