CN118275682A - Allergen detection method based on immunochromatography treatment - Google Patents
Allergen detection method based on immunochromatography treatment Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The present application relates to a method for detecting an allergen based on immunochromatography treatment. The present application provides an immunochromatographic-treated allergen detection method which is specially used for detecting an allergen in a liquid sample such as a wiping solution or washing water and which can detect an allergen rapidly and highly accurately without performing an extraction treatment or a heating treatment, and an allergen detection kit which can be used for the method. An allergen detection method comprises subjecting a liquid sample to immunochromatography in the presence of Sodium Dodecyl Sulfate (SDS).
Description
The application is a divisional application of Chinese patent application No.201780005509.7 (PCT application No. PCT/JP 2017/001473) with the application of 18 days of 1 month of 2017 and the name of 'detection method of allergen based on immunochromatography treatment'.
Technical Field
The present invention relates to a method for detecting an allergen, characterized in that a liquid sample is subjected to immunochromatographic treatment in the presence of sodium dodecyl sulfate (SDS; sodium dodecyl sulfate), and a kit for use in the detection method.
Background
Currently, 1 out of every 3 is said to have some allergic disease. In particular, food allergy is a detrimental immune response caused by ingestion of a sensitizer (hereinafter referred to as a food allergen) contained in food, and causes dermatitis, asthma, digestive tract diseases, anaphylactic shock, and the like, and there is a serious problem in medicine and in food industry due to the increase of patients with such food allergy. Anaphylactic shock and other bodily reactions are sometimes fatal and require treatment to prevent them. The necessity of transmitting information to consumers by labeling or the like has also increased, and the FAO/WHO joint food standards committee (japanese "contract food specification committee") has been searching for a labeling method suitable for the regulations of various countries (1999 month) in which foods containing 8 raw materials known as allergic substances need to be labeled to contain the allergic substances. In japan, a labeling method (performed from 4 th 2002) for 24 kinds of foods in which allergic symptoms of excessive symptoms are actually generated is prescribed in consideration of the degree and frequency of conventional health hazards and the like.
In order to rapidly and easily detect an allergen, an immunoassay method for detecting a substance to be detected including a specific antigen or antibody by utilizing an antigen-antibody specific reaction is generally used as a simple immunoassay method in which a substance to be detected in a sample is bound to an antibody or antigen sensitized with microparticles by an immune reaction, and the state of aggregation of microparticles generated by the binding is measured, in particular, from the viewpoint of being able to be judged visually.
In addition, a radioimmunoassay, an enzyme immunoassay, or a fluorescent immunoassay has been used in which an antibody or an antigen labeled with a labeling substance containing a radioisotope, an enzyme, or a fluorescent substance is bound to a substance to be detected in a sample by an immune reaction, and the bound labeling substance is measured. Among these immunoassays, competitive type reactions and sandwich type reactions have been widely used. Among these, as a so-called sandwich-type reaction assay, immunochromatography is known (for example, see patent document 1), and various allergen detection kits having high specificity due to an antigen-antibody reaction have been sold.
On the other hand, it is considered that unintentional mixing of a specific raw material into food is one of the causes of accidents related to food allergy, and in order to control so-called "contamination" of allergens, importance of manufacturing apparatuses and management of raw materials is strongly recognized, and wiping kits usable in food manufacturing facilities are also commercially available.
Prior art literature
Patent literature
Patent document 1: japanese patent laid-open No. 5-010950
Disclosure of Invention
Problems to be solved by the invention
The inventors of the present application have developed an immunochromatography method or the like for: for a sample to be tested such as food, an allergen is detected by extracting with an anionic surfactant and thiosulfate, or an anionic surfactant and nonionic surfactant, and/or by heating, and then spreading on a spreading support with a spreading solution, and then collecting colloidal gold. Such a method is known as a very high-precision method, and in a food manufacturing place where a wiping solution, washing water, or the like of an apparatus or the like is used as a sample to be inspected, there are many types of samples to be inspected, and it is necessary to inspect the samples for every operation day of the apparatus or the like, and therefore, the above-mentioned extraction process and heating process become one of the causes of lowering the work efficiency. The present application addresses the problem of providing an immunochromatographic-treated allergen detection method that is dedicated to detection of an allergen in a liquid sample such as a wiping solution or washing water and that can rapidly and highly accurately detect an allergen without performing the extraction/heating treatment, and an allergen detection kit that can be used in the method.
Means for solving the problems
The inventors of the present application have studied whether or not an allergen can be detected easily by using a solvent contained in a commercially available wiping kit, but in the case where an immunochromatographic treatment is performed by developing only PBS (phosphate buffered saline: phosphate buffered saline) contained in a commercially available wiping kit on a development support, an allergen in PBS may not be detected. Accordingly, the inventors have next studied whether or not an allergen can be detected by adding an anionic surfactant, thiosulfate, nonionic surfactant, or the like, which were used as an extract in a conventional detection kit, to PBS, and as a result, have confirmed that an allergen can be detected by performing immunochromatography treatment using a solution obtained by adding only SDS to PBS. Furthermore, it was confirmed that if SDS was added, the allergen could be detected even when an aqueous solution other than PBS was used, and even when an immunochromatographic test strip (strip) in which SDS had been immobilized in advance was used, the allergen could be detected, and the present application was completed.
Namely, the present invention is as follows.
(1) A method for detecting an allergen, characterized in that an immunochromatographic treatment is performed on a liquid sample in the presence of Sodium Dodecyl Sulfate (SDS).
(2) The method for detecting an allergen according to (1) above, wherein the liquid sample is a wiping solution or washing water.
(3) The method for detecting an allergen according to (1) or (2) above, wherein the immunochromatographic treatment is performed using a liquid sample without performing an extraction treatment or a heating treatment.
(4) The method for detecting an allergen according to any one of (1) to (3) above, wherein the SDS concentration is 0.05 to 2.0%.
(5) The method for detecting an allergen according to any one of (1) to (4) above, wherein the liquid sample further comprises at least 10% Fetal Bovine Serum (FBS).
(6) The method for detecting an allergen according to any one of (1) to (5) above, wherein the liquid sample further comprises sodium thiosulfate, sodium sulfite or ascorbic acid.
(7) An allergen detection kit comprising a development support and a solvent for preparing a liquid sample, wherein the development support is formed by immobilizing a labeled antibody having a label bound thereto for a monoclonal antibody capable of recognizing both a modified allergen and an unmodified allergen and a monoclonal antibody having a different epitope recognized from the labeled antibody having a label bound thereto for recognizing both a modified allergen and an unmodified allergen, respectively, at a predetermined position.
(8) The allergen detection kit according to the above (7), further comprising FBS.
(9) The allergen detection kit according to the above (8), further comprising sodium thiosulfate, sodium sulfite or ascorbic acid.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the present invention, it is possible to detect an allergen contained in a liquid sample such as washing water or a wiping solution in a food production site without performing extraction and heating treatment of the sample.
Detailed Description
The method for detecting an allergen of the present invention is not particularly limited as long as it is a method for detecting an allergen by subjecting a liquid sample to immunochromatography in the presence of SDS (which is one of anionic surfactants), and the immunochromatography is the following: a metal colloid-labeled antibody such as a colloidal gold-labeled antibody, a colloidal platinum-labeled antibody, or a colloidal silver-labeled antibody, a latex colloid-labeled antibody using latex colored particles containing an organic polymer such as polystyrene, a styrene-butadiene copolymer, or a labeled antibody using a label such as an enzyme-labeled antibody such as peroxidase, alkaline phosphatase, or β -D-galactosidase, is bound to a monoclonal antibody capable of recognizing both the modified allergen and the unmodified allergen, and a monoclonal antibody recognizing an epitope different from the colloidal gold-labeled antibody is used as a stationary phase, and a complex of a specific allergen and the labeled antibody is moved to specifically bind to the monoclonal antibody of the stationary phase, thereby qualitatively or quantitatively detecting an allergen in a liquid sample.
The kit for detecting an allergen for immunochromatography of the present invention is not particularly limited as long as it is a kit having the following characteristics: the liquid sample preparation device comprises a development support and a solvent for preparing a liquid sample, wherein the development support is formed by immobilizing a labeled antibody having the above-mentioned label bound to a monoclonal antibody capable of recognizing both a modified allergen and an unmodified allergen, and a monoclonal antibody having an epitope different from the labeled antibody having the above-mentioned label bound thereto and recognizing the same, respectively, at a predetermined position. The detection kit may further include a wiping section for collecting a liquid sample, and it is desirable to have accuracy and stability that satisfy practical use even when stored for 1 year or more at normal temperature from the time of manufacture.
The liquid sample in the method of the present invention is not particularly limited as long as it is a liquid in which there is a possibility of containing an allergen, and is preferably a solution in which there is a possibility of containing a food allergen in a food manufacturing site. Specifically, there may be mentioned: washing water for washing devices used for manufacturing food and the like; a rinse liquid used for removing the washing water; a wiping liquid obtained by wiping, with a (wiping) solvent, a dried product of the washing water, a dried product of the rinsing liquid, a raw material used for producing a food or a scattered product thereof, a residue remaining on a device used for producing the food, a residue such as a precipitate in a food production process, a packaging paper for packaging a food, a residue in a packaging container, or the like; and a wiping solution obtained by dissolving the wiping solution in a solvent; etc.
The solvent in the liquid sample may be suitably selected from water such as water, pure water, and an aqueous solvent such as (physiological) saline and PBS, or may be a mixture of 2 or more of them, for example, may be wiped with water and dissolved in water, may be wiped with (physiological) saline and dissolved in water, may be wiped with PBS and dissolved in water, may be wiped with water and dissolved in (physiological) saline, may be wiped with (physiological) saline and dissolved in (physiological) saline, may be wiped with PBS and dissolved in (physiological) saline, may be wiped with water and dissolved in PBS, may be wiped with (physiological) saline and dissolved in PBS, and may be wiped with (physiological) saline and dissolved in PBS. Further, a food containing an allergen other than the allergen to be detected may be mixed.
As the immunochromatographic treatment in the presence of SDS in the present invention, a treatment for detecting an allergen by subjecting a liquid sample containing SDS to an immunochromatographic strip without performing an extraction treatment using an extraction solution and/or without performing a heating treatment is preferable, and specifically, the following treatment (first treatment) may be mentioned: immersing a sample of an immunochromatographic test strip in the liquid sample containing SDS with a carrier portion, whereby a specific allergen in the liquid sample containing SDS is bound to a colloidal gold-labeled antibody recognizing the specific allergen, thereby forming an antigen-antibody complex; the formed antigen-antibody complex moves on the development support on the immunochromatographic test strip due to capillary phenomenon or the like; capturing the antigen-antibody complex at a predetermined position where a monoclonal antibody (which recognizes a different epitope from the colloidal gold-labeled antibody and recognizes the specific allergen modified or unmodified) is immobilized; the specific allergen is detected by a colored line which is developed by aggregation of the colloid Jin Zaishang at the predetermined position.
In addition, as another embodiment, the following process (second process) is given: for the liquid sample, SDS is dissolved in the development support by subjecting the liquid sample to an SDS-integrated immunochromatographic strip having SDS immobilized on the development support; binding a specific allergen in the liquid sample with a colloidal gold-labeled antibody recognizing the specific allergen to form an antigen-antibody complex; the formed antigen-antibody complex moves on the development support on the immunochromatographic test strip due to capillary phenomenon or the like; capturing the antigen-antibody complex at a predetermined position where a monoclonal antibody (which recognizes a different epitope from the colloidal gold-labeled antibody and recognizes the specific allergen modified or unmodified) is immobilized; the specific allergen is detected by a colored line which is developed by aggregation of colloidal gold at the predetermined position.
The liquid sample volume to be tested in the immunochromatographic test strip is preferably 0.01 to 1mL, more preferably 0.05 to 0.5mL, still more preferably 0.075 to 0.125mL, and still more preferably 0.08 to 0.12mL.
The method for producing the colloidal gold-labeled antibody in which the colloidal gold is bound to the monoclonal antibody includes a conventionally known method, and is not particularly limited, and examples thereof include the following methods: to a colloidal gold solution obtained by adjusting the pH to 9.0 with a 0.2M potassium carbonate solution, a solution obtained by dissolving a monoclonal antibody in a 2mM boric acid buffer (pH 9.0) was added, and the reaction was carried out at room temperature for 30 minutes, followed by addition of a 10% BSA solution, further reaction was carried out for 15 minutes, and centrifugation was carried out. The colloidal gold-labeled antibody carrier can be produced by applying the colloidal gold-labeled antibody produced as described above to a conjugate pad (conjugate pad) made of, for example, glass wool (glass wool) and drying the same.
The development support can be produced by linearly coating a buffer solution containing a monoclonal antibody which recognizes an epitope different from that of a colloidal gold-labeled antibody and recognizes a modified allergen and an unmodified allergen on, for example, a nitrocellulose membrane, drying the coated nitrocellulose membrane, and then blocking the coated nitrocellulose membrane.
Examples of the sample carrier portion capable of carrying the liquid sample include a sample pad made of glass wool. For example, the immunochromatographic test strip can be produced by sequentially connecting the sample carrier section, the colloidal gold-labeled antibody carrier, the development support, and preferably an absorber such as an absorbent pad that absorbs the development liquid at the other end of the development support.
In the first treatment, the concentration of SDS is exemplified by 0.05 to 2.0%, preferably 0.05 to 1.0%, preferably 0.075 to 0.75%, more preferably 0.1 to 0.5%, and even more preferably 0.15 to 0.3% based on the final concentration in the liquid sample. In the present invention, the concentration (%) represents w/v%.
In the second treatment, the concentration of SDS is preferably 0.05 to 2%, more preferably 0.125 to 1%, and even more preferably 0.1 to 0.5% based on the volume of the liquid sample. The immunochromatographic test strip to which SDS is immobilized can be used by spotting (spot) on a sample carrier portion of the immunochromatographic test strip and drying the spot to integrate the sample carrier portion with the immunochromatographic test strip.
In the method of the present invention, it is preferable to further add fetal bovine serum (FBS; fetal Bovine Serum) as an antibody-protecting agent to the liquid sample, and the concentration of FBS is preferably 10 to 50%, more preferably 20 to 40%, still more preferably 25 to 35%, and less than 10% of the liquid sample, and it is undesirable that a nonspecific reaction is likely to occur. In addition, the sample may be prepared by suspending, opacifying or dissolving various additives such as a preservative and an inorganic salt in a liquid sample. In addition, the FBS may be used integrally with an immunochromatographic test strip instead of being added to a liquid sample. In this case, the immunochromatographic test strip is preferably immobilized by spotting and drying the same at a concentration of 10 to 50% of the liquid sample on a binding pad to which the colloidal gold-labeled antibody is immobilized.
In the method of the present invention, the liquid sample may contain sodium thiosulfate, sodium sulfite or ascorbic acid, and when the solvent is tap water, sodium thiosulfate, sodium sulfite or ascorbic acid is preferably added. The concentration of sodium thiosulfate in the liquid sample is exemplified by 0.05 to 10%, preferably 0.08 to 5%, more preferably 0.5 to 3%, and even more preferably 1 to 2%. The concentration of sodium sulfite in the liquid sample is exemplified by 0.05 to 10%, preferably 0.08 to 7.5%, more preferably 0.1 to 5%, and still more preferably 1 to 5%. The concentration of ascorbic acid in the liquid sample is preferably 0.005 to 1%, more preferably 0.008 to 0.5%, still more preferably 0.01 to 0.25%, and still more preferably 0.075 to 0.125%.
The allergen in the present invention may be suitably exemplified by food allergens contained in foods, and specifically, allergens contained in eggs, casein, whey, wheat, buckwheat, peanut, soybean, sesame, crustaceans, and the like may be exemplified.
Examples of the allergen contained in the egg include ovalbumin, ovotransferrin (ovotransferrin), ovomucoid, and the like, and ovalbumin is preferable in that the allergen is present in the whole egg and the content thereof is the largest. The allergen in casein may be αs1 casein as a main protein of casein, and the allergen in whey may be β lactoglobulin as a main protein of whey. Examples of the allergen contained in wheat include gliadin, which is a main protein of wheat. Examples of the allergen contained in the buckwheat include buckwheat allergens such as proteins having molecular weights of 24kDa and 76kDa, which are major proteins of buckwheat. Examples of the allergen contained in the peanut include Ara h1, which is a major protein of peanut. Examples of the allergen contained in soybean include soybean 7S globulin, which is a main allergen of soybean. Examples of the allergen contained in sesame include sesame 11S globulin, which is a major allergen of sesame. Examples of the allergen contained in the crustacean include tropomyosin.
Examples of the antibody used for detecting the allergen in the immunochromatographic treatment include an antibody specifically recognizing each allergen such as ovalbumin, αs1 casein, βlactoglobulin, wheat gliadin, buckwheat allergen, ara h1, soybean 7S globulin, sesame 11S globulin, or tropomyosin allergen contained in crustaceans, and a combination of 2 monoclonal antibodies recognizing different epitopes in each allergen is preferable.
Examples of the antibody for detecting an allergen contained in the egg include an anti-ovalbumin monoclonal antibody PDOA3 produced by a hybridoma (FERM BP-11235) and an anti-ovalbumin monoclonal antibody PDOA4 produced by a hybridoma (FERM BP-11236). Hybridomas (FERM BP-11235) and hybridomas (FERM BP-11236) were deposited in the national institute of technology for evaluating products by independent administration (Chamber 2-5-8 of Fusarium on the Ministry of Mujin, qianye, japan) (Chamber of technology for evaluating products by independent administration, faberian office, faberian (Faberian name: chamom 2-5-8 of Fusarium on the Shanje, qianye, qianjin, japan) (22 nd of year 2010).
Examples of the antibody for detecting an allergen contained in casein include an anti- αs1 casein monoclonal antibody Pas1CN1 produced by hybridoma (FERM BP-10263) and an anti- αs1 casein monoclonal antibody Pas1CN2 produced by hybridoma (FERM BP-10264). Hybridomas (FERM BP-10263) and hybridomas (FERM BP-10264) were deposited with the national institute of technology for human product evaluation, patent biological Collection (24, 2, 2005).
Examples of the antibody for detecting an allergen contained in the whey include an anti-beta-lactoglobulin monoclonal antibody PβLG3 produced by a hybridoma (FERM BP-11237) and an anti-beta-lactoglobulin monoclonal antibody PβLG4 produced by a hybridoma (FERM BP-11238). Hybridomas (FERM BP-11237) and hybridomas (FERM BP-11238) were deposited with the national institute of technology for human product evaluation, patent biological collection (22. 2010).
Examples of the antibody for detecting an allergen contained in wheat include a combination of an anti-gliadin monoclonal antibody PGL1 produced by a hybridoma (FERM BP-10267) and an anti-gliadin monoclonal antibody PGL2 produced by a hybridoma (FERM BP-10268). Hybridomas (FERM BP-10267) and hybridomas (FERM BP-10268) were deposited with the national institute of technology for human product evaluation, patent biological Collection (24, 2, 2005).
Examples of the antibody for detecting an allergen contained in the buckwheat include an anti-24 kDa protein monoclonal antibody PBW5 produced by a hybridoma (FERM BP-11241) and an anti-76 kDa protein monoclonal antibody PBW2 produced by a hybridoma (FERM BP-10273). The hybridoma (FERM BP-11241) was deposited with the national institute of advanced technology for human product evaluation (national institute of patent and biological sciences, 2.2010, 22), and the hybridoma (FERM BP-10273) was deposited with the national institute of advanced technology for human product evaluation (national institute of patent and biological sciences, 2.2005, 24).
Examples of the anti-Arab 1 monoclonal antibody include anti-Arab 1 protein monoclonal antibody PAh1-5 produced by hybridoma (FERM BP-11240) and anti-Arab 1 protein monoclonal antibody PAh1-4 produced by hybridoma (FERM BP-11239). Hybridomas (FERM BP-11240) and hybridomas (FERM BP-11239) were deposited with the national institute of technology for human product evaluation, patent biological collection (22. 2010).
Examples of the anti-soybean 7S globulin monoclonal antibody include PDSY1 produced by hybridoma (NITE BP-02039) and PDSY2 produced by hybridoma (NITE BP-02040). Hybridomas (NITE BP-02039) and hybridomas (NITE BP-02040) were deposited at the national institute of technology for human product evaluation (NITE) patent microbiological deposit (NPMD) (address: fusarium Makino, general Fusarium, no. 2-5-8122, qianye, mujingzu, 5, 7 days 2015).
Examples of the anti-sesame 11S globulin monoclonal antibody include PDSE1 produced by hybridoma (NITE BP-02041) and PDSE2 produced by hybridoma (NITE BP-02042). The hybridoma (NITE BP-02041) and the hybridoma (NITE BP-02042) were deposited at NITE NPMD at 5.5.7.
As the anti-crustacean tropomyosin monoclonal antibody, PDTM1 produced by hybridoma (NITE BP-02173) and PDTM2 produced by hybridoma (NITE BP-02174) are suitably mentioned. The hybridoma (NITE BP-02173) and the hybridoma (NITE BP-02174) were deposited at NITE NPMD at 12-9-2015.
The detection accuracy in the method for detecting an allergen of the present invention is, for example, 0.5ppm or more, preferably 0.1ppm or more, more preferably 0.05ppm or more, and still more preferably 0.01ppm or more, based on the concentration of the food protein in the liquid sample.
The solvent used in the detection kit for an allergen for immunochromatography may, for example, be water, (physiological) saline or PBS, preferably PBS. In the above-mentioned kit for detecting an allergen for immunochromatography, it is preferable that FBS is contained in a concentration of 10 to 50% of a liquid sample, and sodium thiosulfate, sodium sulfite or ascorbic acid may be contained.
The present invention will be described in more detail with reference to examples below, but the technical scope of the present invention is not limited to these examples.
Example 1
(Preparation of immunochromatographic test strip for detection of foods)
And preparing immunochromatography test strips for detecting foods corresponding to the foods.
(1) Preparation of immunochromatography test strip for casein detection
1) Preparation of colloidal gold-labeled antibody
A solution of the Pas1CN1 monoclonal antibody was prepared with 2mM boric acid buffer (pH 9.0) to 1mg/mL. To 5mL of a colloidal gold solution (manufactured by Sigma Co., ltd.) having a pH adjusted to 9.0 with 0.2M potassium carbonate solution, 500. Mu.L of a Pas1CN1 monoclonal antibody solution was added, and the reaction was carried out at room temperature for 30 minutes, then 635. Mu.L of a 10% BSA solution was added, and the reaction was further carried out for 15 minutes. Centrifugation was performed and adjusted with 1% bsa solution such that OD 525 = 1.0. The resultant was applied to a glass wool bonding pad so as to be 68. Mu.L/cm 2, and dried.
2) Preparation of antibody immobilized membrane
A solution of the Pas1CN2 monoclonal antibody was prepared in PBS to 4mg/mL, applied to nitrocellulose membrane in a straight line, and dried. Then, blocking was performed with TBS containing 0.1% bovine gelatin at 37℃for 1 hour, followed by washing with TBS, and drying was performed.
3) Assembly of immunochromatographic test strips
In addition to the antibody-immobilized membrane, a glass wool sample pad as a sample carrier portion and a glass wool absorbent pad for absorbing a liquid sample were separately prepared, and the sample pad, the antibody-immobilized membrane, and the absorbent pad were bonded in this order to prepare an immunochromatographic test strip.
(2) Preparation of immunochromatography test strip for beta-lactoglobulin detection
An immunochromatographic test strip for detecting beta-lactoglobulin was produced in the same manner as in the production of the immunochromatographic test strip for detecting casein (1) above, except that a P.beta.LG3 monoclonal antibody solution was produced in the production of 1) the colloidal gold-labeled antibody and a P.beta.LG4 monoclonal antibody solution was produced in the production of 2) the antibody-immobilized membrane.
(3) Preparation of immunochromatography test strip for detecting ovalbumin
An immunochromatographic test strip for ovalbumin detection was produced in the same manner as in the production of the immunochromatographic test strip for casein detection (1) above, except that a monoclonal antibody solution of PDOA3 was produced in the production of 1) a colloidal gold-labeled antibody and a monoclonal antibody solution of PDOA4 was produced in the production of 2) an antibody-immobilized membrane.
(4) Preparation of immunochromatography test strip for wheat gliadin detection
An immunochromatographic test strip for wheat gliadin detection was produced in the same manner as the production of the immunochromatographic test strip for casein detection (1) above, except that a PGL1 monoclonal antibody solution was produced in the production of 1) a colloidal gold-labeled antibody and a PGL2 monoclonal antibody solution was produced in the production of 2) an antibody-immobilized membrane.
(5) Preparation of immunochromatography test strip for detecting buckwheat allergen
The immunochromatographic test strip for detecting buckwheat-allergen was prepared in the same manner as the preparation of the immunochromatographic test strip for detecting casein in the above-mentioned (1), except that the preparation of the monoclonal antibody solution of PBW5 in the preparation of the colloidal gold-labeled antibody and the preparation of the monoclonal antibody solution of PBW2 in the preparation of the antibody-immobilized membrane in the above-mentioned (2).
(6) Preparation of immunochromatography test strip for peanut Ara h1 protein detection
An immunochromatographic test strip for detecting peanut Ara h1 protein was produced in the same manner as the production of the immunochromatographic test strip for detecting casein in (1) above, except that a monoclonal antibody solution of PAh1-5 was produced in the production of 1) a colloidal gold-labeled antibody and a monoclonal antibody solution of PAh1-4 was produced in the production of 2) an antibody-immobilized membrane.
(7) Preparation of immunochromatography test strip for soybean 7S globulin detection
The immunochromatographic test strip for soybean 7S globulin detection was prepared in the same manner as in the preparation of the immunochromatographic test strip for casein detection (1) above, except that a PDSY-2 monoclonal antibody solution was prepared in the preparation of 1) a colloidal gold-labeled antibody and a PDSY-1 monoclonal antibody solution was prepared in the preparation of 2) an antibody-immobilized membrane.
(8) Preparation of immunochromatography test strip for sesame 11S globulin detection
The immunochromatographic test strip for soybean 7S globulin detection was prepared in the same manner as the preparation of the immunochromatographic test strip for casein detection (1) above, except that the monoclonal antibody solution for PDSE2 was prepared in the preparation of 1) the colloidal gold-labeled antibody and the monoclonal antibody solution for PDSE1 was prepared in the preparation of 2) the antibody-immobilized membrane.
(9) Preparation of immunochromatography test strip for detecting crustacean proteins
An immunochromatographic test strip for detecting crustacean proteins was produced in the same manner as in the production of the immunochromatographic test strip for detecting casein (1) above, except that a PDTM-2 monoclonal antibody solution was produced in the production of 1) a colloidal gold-labeled antibody and a PDTM-1 monoclonal antibody solution was produced in the production of 2) an antibody-immobilized membrane.
[ Production of liquid sample ]
In this example, the following food proteins were prepared for detection of each food allergen.
(Preparation of food protein 1)
(Preparation of unheated food protein)
1) Preparation of unheated egg proteins
The unheated egg protein was prepared from a powder prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
2) Preparation of unheated casein
The unheated casein protein was prepared from a powder prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
3) Preparation of unheated whey
Unheated whey proteins were prepared from powders prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
4) Preparation of unheated wheat protein
Unheated wheat protein was prepared from powder prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
5) Preparation of unheated buckwheat protein
Unheated buckwheat protein was prepared from a powder prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
6) Preparation of unheated peanut proteins
Unheated peanut proteins were prepared from powders prepared according to the standard method described in "inspection method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
7) Preparation of unheated soy proteins
Unheated soybean protein is prepared from a powder obtained by pulverizing soybean with a pulverizer (Miller, japanese "chemical") and defatting with acetone.
8) Preparation of unheated sesame protein
Unheated sesame protein is prepared from a powder obtained by pulverizing sesame with a pulverizer (Miller) and degreasing with acetone.
9) Preparation of unheated crustacean proteins
The crustacean protein was prepared from a powder prepared according to the standard method described in "examination method for foods containing allergic substances (reference) (2014, 3, 26, japan consumer agency)".
(Preparation of food protein 2)
(Preparation of heated food protein)
The above unheated food proteins were heated at 75℃for 30 minutes, at 100℃for 10 minutes, and at 120℃for 4 minutes, respectively, to obtain products, which were designated 75℃egg protein, 100℃egg protein, 120℃egg protein, 75℃casein protein, 100℃casein protein, 120℃casein protein, 75℃whey protein, 100℃whey protein, 120℃whey protein, 75℃wheat protein, 100℃wheat protein, 120℃wheat protein, 75℃buckwheat protein, 100℃buckwheat protein, 120℃buckwheat protein, 75℃peanut protein, 100℃peanut protein, 120℃peanut protein, 75℃soybean protein, 100℃soybean protein, 120℃soybean protein, 75℃sesame protein, 100℃sesame protein, 75℃sesame protein, 75℃crustacean protein, 100℃crustacean protein, 120℃crustacean protein, and food products of 75℃food protein, 100℃food protein, 120℃food protein were prepared.
Reference example 1
In the detection of food allergens, it has been studied whether or not the detection of allergens can be performed without any extraction treatment or heat treatment by using only a solvent of a conventional detection method (kit). The unheated food proteins and the heated food proteins were dissolved in 10mL of PBS so that the final concentrations became 0.1ppm, respectively, to obtain 36 kinds of liquid sample groups 1. FBS was added to each sample of the liquid sample group 1 so that the final concentration of FBS became 30%, 0.1mL of the resulting solution was prepared, and the immunochromatographic test strip for food detection was used to test the immunochromatographic treatment, and the result was determined after 20 minutes. The results are shown in Table 1.
TABLE 1
(Results)
As shown in Table 1, when unheated egg proteins were used; heating egg proteins at 75deg.C, 100deg.C and 120deg.C; and no allergen could be detected in the model liquid sample of unheated sesame protein.
Reference example 2
In the case where polyoxyethylene sorbitan monolaurate (Tween 20), SDS, and sodium thiosulfate, which are known as one of the nonionic surfactants constituting the heating extract in the conventional method, are added to a liquid sample, it has been studied whether or not they contribute to the detection of an allergen without heat treatment. The unheated food proteins and the heated food proteins were dissolved in 10mL of PBS so that the final concentrations became 0.5ppm, respectively, to obtain 36 kinds of liquid sample groups 2. To the liquid sample group 2, tween20, SDS and sodium thiosulfate were added so that the final concentrations became 0.2% Tween20, 0.5% SDS and 0.1% sodium thiosulfate, 36 liquid sample groups to which three kinds of liquid samples were added were prepared, FBS was further added so that the final concentration of FBS became 30%, and the results were determined after 20 minutes by using corresponding immunochromatographic test strips for food detection. The results are shown in Table 2.
TABLE 2
(Results)
As shown in table 2, even in the case where the extraction and heating treatment were not performed, the egg proteins were all included without heating in the case where 0.2% tween20, 0.5% sds, and 0.1% sodium thiosulfate were added; heating egg proteins at 75deg.C, 100deg.C and 120deg.C; and unheated sesame protein, the allergens can be detected in each of the three liquid sample groups of 36 samples.
Example 2
Whether or not the above Tween20, SDS, and sodium thiosulfate alone were added to a liquid sample without performing extraction and heating treatment contributed to the detection of an allergen using an immunochromatographic test strip was studied. An unheated egg protein was used as a food protein, and dissolved in 10mL of PBS at a final concentration of 0.5ppm to prepare an unheated egg liquid sample. 4 parts of the unheated egg liquid samples were prepared, and the unheated egg liquid samples to which Tween20 was added, the unheated egg liquid samples to which SDS was added, and the unheated egg liquid samples to which sodium thiosulfate was added were prepared so that the final concentrations became 0.2% Tween20, 0.5% SDS, and 0.1% sodium thiosulfate, respectively. In addition, as a positive control, all of Tween20, SDS, and sodium thiosulfate were added to the unheated egg liquid sample. Further, FBS was added so that the final concentration of FBS became 30%, and the immunochromatographic test strip for food detection was used to test the immunochromatographic treatment, and the result was judged after 20 minutes. The results are shown in Table 3.
TABLE 3
(Results)
As shown in table 3, ovalbumin could be detected when SDS was added to the liquid sample at a final concentration of 0.5%, but no allergen could be detected when Tween20 or sodium thiosulfate was added alone.
Example 3
[ Optimal concentration of SDS ]
The optimum concentration of SDS in liquid samples was studied. 9 kinds of the above unheated food proteins were used as food proteins, and dissolved in 10mL of PBS so that the final concentration became 0.5ppm, respectively, to obtain an unheated food liquid sample group consisting of 9 kinds. SDS was added to each sample of the unheated food liquid sample group so that the final concentration became 0.1, 0.25, 0.5, 1.0 and 5.0%, and further FBS was added so that the final concentration of FBS became 30%, respectively, and the corresponding immunochromatographic test strip for food detection was used to test immunochromatographic treatment. The results are shown in Table 4.
TABLE 4
(Results)
As shown in Table 4, allergens were detected in all of the unheated food liquid samples at final SDS concentrations of 0.1, 0.25, and 0.5% in the liquid samples. In particular, the following results were confirmed: the detection sensitivity was particularly improved in casein, wheat and peanut when the concentration of SDS was 0.1%, the detection sensitivity was particularly improved in egg and whey when the concentration of SDS was 0.25%, and the detection sensitivity was particularly improved in sesame when the concentration of SDS was 0.5%. However, no allergen was detected in whey, buckwheat and soybean at a final concentration of 1% and no allergen was detected in all unheated food liquid samples at a final concentration of 5% SDS.
Example 4
(Study of wiping liquid 1)
In consideration of actual plant equipment, studies have been made on whether or not an allergen can be detected by using physiological saline, ham (japanese "harpoon") extract, or pure water instead of PBS. The above 9 types of unheated food proteins were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water so that the final concentration became 0.5ppm, respectively, to prepare 9 types of unheated food/physiological saline liquid sample groups 1, 9 types of unheated food/ham extract liquid sample groups 1, and 9 types of unheated food/pure water liquid sample groups 1. 9 kinds of 100℃heated food proteins were dissolved in 10mL of physiological saline, 10mL of ham extract and 10mL of pure water so that the final concentration became 0.5ppm, respectively, to prepare 9 kinds of 100℃heated food/physiological saline liquid sample groups 1, 9 kinds of 100℃heated food/ham extract liquid sample groups 1 and 9 kinds of 100℃heated food/pure water liquid sample groups 1. Further, SDS was added to each sample so that the final concentration became 0.25%, and further FBS was added so that the final concentration of FBS became 30%, and the result was determined after 20 minutes by using the corresponding immunochromatographic test strip for food detection. Samples obtained by mixing the above 9 unheated food proteins and the above 9 heated food proteins at 100℃in SDS/PBS were used as positive controls. The results are shown in Table 5. The ham extract was prepared by the following method: ham was supplied to the mixer at a weight of 2% with respect to physiological saline, and then filtered.
TABLE 5
(Results)
As shown in table 5, it was confirmed that even when PBS was replaced with physiological saline, ham extract, and pure water, if 0.25% sds was added to the developing solution, the allergen could be detected in all liquid samples of food proteins which were not heated or heated at 100 ℃.
Example 5
(Study of wiping liquid 2)
The 9 unheated food proteins in the above (study 1 of a wiping solution) were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water to prepare 9 unheated food/physiological saline liquid sample groups 2, 9 unheated food/ham extract liquid sample groups 2, and 9 unheated food/pure water liquid sample groups 2, respectively, and the 9 100℃heated food proteins were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water to obtain 9 100℃heated food/physiological saline liquid sample groups 2, 9 100℃heated food/ham extract liquid sample groups 2, and 9 100℃heated food/pure water liquid sample groups 2, respectively, so that the final concentration became 0.1 ppm. Further, SDS was added to each sample so that the final concentration became 0.25%, and FBS was added so that the final concentration of FBS became 30%, respectively, to conduct the same study as in example 4. The results are shown in Table 6.
TABLE 6
(Results)
As shown in Table 6, it was confirmed that even when the concentration of each food protein was 0.1ppm, if 0.25% SDS was added to the liquid sample in the case where the solvent of the liquid sample was replaced with normal saline, ham extract or pure water, the allergen could be detected in the liquid sample of all the above-mentioned food proteins which were not heated or heated at 100 ℃.
Example 6
(Study of wiping liquid 3)
The 9 unheated food proteins in the above (study 1 of a wiper) were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water to give 9 unheated food/physiological saline liquid sample groups 3, 9 unheated food/ham extract liquid sample groups 3, and 9 unheated food/pure water liquid sample groups 3, respectively, at a final concentration of 0.05ppm, and the 9 100℃heated food proteins were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water, respectively, to give 9 100℃heated food/physiological saline liquid sample groups 3, 9 100℃heated food/ham extract liquid sample groups 3, and 9 100℃heated food/pure water liquid sample groups 3, respectively. Further, SDS was added to each sample so that the final concentration became 0.25%, and FBS was added so that the final concentration of FBS became 30%, respectively, to conduct the same study as in example 4. The results are shown in Table 7.
TABLE 7
(Results)
As shown in Table 7, even in the case where the food concentration in the liquid sample was 0.05ppm, allergens could be detected in eggs, casein, whey, wheat, buckwheat, peanuts, soybeans, sesame. No allergen could be detected from a liquid sample comprising crustacean proteins.
Example 7
(Study of wiping liquid 4)
The 9 unheated food proteins in the above (study 1 of a wiper) were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water to give 9 unheated food/physiological saline liquid sample groups 4, 9 unheated food/ham extract liquid sample groups 4, and 9 unheated food/pure water liquid sample groups 4, respectively, at a final concentration of 0.01ppm, and the 9 100℃heated food proteins were dissolved in 10mL of physiological saline, 10mL of ham extract, and 10mL of pure water, respectively, to give 9 100℃heated food/physiological saline liquid sample groups 4, 9 100℃heated food/ham extract liquid sample groups 4, and 9 100℃heated food/pure water liquid sample groups 4, respectively. Further, SDS was added to each sample so that the final concentration became 0.25%, and FBS was added so that the final concentration of FBS became 30%, respectively, to conduct the same study as in example 4. The results are shown in Table 8.
TABLE 8
(Results)
As shown in Table 8, even when the food concentration in the liquid sample was 0.01ppm, allergens could be detected in eggs, wheat, buckwheat, sesame.
Example 8
The same study as in example 4 was performed using immunochromatographic test strips for food detection corresponding to respective foods, by adding SDS to 10mL of PBS, physiological saline, ham extract, and pure water, each of which did not contain any food protein, at a final concentration of 0.25%, and further adding FBS at a final concentration of 30%. The results are shown in Table 9.
TABLE 9
(Results)
At a food protein concentration of 0ppm, the results were negative in all the items. Therefore, it was confirmed that each food protein (allergen) was not detected in the method of the present invention.
Example 9
(Study of washing Water 1)
In consideration of the actual food factory equipment, studies were made on whether or not an allergen could be detected in the case of using tap water washing equipment. 6 liquid samples of 10mL were prepared by dissolving unheated egg proteins in pure water or tap water at final concentrations of 0.1ppm, 0.05ppm, and 0.01ppm, respectively. Further, a sample obtained by adding SDS to 10mL of purified water and tap water containing no egg protein so that the final concentration became 0.25% was prepared as a negative control (0 ppm). SDS was added to each sample so that the final concentration became 0.25%, and further FBS was added so that the final concentration of FBS became 30%, and the result was determined after 20 minutes by using an immunochromatographic test strip for egg detection. The results are shown in Table 10.
TABLE 10
(Results)
Table 10 shows that the concentration of unheated egg protein was positive at 0.1ppm, 0.05ppm and 0.01ppm for pure water, but positive at only 0.1ppm for tap water, confirming the decrease in sensitivity in tap water.
Example 10
(Study of washing Water 2)
An investigation was made as to whether an allergen could be detected when sodium thiosulfate, sodium sulfite or ascorbic acid was added to tap water. To tap water containing 5%, 2%, 1%, 0.1% sodium thiosulfate, tap water containing 5%, 2%, 1%, 0.1% sodium sulfite, tap water containing 0.1%, 0.01% ascorbic acid were added unheated egg proteins at a final concentration of 0.1ppm, 0.05ppm, 0.01ppm, respectively, to prepare 30 kinds of 10mL liquid samples. As a negative control (0 ppm), 10mL of tap water containing no egg protein, tap water containing 5%, 2%, 1%, 0.1% sodium thiosulfate, tap water containing 5%, 2%, 1%, 0.1% sodium sulfite, tap water containing 0.1%, 0.01% ascorbic acid were used. SDS was added to each sample so that the final concentration became 0.25%, and further FBS was added so that the final concentration of FBS became 30%, and the result was determined after 20 minutes by using an immunochromatographic test strip for egg detection. The results are shown in Table 11.
TABLE 11
(Results)
Table 11 shows that when sodium thiosulfate, sodium sulfite or ascorbic acid was added to tap water, the results were positive when egg proteins were not heated at 0.05 ppm. Further, it was confirmed that: when 1% or 2% sodium thiosulfate was added, when 1%, 2% or 5% sodium sulfite was added, and when 0.1% ascorbic acid was added, 0.01ppm egg allergen was detected, and the detection sensitivity was equivalent to that of pure water shown in table 10. In addition, it was confirmed that when the unheated egg proteins were not present, all of the egg proteins showed negative results and were not detected.
Example 11
(Study of Integrated kit)
SDS was applied to a sample pad at the tip of an immunochromatographic test strip for detecting ovalbumin so as to be 0.125 to 1% of the capacity of the sample to be subjected to immunochromatography (0.1 mL), FBS was applied so as to be 30% of the capacity of the sample to be subjected to immunochromatography (0.1 mL), and dried, and then 0.1mL of PBS containing 0.5ppm of unheated ovalbumin was supplied to the test. The results are shown in Table 12.
TABLE 12
| SDS(％) | 0.125 | 0.250 | 0.500 | 1.000 |
| Determination of | + | + | + | + |
(Results)
Table 12 shows that the positive results were found in 0.125%, 0.250%, 0.500% and 1.000%.
Industrial applicability
The method for detecting an allergen of the present invention, which can rapidly and highly accurately detect an allergen contained in washing water or rinsing liquid in food manufacturing facilities, and the kit for detecting an allergen of the present invention, which can be used for the detection method, are useful in the food industry.
Claims (3)
1. A method for detecting an allergen, characterized in that an immunochromatographic treatment is performed on a wiping solution or wash water using 2 monoclonal antibodies recognizing different epitopes without performing an extraction treatment and without performing a heating treatment, in the presence of Sodium Dodecyl Sulfate (SDS) and in the absence of thiosulfate and a nonionic surfactant.
2. The method for detecting an allergen according to claim 1, wherein the concentration of SDS is 0.05 to 2.0%.
3. The method for detecting an allergen according to claim 1, wherein at least 10% of Fetal Bovine Serum (FBS) is further added to the wiping solution or the washing water.
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| PCT/JP2017/001473 WO2017126525A1 (en) | 2016-01-19 | 2017-01-18 | Method for allergen detection by immunochromatographic assay |
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| JP2022076838A (en) * | 2020-11-10 | 2022-05-20 | プリマハム株式会社 | Allergen detection kit |
| CN112526142A (en) * | 2020-11-25 | 2021-03-19 | 杭州福德敏生物技术有限公司 | Egg immunofluorescence detection test strip and application and detection method thereof |
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| JP4690928B2 (en) * | 2006-04-04 | 2011-06-01 | プリマハム株式会社 | Allergen detection method by immunochromatography |
| JP5133663B2 (en) * | 2007-11-30 | 2013-01-30 | 森永製菓株式会社 | Food component extraction method, food inspection method, and food inspection kit |
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