WO2019009472A1 - Marker for determining presence of arachis hypogaea in food and use thereof - Google Patents

Marker for determining presence of arachis hypogaea in food and use thereof Download PDF

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Publication number
WO2019009472A1
WO2019009472A1 PCT/KR2017/013320 KR2017013320W WO2019009472A1 WO 2019009472 A1 WO2019009472 A1 WO 2019009472A1 KR 2017013320 W KR2017013320 W KR 2017013320W WO 2019009472 A1 WO2019009472 A1 WO 2019009472A1
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antibody
peanut
food
present
peanuts
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PCT/KR2017/013320
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French (fr)
Korean (ko)
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심원보
김솔아
이정은
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경상대학교산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • G01N33/02—Food
    • 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
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to a marker for discriminating whether or not a peanut is incorporated in a food and a use thereof.
  • Food allergy is a disease caused by immunoglobulin E (IgE) -mediated or non-immunoglobulin-mediated hypersensitivity to dietary protein, as well as digestive organs, skin, respiratory or cardiovascular Is a relatively common allergic disease that manifests various symptoms. It is a harmless food for normal people, but it refers to the excessive immune response of the human body to a specific protein of the food when it is ingested by a specific person. The incidence of food allergy is reported to be about 8% in children younger than 3 years, and about 2% in adults.
  • IgE immunoglobulin E
  • the amount of food a person consumes for a lifetime is about 2 to 3 tons and is exposed to a wide variety of foods, but the human gastrointestinal immune system has evolved to cause immunological tolerance to most dietary proteins, For very limited types of food, sensitization to dietary proteins occurs and ultimately allergic symptoms are induced.
  • Food allergens are defined as components of foods that induce the production of immunoglobulin E and cause an immediate hypersensitivity reaction, as well as inhalation and contact food allergens. Most major allergens in food are water-soluble glycoproteins with a molecular weight of about 10,000-60,000 Da. Unlike inhalational allergens, they are likely to be transformed by digestive enzymes, bacteria, toxins, etc. during their passage through the gastrointestinal tract, Anaphylaxis reactions as well as delayed type reactions (delayed type reaction) are often caused. Common foods that cause allergic reactions by immunoglobulin E include eggs, milk, beans, peanuts, nuts, buckwheat, wheat, fish, and shellfish.
  • the peanut Arachis hypogaea
  • the peanut is one of the food allergens that can cause severe systemic symptoms and lethal anaphylaxis. Unlike other foods, the natural loss does not tend to last for a lifetime.
  • a hybridoma cell capable of producing a monoclonal antibody capable of specifically binding to a peanut thermostable water-soluble protein Ara h1 was prepared, and a monoclonal antibody produced from the hybridoma cell was used for heat treatment It was confirmed that it is possible to quickly analyze the presence of peanuts in the mixture of food raw materials and processed foods.
  • Japanese Patent No. 3853692 discloses an antibody for testing peanut components, an assay method and an assay kit, and Korean Patent No. 1223256 discloses a method for reducing allergenicity of peanut and a peanut with reduced allergenicity.
  • the marker for discriminating the presence or absence of peanuts in the food of the present invention and the use thereof are not described.
  • the present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a hive producing a monoclonal antibody which specifically binds to a thermostable water-soluble protein of a peanut using a heat- Lidomaju was prepared.
  • Immunoassay using the monoclonal antibody of the present invention showed that the presence or absence of peanut contamination can be quickly determined by the monoclonal antibody of the present invention regardless of whether or not heat treatment is applied to all food samples containing peanut
  • the present invention has been completed.
  • the present invention provides a marker for discriminating the presence or absence of peanuts in a food containing a peanut-derived Ara h1.
  • the present invention also provides a composition for determining the presence or absence of peanut in a food comprising an antibody that specifically binds to peanut-derived Ara h1.
  • the present invention provides a kit for judging whether or not a peanut is contained in a food comprising an antibody specifically binding to a pean-derived Ara h1.
  • the present invention provides a method for determining whether a peanut is incorporated in a food, comprising the step of detecting an antigen-antibody complex formed by contacting an antibody that specifically binds to peanut-derived Ara h1 with a test sample.
  • the present invention provides a monoclonal antibody produced by a hybridoma cell line of deposit number KCTC 13295BP and specifically binding to peanut-derived Ara h1.
  • the present invention provides a hybridoma cell line having the accession number KCTC 13295BP.
  • the monoclonal antibody of the present invention reacts specifically with Ara h1 among heat stable water soluble proteins in peanuts, and can effectively analyze peanuts incorporated in intentional unheated foods manufactured and processed by heat treatment. Further, through the present invention, information on the presence or absence of peanuts in foods can be provided to consumers to prevent food allergy induction, and it can be utilized to improve food manufacturing and processing, thereby contributing to securing food stability It is expected.
  • Figure 1 shows the results of SDS-PAGE of heat-stable proteins extracted from non-heat and heat-treated peanuts.
  • Lane 1 size marker
  • lane 2 green peanut sample extracted by non-heat treatment method
  • lane 3 green peanut sample extracted by heat treatment
  • lane 4 boiled peanut sample extracted by non-heat treatment method
  • lane 5 boiled peanut sample extracted by heat treatment
  • Lane 6 peanut samples extracted by non-heat treatment method
  • lane 7 roasted peanut samples extracted by heat treatment
  • lane 8 peanut samples extracted by non-heat treatment method
  • lane 9 peanut samples extracted by heat treatment.
  • FIG. 2 is a result of indirect ELISA measurement of anti-serum antibody levels of mice immunized with a heat-stable water-soluble protein extracted from roasted peanut and boiled peanut.
  • FIG. 3 is a graph showing the distribution of peanut (lane 2), boiled peanut (lane 3), peanut butter (lane 4), walnut (lane 5), almond (A and B) and ELISA (C) analysis of the heat-stable proteins extracted from soybean (lane 6), cashew nut (lane 7), red bean (lane 8) and soybean (lane 9).
  • Lane 1 Size marker.
  • Fig. 4 is a Western blot result for confirming the reaction of BP 3A1-12, a monoclonal antibody against Ara h1, a heat-stable protein in peanuts.
  • Lane 1 Size marker
  • Lane 2 Commercial Ara h1 protein
  • Lane 3 Roasted peanuts
  • Lane 4 Boiled peanuts
  • Lane 5 Peanut butter.
  • FIG. 5 shows the results of indirect ELISA measurement of the presence of peanuts in various processed foods using the antibody of the present invention.
  • Original Hybridoma cell culture, 1/10 diluted: Hybridoma cell culture diluted to 1/10.
  • 1/100 diluted Hybridoma cell culture diluted to 1/100.
  • A BP 3A1-12 antibody,
  • B RP 4C12-10 antibody.
  • FIG. 6 shows the results of measurement of the detection of the peptone protein content of the monoclonal antibodies (BP 3A1-12 and RP 4C12-10) of the present invention by indirect ELISA.
  • the present invention provides a marker for determining the presence or absence of a peanut in a food containing ara h1 from a peanut ( Arachis hypogaea ).
  • the Ara h1 is a seed storage protein or vicilin and is one of the peanut allergens.
  • Ara h1 which is resistant to heat and digestive enzymes, forms a stable trimmer structure due to the interaction between monomers, and there is an epitope (antigenic determinant) capable of binding immunoglobulin E at the contact site do.
  • an epitope antigenic determinant
  • heat treatment is applied to Ara h1
  • the immunoglobulin E binding epitope buried in the three-dimensional structure due to the protein modification is exposed, and the exposed epitope binds with immunoglobulin E to induce an allergic reaction.
  • the peanut-derived Ara h1 protein is detected in the food, it can be judged that the peanut is incorporated.
  • the marker may be used in the same manner as the marker composition for determining whether peanuts are mixed in the food.
  • the present invention also provides a composition for determining the presence or absence of peanut in a food comprising an antibody that specifically binds to peanut-derived Ara h1.
  • the composition includes an antibody that specifically binds to Ara h1 derived from peanuts as an active ingredient, and can determine the presence or absence of peanuts in the food using the antibody.
  • antibody is meant to include all forms as long as it specifically binds to a marker for discriminating the presence or absence of peanuts in the food of the present invention.
  • a monoclonal antibody polyclonal antibody, multispecific antibody (i.e., an antibody recognizing two or more antigens or two or more epitopes, such as a bispecific antibody, etc.)
  • Recombinant antibodies chemically modified antibodies that have the ability to specifically bind to a recognition marker.
  • fragments of the antibody include Fab, F (ab ') 2, scFv (an antibody in which a heavy or light chain Fv is linked by a suitable linker), Fv, Fab / c Means an antibody fragment obtained by introducing and expressing a gene for an antibody fragment or fragment obtained by treating with a protein cleaving enzyme such as papain or pepsin into a host cell by a gene recombination method.
  • the globulin type of the antibody may be any one of IgG, IgM, IgA, IgE, and IgD, provided that the globulin type of the antibody specifically binds to the marker for discriminating the presence or absence of peanuts in the food of the present invention.
  • the monoclonal antibody may preferably be produced by a hybridoma cell line having the accession number KCTC 13295BP, but is not limited to this.
  • the food may include, or is not limited to, all foods that contain or are expected to contain peanuts, preferably processed products such as peanuts, roasted peanuts, boiled peanuts, fried peanuts or butter peanuts.
  • the butter peanut refers to a product processed like a jam that is generally commercially available in the United States.
  • the present invention provides a kit for judging whether or not a peanut is contained in a food comprising an antibody specifically binding to a pean-derived Ara h1.
  • the antibody may include any type of antibody that specifically binds to the Ara h1 from the peanut as described above.
  • the antibody may be a monoclonal antibody that specifically binds to the Ara h1 from the peanut. More preferably, May be a monoclonal antibody produced by a hybridoma cell line with the accession number KCTC 13295BP, but is not now limited.
  • Kit systems for use with the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography methods and split-split immune assay devices, flow-through devices, and the like.
  • the kit of the present invention is not particularly limited as long as it is used in a kit, but if it is an ELISA, for example, it may be a solid-phase supporter; A monoclonal antibody of the present invention; And an enzyme-linked immunosorbent assay (ELISA) reaction solution containing an enzyme-labeled antibody solution for reaction with an antigen and a coloring solution indicating an enzyme reaction.
  • ELISA enzyme-linked immunosorbent assay
  • the present invention provides a method for determining whether a peanut is incorporated in a food, comprising the step of detecting an antigen-antibody complex formed by contacting an antibody that specifically binds to peanut-derived Ara h1 with a test sample.
  • antigen-antibody complex in the present invention refers to a conjugate of Ara h1, a peanut thermostable water-soluble protein in a sample, and a monoclonal antibody according to the present invention, ),
  • An electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method It can be detected by any method. However, various applications and applications are possible without being limited thereto.
  • markers can be used for detecting an antigen-antibody complex.
  • Specific examples include, but are not limited to, enzymes, chromophores, ligands, luminescent materials, microparticles, and radioactive isotopes.
  • Examples of preferred enzymes used as the detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase or? -Lactamase, and preferable examples of the minerals include quantum dots, Eu 3+ , Eu 3+ chelate, or cryptate.
  • Preferred ligands include biotin derivatives.
  • Preferred emitters are acridinium esters or isoluminol derivatives.
  • Preferred microparticles include colloidal gold or There are colored latexes and preferred radioisotopes include, but are not limited to, 7 Co, 3 H, 125 I or 125 I Bolton Hunter reagents.
  • the method for detecting the antigen-antibody complex can be preferably detected using enzyme immunoassay (ELISA), but is not limited thereto.
  • Enzyme immunosorbent assays include direct ELISA using an antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attachment to a solid support
  • indirect sandwich ELISA using secondary antibodies include direct sandwich ELISA using an antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attachment to a solid support
  • the antibody may have a detection label, and when it does not have a detection label, the antibody can be captured and can be confirmed by treating another antibody having the detection label.
  • the present invention provides a monoclonal antibody that is produced by a hybridoma cell line with accession number KCTC 13295BP and specifically binds to pean-derived Ara h1.
  • monoclonal antibody means a highly specific antibody that is indicated in the art as a single antigenic site.
  • Monoclonal antibodies may also be referred to as monoclonal antibody, monoclonal antibody or monoclonal antibody.
  • monoclonal antibodies are directed against a single determinant on the antigen.
  • Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and also have other advantages that are not contaminated by other immunoglobulins because they are produced by hybridoma cultures.
  • the monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from the culture medium or the plural liquids using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.
  • a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.
  • the monoclonal antibody of the present invention was prepared from the hybridoma cell line BP 3A1-12, KCTC 13295BP deposited on Jul. 04, 2017 at the BRC.
  • the antibody of the present invention comprises (1) mixing peanuts with 0.5 M sodium chloride in a weight ratio of 1: 2; (2) reacting the peanut mixed with sodium chloride in boiling water for 15 minutes to extract a thermostable water-soluble protein from the peanut; (3) dialyzing the extracted thermostable water-soluble protein into a phosphate buffer, and injecting it into a mouse; (4) forming an antibody in the mouse against the injected thermostable water-soluble protein; (5) developing a hybridoma cell for monoclonal antibody production using spleen cells of a mouse in which an antibody is formed.
  • the method for producing a monoclonal antibody specific to a water-soluble protein of the peanut is disclosed.
  • peanut was mixed with 0.5 M sodium chloride at a weight ratio of 1: 2 and boiled for 15 minutes to extract a thermostable water-soluble protein.
  • the immunogen was injected into a mouse, and a spleen cell of an immunized mouse showing a high antibody titer was fused to a mouse-derived SP2 / 0 myeloma cell, which is a cancer cell, to prepare a hybridoma cell line.
  • the monoclonal antibody of the present invention was finally selected from the hybridoma cell line BP 3A1-12 and confirmed to be a monoclonal antibody that specifically binds to Ara h1 from peanuts.
  • the monoclonal antibody of the present invention was identified as BP 3A1-12 Respectively.
  • the present invention provides a hybridoma cell line wherein the accession number for producing said monoclonal antibody is KCTC 13295BP.
  • the hybridoma cell line of the present invention was deposited at KCTC 13295BP on Jul. 04, 2017 at the BRC, Korea Research Institute of Bioscience and Biotechnology.
  • the hybridoma cell line is a cell formed by fusion of an antibody-producing cell and a mouse-derived myeloma cell, and the hybridoma cell can continuously supply the antibody of the present invention.
  • thermostable soluble protein extracted from peanuts was used as an immunogen. Specifically, a 0.5 M sodium chloride (NaCl) solution was added to finely ground soybeans The mixture was cooled to room temperature and centrifuged at 3,220 x g for 15 min at 4 ° C. Then, the supernatant was collected by using Whatman No. 1. 1 filter paper, and the protein was extracted by dialyzing for 3 days while changing the phosphate buffered saline (PBS, pH 7.4) solution. The dialyzed protein crude extract was used as an immunogen. Protein concentrations of crude extracts were determined using a Quick start TM Bradford protein assay dye (Bio-Rad Laboratories Inc., USA).
  • the protein crude extract (100 ⁇ g / 100 ⁇ l PBS) was emulsified with the same amount (100 ⁇ l) of Freund's complete adjuvant, and the emulsified immunoglobulin (200 ⁇ l / mouse) was intraperitoneally injected into 7-week old BALB / . Subsequently, the protein crude extract was emulsified with an incomplete Freund ' s incomplete adjuvant at 2-week intervals, followed by second and third injection, and fourth injection of protein crude extract (200 ⁇ g / 200 ⁇ l PBS) . After the fourth injection, serum was collected from caudal vein and high antibody titers were confirmed by ELISA.
  • the culture supernatant of the hybridomas was measured by an indirect enzyme linked immunosorbent assay (ID-ELISA) using a plate coated with peanut antigens to determine the absorbance of the monoclonal antibody , MAb).
  • ID-ELISA indirect enzyme linked immunosorbent assay
  • Crude protein of peanuts and other types of nuts is prepared by adding 0.5 M sodium chloride solution to finely crushed powder of each material (raw peanut, roasted peanut, boiled peanut, walnut, almond, cashew nut, Was added to a glass beaker, and the mixture was extracted with boiling water for 15 minutes. After cooling at room temperature, the mixture was centrifuged at 3,220 x g for 15 minutes at 4 ° C. 1 filter paper, and the filtrate was stored frozen until the experiment.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the samples (3 ⁇ g per well) and protein markers (5 ⁇ l per well) (Prestained Broad Range Protein Maker, Bio-Rad, USA) were loaded on 5% stacking gel (pH 6.8) .
  • Electrophoresis was performed for the Mini-PROTEAN ® tetra electrophoresis cell ( Bio-Rad) and PowerPac TM Basic using (Bio-Rad) (1 hour and 30 minutes at 150V).
  • the gel was stained with a staining solution (EZblue TM gel staining reagent, Sigma) and discolored with water. Another gel was used for western blot analysis.
  • the proteins separated from the 12% separating gel were transferred to a nitrocellulose membrane (Bio-Rad) for 1 h at 100 V using a MiniTrans-Blot unit (Bio-Rad).
  • the membrane was blocked with blocking solution (3% skim milk) at 37 ° C for 1 hour and then washed 3 times with PBST.
  • the membrane was incubated at 37 ° C for 1 hour in a hybridoma cell culture (1: 4 in 3% skim milk) producing antibody, washed 4 times with PBST, and then incubated with a goat anti-mouse IgG alkali And reacted with a goat anti-mouse IgG alkaline phosphatase conjugate (Bio-Rad).
  • the membrane was illuminated by adding ECL (PerkinElmer, USA) and confirmed by an image analyzer.
  • ECL PerkinElmer, USA
  • an image analyzer In order to analyze whether the monoclonal antibody of the present invention binds to Ara h1, commercially available Ara h1 protein (20 ⁇ g / ml, INDOOR Biotechnologies, USA) was purchased and the binding force was analyzed.
  • Example 1 Preparation of a thermal stable-soluble protein in a peanut as an immunogen
  • the protein expression (lane 2) of the non-heat-treated peanut samples and the protein (lane 3) expression of the heat-treated peanut samples were significantly different from those of the non-heat treated peanuts by SDS-PAGE analysis And in all other samples, the main protein band appeared in the range of 15-100 kDa. As a result, it was confirmed that a thermostable water-soluble protein was present in peanuts extracted by the heat treatment method (FIG. 1).
  • the 3A1-12 antibody of the present invention was confirmed to be an antibody reactive with Ara h1. Since the molecular weight of Ara h1 currently known is 66 kDa, the target protein binding to the antibody of the present invention is identified as Ara h1.
  • Indirect ELISA was performed using the BP 3A1-12 and RP 4C12-10 antibodies of the present invention to determine the presence or absence of peanuts in the processed foods of Table 1 below.
  • the BP 3A1-12 and RP 4C12-10 antibodies of the present invention not only reacted to roasted peanut, boiled peanut and peanut butter, , Only the processed foods containing the peanut of Table 1 as a main component showed a specific reaction. In particular, it was confirmed that even when the concentration of the sample is diluted to 1/10, it can be detected.
  • the sensitivity of the BP 3A1-12 and RP 4C12-10 antibodies of the present invention was determined according to various peanut extract contents (0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100% Were measured by indirect ELISA.
  • the antibody of the present invention can detect the peanut protein at a concentration of 0.0001% (w / v) in the roasted peanut, boiled peanut and peanut butter extract (FIG. 6).
  • the monoclonal antibody produced in the hybridoma cells developed in the present invention reacted very specifically with the peanut protein, particularly the heat-stable protein, and it was found that the processed food containing various food additives, It is predicted that it will be useful for the future development of immune sensor or biosensor since it can quickly discriminate the presence of peanuts.

Abstract

The present invention relates to a marker for determining the presence of Arachis hypogaea 1 (Ara h1) derived from Arachis hypogaea in food containing Arachis hypogaea and a use thereof. A monoclonal antibody of the present invention, which is produced by a hybridoma cell line of deposit number KCTC 13295BP, and which specifically binds to Arachis hypogaea-derived Ara h1, can effectively analyze Arachis hypogaea mixed in food.

Description

식품 내 땅콩 혼입 여부 판별용 마커 및 이의 용도Markers for the determination of the presence of peanuts in food and their uses
본 발명은 식품 내 땅콩 혼입 여부 판별용 마커 및 이의 용도에 관한 것이다.The present invention relates to a marker for discriminating whether or not a peanut is incorporated in a food and a use thereof.
식품 알레르기(food allergy)는 식이단백질(dietary protein)에 대한 면역글로불린 E(immunoglobulin E, IgE)-매개성 혹은 비면역글로불린-매개성 과민반응에 의하여 소화기뿐만 아니라 피부, 호흡기, 심혈관계 등의 장기에 다양한 증상을 나타내는 비교적 흔한 알레르기 질환으로, 정상인에게는 무해한 식품이지만, 특정인이 섭취하였을 경우 그 식품의 특정 단백질에 대한 인체의 과도한 면역반응이 일어나는 것을 말한다. 식품 알레르기의 발생 빈도는 3세 이하의 소아의 경우는 약 8%, 성인의 경우는 약 2% 내외로 보고되고 있다. 한 사람이 일생동안 섭취하는 식품의 양은 약 2~3톤에 달하고 매우 다양한 종류의 식품에 노출되지만, 인간의 위장관 면역계는 대부분의 식이단백질에 대한 면역관용(immunological tolerance)이 일어나도록 진화되어 왔으며, 극히 제한된 종류의 식품에 한하여, 식이단백질에 대한 감작(sensitization)이 일어나고 궁극적으로 알레르기 증상이 유발된다.Food allergy is a disease caused by immunoglobulin E (IgE) -mediated or non-immunoglobulin-mediated hypersensitivity to dietary protein, as well as digestive organs, skin, respiratory or cardiovascular Is a relatively common allergic disease that manifests various symptoms. It is a harmless food for normal people, but it refers to the excessive immune response of the human body to a specific protein of the food when it is ingested by a specific person. The incidence of food allergy is reported to be about 8% in children younger than 3 years, and about 2% in adults. The amount of food a person consumes for a lifetime is about 2 to 3 tons and is exposed to a wide variety of foods, but the human gastrointestinal immune system has evolved to cause immunological tolerance to most dietary proteins, For very limited types of food, sensitization to dietary proteins occurs and ultimately allergic symptoms are induced.
식품 알레르기의 발생 여부는 개별 식품의 항원에 대한 면역관용이 성공적으로 확립되었는지 여부에 기인한다. 그러나 모든 식품이 알레르기를 일으키는 빈도와 정도가 동일한 것이 아니며, 이는 식이단백질의 알레르기 항원성 존재 여부와 깊은 관련이 있기 때문에 흔히 알레르기를 일으키는 식품이 한정되어 있다.Whether or not the food allergy occurs is due to whether the immunity tolerance of the individual food to the antigen has been successfully established. However, not all foods have the same degree of frequency with which they cause allergies, which is often related to the presence of allergenicity of dietary proteins, so there are limited foods that cause allergies.
식품 알레르겐은 면역글로불린 E의 생산을 유도하여 즉각적인 과민반응을 일으키는 식품의 성분으로 정의되며, 입으로 섭취되는 식품 외에도 흡입성과 접촉성 식품 알레르겐도 포함된다. 식품의 주요 알레르겐 대부분은 수용성 당단백으로 분자량은 약 10,000-60,000Da이고, 흡입성 알레르겐과는 달리 위장관을 통과하는 동안 소화효소, 세균, 독소 등에 의해 변형될 가능성이 많으며, 장 점막에 머무르는 시간이 길어서 아나필락시스(anaphylaxis) 반응뿐만 아니라 지연형 반응(delayed type reaction)을 일으키는 경우도 많다. 면역글로불린 E에 의한 알레르기 반응을 일으키는 흔한 음식물로는 계란, 우유, 콩, 땅콩, 견과류, 메밀, 밀, 생선, 어패류 등이 있다. 이외에 음식물 보존제, 향신료, 색소 등에 의해서도 이상반응이 일어날 수 있으나, 이러한 경우는 면역학적 기전에 의한 경우가 아니므로 별도로 취급되어야 한다. 소아의 경우는 우유, 계란, 땅콩, 대두, 밀 등이 가장 흔한 식품 알레르겐으로 약 90% 이상의 환자에서 감작되어 있으며, 연령이 증가할수록 생선, 갑각류, 견과류(호두, 잣, 밤) 등이 문제가 된다.. Food allergens are defined as components of foods that induce the production of immunoglobulin E and cause an immediate hypersensitivity reaction, as well as inhalation and contact food allergens. Most major allergens in food are water-soluble glycoproteins with a molecular weight of about 10,000-60,000 Da. Unlike inhalational allergens, they are likely to be transformed by digestive enzymes, bacteria, toxins, etc. during their passage through the gastrointestinal tract, Anaphylaxis reactions as well as delayed type reactions (delayed type reaction) are often caused. Common foods that cause allergic reactions by immunoglobulin E include eggs, milk, beans, peanuts, nuts, buckwheat, wheat, fish, and shellfish. In addition, adverse reactions may occur with food preservatives, spices, and pigments, but these cases are not due to immunological mechanisms and should be handled separately. In children, milk, eggs, peanuts, soybeans and wheat are the most common food allergens. More than 90% of them are sensitized. As age increases, fish, crustaceans, nuts (walnuts, pine nuts, do..
땅콩(Arachis hypogaea)은 심한 전신증상 및 치명적인 아나필락시스를 유발할 수 있는 식품 알레르겐 중 하나로, 다른 식품과 달리 자연 소실이 잘 되지 않아 평생 지속되는 경향을 보인다.The peanut ( Arachis hypogaea ) is one of the food allergens that can cause severe systemic symptoms and lethal anaphylaxis. Unlike other foods, the natural loss does not tend to last for a lifetime.
식품 알레르기에 대한 예방 및 치료방법은 특별히 없으며, 이를 예방하는 가장 확실하고 유일한 방법은 식품 알레르기를 일으키는 원인 식품을 식단에서 제거하여 섭취하지 않는 것이다. 원인 식품의 제거식이(elimination diet)는 단지 자연 상태의 원인 식품만을 금식하는 것이 아니라, 원인 식품이 어떤 형태로든 소량이라도 포함되어 있는 식품 또는 제조·가공된 가공식품 모두를 철저히 금식해야 하고, 원인 식품과 교차 항원성이 있는 식품도 금식하는 것이 바람직하다. 즉, 땅콩 알레르기 환자의 경우에는 기타의 견과류(호두, 잣, 밤, 헤이즐넛 등)도 금식할 필요가 있다. There is no particular way to prevent and treat food allergies, and the most obvious and unique way to prevent them is to avoid eating foods that cause food allergy from the diet. Cause The elimination diet of food should not only fast food that is the cause of the natural state but the food which contains the small amount of the causal food or the processed food which is produced or processed thoroughly, And foods having cross-antigenicity are also preferably fasted. In other words, in case of peanut allergy, other nuts (walnuts, pine nuts, chestnuts, hazelnuts, etc.) need to fast.
따라서, 식품 알레르기를 유발하는 원인 식품을 분석하는 것이 매우 중요하고, 이를 통해 식품의 원재료뿐만 아니라 제조·가공된 가공식품에 알레르기 유발 성분을 정확하게 표시하여 미리 확인하는 것이 중요하다.Therefore, it is very important to analyze the causative food causing allergies to food, and it is important to precisely mark and display allergen-inducing ingredients in raw foods of the food as well as processed and processed foods.
이에 본 발명에서는 땅콩 열 안정성 수용성 단백질인 Ara h1에 특이적으로 결합할 수 있는 단일클론항체를 생산할 수 있는 하이브리도마 세포를 제조하였고, 상기 하이브리도마 세포로부터 생산되는 단일클론항체를 사용하여 열처리된 식품 원·부재료의 혼합물 및 가공식품 내에 땅콩 혼입여부를 신속하게 분석할 수 있음을 확인하였다.Therefore, in the present invention, a hybridoma cell capable of producing a monoclonal antibody capable of specifically binding to a peanut thermostable water-soluble protein Ara h1 was prepared, and a monoclonal antibody produced from the hybridoma cell was used for heat treatment It was confirmed that it is possible to quickly analyze the presence of peanuts in the mixture of food raw materials and processed foods.
한편, 일본등록특허 제3853692호에는 '땅콩 성분 검사용 항체, 검사 방법 및 검사용 키트'가 개시되어 있고, 한국등록특허 제1223256호에는 '땅콩의 알레르기성 저감 방법 및 알레르기성이 저감된 땅콩'이 개시되어 있으나, 본 발명의 식품 내 땅콩 혼입 여부 판별용 마커 및 이의 용도에 대해서는 기재된 바가 없다.Japanese Patent No. 3853692 discloses an antibody for testing peanut components, an assay method and an assay kit, and Korean Patent No. 1223256 discloses a method for reducing allergenicity of peanut and a peanut with reduced allergenicity. However, the marker for discriminating the presence or absence of peanuts in the food of the present invention and the use thereof are not described.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명에서는 땅콩 내에 존재하는 열 알정성 수용성 단백질을 항원으로 사용하여, 땅콩의 열 안정성 수용성 단백질에 특이적으로 결합하는 단일클론항체를 생산하는 하이브리도마주를 제조하였다. 또한, 본 발명의 상기 단일클론항체를 이용하여 면역분석을 수행한 결과, 땅콩이 함유된 모든 식품시료의 경우 열처리 유무에 관계없이 본 발명의 단일클론항체에 의해 땅콩 혼입 유무가 신속하게 판별될 수 있음을 확인함으로써, 본 발명을 완성하였다.SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide a hive producing a monoclonal antibody which specifically binds to a thermostable water-soluble protein of a peanut using a heat- Lidomaju was prepared. Immunoassay using the monoclonal antibody of the present invention showed that the presence or absence of peanut contamination can be quickly determined by the monoclonal antibody of the present invention regardless of whether or not heat treatment is applied to all food samples containing peanut The present invention has been completed.
상기 과제를 해결하기 위해, 본 발명은 땅콩 유래 Ara h1을 포함하는 식품 내 땅콩 혼입 여부 판별용 마커를 제공한다.In order to solve the above problems, the present invention provides a marker for discriminating the presence or absence of peanuts in a food containing a peanut-derived Ara h1.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 조성물을 제공한다.The present invention also provides a composition for determining the presence or absence of peanut in a food comprising an antibody that specifically binds to peanut-derived Ara h1.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 키트를 제공한다.In addition, the present invention provides a kit for judging whether or not a peanut is contained in a food comprising an antibody specifically binding to a pean-derived Ara h1.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 검체 시료와 접촉시켜 형성된 항원-항체 복합체를 검출하는 단계를 포함하는 식품 내 땅콩 혼입 여부의 판별방법을 제공한다.In addition, the present invention provides a method for determining whether a peanut is incorporated in a food, comprising the step of detecting an antigen-antibody complex formed by contacting an antibody that specifically binds to peanut-derived Ara h1 with a test sample.
또한, 본 발명은 기탁번호 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산되고, 땅콩 유래 Ara h1에 특이적으로 결합하는 단일클론항체를 제공한다.In addition, the present invention provides a monoclonal antibody produced by a hybridoma cell line of deposit number KCTC 13295BP and specifically binding to peanut-derived Ara h1.
또한, 본 발명은 기탁번호가 KCTC 13295BP인 하이브리도마 세포주를 제공한다.In addition, the present invention provides a hybridoma cell line having the accession number KCTC 13295BP.
본 발명의 단일클론항체는 땅콩 내 열 안정성 수용성 단백질 중 Ara h1에 특이적으로 반응하는 것으로, 열처리에 의해 제조·가공된 의도적 미표기 식품에 혼입되어 있는 땅콩을 효과적으로 분석할 수 있다. 또한, 본 발명을 통하여, 식품 내 땅콩의 함유 유무에 관한 정보를 소비자에게 제공하여 식품 알레르기 유발을 방지할 수 있고, 식품 제조 및 가공 과정을 개선시키기 위해 활용됨으로써, 식품 안정성 확보에 기여할 수 있을 것으로 기대된다.The monoclonal antibody of the present invention reacts specifically with Ara h1 among heat stable water soluble proteins in peanuts, and can effectively analyze peanuts incorporated in intentional unheated foods manufactured and processed by heat treatment. Further, through the present invention, information on the presence or absence of peanuts in foods can be provided to consumers to prevent food allergy induction, and it can be utilized to improve food manufacturing and processing, thereby contributing to securing food stability It is expected.
도 1은 비열처리(Non-Heat) 및 열처리(Heat) 된 땅콩에서 추출한 열 안정성 단백질의 SDS-PAGE 결과이다. 레인 1: 크기 마커, 레인 2: 비열처리 방법으로 추출한 생땅콩 시료, 레인 3: 열처리에 의해 추출한 생땅콩 시료, 레인 4: 비열처리 방법으로 추출한 삶은 땅콩 시료, 레인 5: 열처리에 의해 추출한 삶은 땅콩 시료, 레인 6: 비열처리 방법으로 추출한 구운 땅콩 시료, 레인 7: 열처리에 의해 추출한 구운 땅콩 시료, 레인 8: 비열처리 방법으로 추출한 땅콩잼 시료, 레인 9: 열처리에 의해 추출한 땅콩잼 시료.Figure 1 shows the results of SDS-PAGE of heat-stable proteins extracted from non-heat and heat-treated peanuts. Lane 1: size marker, lane 2: green peanut sample extracted by non-heat treatment method, lane 3: green peanut sample extracted by heat treatment, lane 4: boiled peanut sample extracted by non-heat treatment method, lane 5: boiled peanut sample extracted by heat treatment, Lane 6: peanut samples extracted by non-heat treatment method, lane 7: roasted peanut samples extracted by heat treatment, lane 8: peanut samples extracted by non-heat treatment method, lane 9: peanut samples extracted by heat treatment.
도 2는 구운 땅콩(roasted peanut) 및 삶은 땅콩(boiled peanut)에서 추출한 열 안정성 수용성 단백질로 면역한 마우스의 항-혈청 항체가를 간접 ELISA법으로 측정한 결과이다.FIG. 2 is a result of indirect ELISA measurement of anti-serum antibody levels of mice immunized with a heat-stable water-soluble protein extracted from roasted peanut and boiled peanut.
도 3은 본 발명에 따른 BP 3A1-12 및 RP 4C12-10 단일클론항체를 이용하여 구운 땅콩(레인 2), 삶은 땅콩(레인 3), 땅콩 버터(레인 4), 월넛(레인 5), 아몬드(레인 6), 캐슈넛(레인 7), 팥(레인 8) 및 대두(레인 9)에서 추출한 열 안정성 단백질에 대한 웨스턴 블롯(A 및 B) 및 ELISA(C) 분석 결과이다. 레인 1: 크기 마커. FIG. 3 is a graph showing the distribution of peanut (lane 2), boiled peanut (lane 3), peanut butter (lane 4), walnut (lane 5), almond (A and B) and ELISA (C) analysis of the heat-stable proteins extracted from soybean (lane 6), cashew nut (lane 7), red bean (lane 8) and soybean (lane 9). Lane 1: Size marker.
도 4는 땅콩 내 열 안정성 단백질인 Ara h1에 대한 단일클론항체인 BP 3A1-12의 반응 여부 확인을 위한 웨스턴 블롯 결과이다. 레인 1: 크기 마커, 레인 2: 시판 Ara h1 단백질, 레인 3: 구운 땅콩, 레인 4: 삶은 땅콩, 레인 5: 땅콩 버터. Fig. 4 is a Western blot result for confirming the reaction of BP 3A1-12, a monoclonal antibody against Ara h1, a heat-stable protein in peanuts. Lane 1: Size marker, Lane 2: Commercial Ara h1 protein, Lane 3: Roasted peanuts, Lane 4: Boiled peanuts, Lane 5: Peanut butter.
도 5는 본 발명의 항체를 이용하여 다양한 가공식품 내에 땅콩 혼입 유무를 간접 ELISA법으로 측정한 결과이다. Original: 하이브리도마 세포 배양액, 1/10 diluted: 하이브리도마 세포 배양액을 10분의 1로 희석. 1/100 diluted: 하이브리도마 세포 배양액을 100분의 1로 희석. (A); BP 3A1-12 항체, (B); RP 4C12-10 항체. FIG. 5 shows the results of indirect ELISA measurement of the presence of peanuts in various processed foods using the antibody of the present invention. Original: Hybridoma cell culture, 1/10 diluted: Hybridoma cell culture diluted to 1/10. 1/100 diluted: Hybridoma cell culture diluted to 1/100. (A); BP 3A1-12 antibody, (B); RP 4C12-10 antibody.
도 6은 본 발명의 단일클론항체(BP 3A1-12 및 RP 4C12-10)의 땅콩 단백질 함량에 따른 검출 여부를 간접 ELISA법으로 측정한 결과이다.FIG. 6 shows the results of measurement of the detection of the peptone protein content of the monoclonal antibodies (BP 3A1-12 and RP 4C12-10) of the present invention by indirect ELISA.
본 발명의 목적을 달성하기 위하여, 본 발명은 땅콩(Arachis hypogaea) 유래 Ara h1을 포함하는 식품 내 땅콩 혼입 여부 판별용 마커를 제공한다.In order to achieve the object of the present invention, the present invention provides a marker for determining the presence or absence of a peanut in a food containing ara h1 from a peanut ( Arachis hypogaea ).
상기 Ara h1은 종자 저장 단백질(seed storage protein) 또는 비실린(vicilin)으로, 땅콩 알레르겐(allergen) 중 하나이다. 열과 소화효소에 저항성을 가진 Ara h1은 단량체(monomer)간의 상호작용에 의하여 안정적인 트리머(trimmer) 구조를 형성하고 있으며, 이들의 접촉부위에 면역글로불린 E가 결합할 수 있는 에피토프(항원결정기)가 존재한다. Ara h1에 열처리를 할 경우, 단백질 변형으로 인해 삼차원적 구조에 묻혀있던 면역글로불린 E 결합 에피토프가 노출되고, 노출된 에피토프는 면역글로불린 E와 결합하여 알레르기 반응이 유도된다.The Ara h1 is a seed storage protein or vicilin and is one of the peanut allergens. Ara h1, which is resistant to heat and digestive enzymes, forms a stable trimmer structure due to the interaction between monomers, and there is an epitope (antigenic determinant) capable of binding immunoglobulin E at the contact site do. When heat treatment is applied to Ara h1, the immunoglobulin E binding epitope buried in the three-dimensional structure due to the protein modification is exposed, and the exposed epitope binds with immunoglobulin E to induce an allergic reaction.
본 발명에서는 땅콩 유래 Ara h1 단백질이 식품 내에서 검출된다면 땅콩이 혼입된 것으로 판정할 수 있다.In the present invention, if the peanut-derived Ara h1 protein is detected in the food, it can be judged that the peanut is incorporated.
상기 마커는 식품 내 땅콩 혼입 여부를 판별하기 위한 마커 조성물과 동일하게 쓰일 수 있다.The marker may be used in the same manner as the marker composition for determining whether peanuts are mixed in the food.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 조성물을 제공한다.The present invention also provides a composition for determining the presence or absence of peanut in a food comprising an antibody that specifically binds to peanut-derived Ara h1.
상기 조성물은 유효성분으로 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하며, 상기 항체를 이용하여 식품 내 땅콩 혼입 여부를 판별할 수 있는 것이다.The composition includes an antibody that specifically binds to Ara h1 derived from peanuts as an active ingredient, and can determine the presence or absence of peanuts in the food using the antibody.
본 명세서에서, "항체"는 본 발명의 식품 내 땅콩 혼입 여부 판별용 마커에 특이적으로 결합하는 것이면 모든 형태의 것을 포함하는 의미이다. 따라서 단일클론항체, 다클론항체, 다중특이적 항체(즉 두 개 이상의 항원 또는 두 개 이상의 에피토프를 인식하는 항체로서 예컨대 이특이적 항체 등을 말함)를 포함하는 것 이외에, 본 발명의 식품 내 땅콩 혼입 여부 판별용 마커에 특이적으로 결합할 수 있는 능력을 보유하는 항체의 단편, 재조합 항체, 화학적으로 수식된 항체를 포함한다. 여기서 항체의 단편의 예로서는 Fab, F(ab')2, scFv(중쇄나 경쇄의 Fv를 적당한 링커로 연결시킨 항체), Fv, Fab/c(1개의 Fab와 완전한 Fc를 가지는 항체), 항체를 단백질 절단 효소 예컨대, 파파인, 펩신으로 처리하여 얻어진 항체단편, 단편에 대한 유전자를 유전자 재조합 방법으로 숙주세포에 도입·발현시켜 얻어지는 항체 단편을 포함하는 의미이다. 상기 항체의 글로불린 유형도 본 발명의 식품 내 땅콩 혼입 여부 판별용 마커에 특이적으로 결합하는 것이면 특별히 한정되지 않는데, 그 글로불린 유형은 IgG, IgM, IgA, IgE, IgD 중 어느 하나일 수 있다.In the present specification, the term " antibody " is meant to include all forms as long as it specifically binds to a marker for discriminating the presence or absence of peanuts in the food of the present invention. Thus, in addition to containing a monoclonal antibody, polyclonal antibody, multispecific antibody (i.e., an antibody recognizing two or more antigens or two or more epitopes, such as a bispecific antibody, etc.) Recombinant antibodies, chemically modified antibodies that have the ability to specifically bind to a recognition marker. Examples of fragments of the antibody include Fab, F (ab ') 2, scFv (an antibody in which a heavy or light chain Fv is linked by a suitable linker), Fv, Fab / c Means an antibody fragment obtained by introducing and expressing a gene for an antibody fragment or fragment obtained by treating with a protein cleaving enzyme such as papain or pepsin into a host cell by a gene recombination method. The globulin type of the antibody may be any one of IgG, IgM, IgA, IgE, and IgD, provided that the globulin type of the antibody specifically binds to the marker for discriminating the presence or absence of peanuts in the food of the present invention.
상기 단일클론항체는 바람직하게는 기탁번호가 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산될 수 있으나, 이제 제한되지 않는다.The monoclonal antibody may preferably be produced by a hybridoma cell line having the accession number KCTC 13295BP, but is not limited to this.
상기 식품이란 땅콩을 함유하거나, 함유할 것으로 예상되는 모든 식품을 포함할 수 있으며, 바람직하게는 생땅콩, 구운 땅콩, 삶은 땅콩, 튀긴 땅콩 또는 버터땅콩 등의 가공제품일 수 있으나, 이에 제한되지 않는다. 상기 버터땅콩은 미국에서 일반적으로 시판되고 있는 잼처럼 가공된 제품을 말한다.The food may include, or is not limited to, all foods that contain or are expected to contain peanuts, preferably processed products such as peanuts, roasted peanuts, boiled peanuts, fried peanuts or butter peanuts. The butter peanut refers to a product processed like a jam that is generally commercially available in the United States.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 키트를 제공한다.In addition, the present invention provides a kit for judging whether or not a peanut is contained in a food comprising an antibody specifically binding to a pean-derived Ara h1.
상기 항체는 전술한 바와 같이 땅콩 유래 Ara h1에 특이적으로 결합하는 모든 형태의 항체를 포함할 수 있으며, 바람직하게는 땅콩 유래 Ara h1에 특이적으로 결합하는 단일클론항체일 수 있으며, 더욱 바람직하게는 기탁번호가 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산되는 단일클론항체일 수 있으나, 이제 제한되지 않는다.The antibody may include any type of antibody that specifically binds to the Ara h1 from the peanut as described above. Preferably, the antibody may be a monoclonal antibody that specifically binds to the Ara h1 from the peanut. More preferably, May be a monoclonal antibody produced by a hybridoma cell line with the accession number KCTC 13295BP, but is not now limited.
본 발명에 사용하기 위한 키트 시스템은 이에 한정되는 것은 아니나, ELISA 플레이트, 딥-스틱 디바이스, 면역크로마토그래피법 및 방사 분할 면역검정 디바이스, 플로우-쓰로우(flow-through) 디바이스 등을 포함한다.Kit systems for use with the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography methods and split-split immune assay devices, flow-through devices, and the like.
상기 본 발명의 키트에는 통상적으로 키트에 사용되는 것이라면 이에 한정되지 않지만, 예를 들어 ELISA를 이용한 것이라면 고체상 지지체; 본 발명의 단일클론 항체; 및 항원과의 반응을 위한 효소표지항체액 및 효소반응을 나타내는 발색액을 함유하는 효소-연관 면역분석용(ELISA) 반응액을 포함할 수 있다. The kit of the present invention is not particularly limited as long as it is used in a kit, but if it is an ELISA, for example, it may be a solid-phase supporter; A monoclonal antibody of the present invention; And an enzyme-linked immunosorbent assay (ELISA) reaction solution containing an enzyme-labeled antibody solution for reaction with an antigen and a coloring solution indicating an enzyme reaction.
또한, 본 발명은 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 검체 시료와 접촉시켜 형성된 항원-항체 복합체를 검출하는 단계를 포함하는 식품 내 땅콩 혼입 여부의 판별방법을 제공한다.In addition, the present invention provides a method for determining whether a peanut is incorporated in a food, comprising the step of detecting an antigen-antibody complex formed by contacting an antibody that specifically binds to peanut-derived Ara h1 with a test sample.
본 발명에서 용어 "항원-항체 복합체"란, 시료 중의 땅콩 열 안정성 수용성 단백질인 Ara h1과 이를 인지하는 본 발명에 따른 단일클론항체의 결합물을 의미하며, 이러한 항원-항체 복합체는 비색법(colormetric method), 전기화학법(electrochemical method), 형광법(fluorimetric method), 발광법(luminometry), 입자계수법(particle counting method), 육안측정법(visual assessment) 및 섬광계수법(scintillation counting method)으로 이루어진 군에서 선택되는 임의의 방법으로 검출할 수 있다. 그러나 반드시 이들로만 제한되지 않고 다양한 응용과 적용이 가능하다.The term " antigen-antibody complex " in the present invention refers to a conjugate of Ara h1, a peanut thermostable water-soluble protein in a sample, and a monoclonal antibody according to the present invention, ), An electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method It can be detected by any method. However, various applications and applications are possible without being limited thereto.
본 발명에서는 항원-항체 복합체를 검출하기 위한 것으로 여러가지 표지체를 사용할 수 있다. 구체적인 예로는 효소, 형광물, 리간드, 발광물, 미소입자, 방사성 동위원소로 이루어진 그룹 중에서 선택될 수 있으며, 반드시 이들로만 한정되는 것은 아니다.In the present invention, various markers can be used for detecting an antigen-antibody complex. Specific examples include, but are not limited to, enzymes, chromophores, ligands, luminescent materials, microparticles, and radioactive isotopes.
검출 표지체로서 사용되는 바람직한 효소로는 아세틸콜린에스테라제, 알칼라인 포스파타제, β-D-갈락토시다제, 호스래디쉬 퍼옥시다제 또는 β-락타마제가 있으며, 바람직한 형광물로는 양자점, 플루오레세인, Eu3+, Eu3+ 킬레이트 또는 크립테이트가 있으며, 바람직한 리간드로는 바이오틴 유도체가 있고, 바람직한 발광물로는 아크리디늄 에스테르 또는 이소루미놀 유도체가 있으며, 바람직한 미소입자로는 콜로이드 금 또는 착색된 라텍스가 있고, 바람직한 방사성 동위원소로는 7Co,3H, 125I 또는 125I-볼톤(Bolton) 헌터(Hunter) 시약이 있으나, 이에 한정하는 것은 아니다.Examples of preferred enzymes used as the detection label include acetylcholinesterase, alkaline phosphatase,? -D-galactosidase, horseradish peroxidase or? -Lactamase, and preferable examples of the minerals include quantum dots, Eu 3+ , Eu 3+ chelate, or cryptate. Preferred ligands include biotin derivatives. Preferred emitters are acridinium esters or isoluminol derivatives. Preferred microparticles include colloidal gold or There are colored latexes and preferred radioisotopes include, but are not limited to, 7 Co, 3 H, 125 I or 125 I Bolton Hunter reagents.
항원-항체 복합체를 검출하는 방법은 바람직하게는 효소면역흡착법(ELISA)을 이용하여 검출할 수 있으나, 이에 한정하는 것은 아니다. 효소면역흡착법은 고체 지지체에 부착된 항원을 인지하는 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 이차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다.The method for detecting the antigen-antibody complex can be preferably detected using enzyme immunoassay (ELISA), but is not limited thereto. Enzyme immunosorbent assays include direct ELISA using an antibody recognizing an antigen attached to a solid support, indirect ELISA using a labeled secondary antibody recognizing the capture antibody in a complex of antibodies recognizing an antigen attached to a solid support, attachment to a solid support A direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antigen, a label which recognizes the antibody after reacting with another antibody recognizing the antigen in the complex of the antibody and the antigen attached to the solid support And indirect sandwich ELISA using secondary antibodies.
상기 항체는 검출 표지체를 가질 수 있으며, 검출 표지체를 가지지 않을 경우는 이들 항체를 포획할 수 있고, 검출 표지체를 가지는 또 다른 항체를 처리하여 확인할 수 있다.The antibody may have a detection label, and when it does not have a detection label, the antibody can be captured and can be confirmed by treating another antibody having the detection label.
또한, 본 발명은 기탁번호가 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산되고, 땅콩 유래 Ara h1에 특이적으로 결합하는 단일클론항체를 제공한다.In addition, the present invention provides a monoclonal antibody that is produced by a hybridoma cell line with accession number KCTC 13295BP and specifically binds to pean-derived Ara h1.
본 발명에서, "단일클론항체(monoclonal antibody)"란 당해 분야에 공지된 용어로서 단일 항원성 부위에 대해서 지시되는 고도의 특이적인 항체를 의미한다. 단일클론항체는 단세포군 항체, 모노클로날 항체 또는 단클론 항체라고 불리기도 한다. 통상적으로, 상이한 에피토프(항원결정기)들에 대해 지시되는 상이한 항체들을 포함하는 다클론항체와는 다르게, 단일클론항체는 항원상의 단일 결정기에 대해서 지시된다. 단일클론항체는 항원-항체 결합을 이용하는 진단 및 분석학적 분석법의 선택성과 특이성을 개선시키는 장점이 있으며, 또한 하이브리도마 배양에 의해 생산되기 때문에 다른 면역글로불린에 의해 오염되지 않는 또 다른 장점을 갖는다.In the present invention, the term " monoclonal antibody " means a highly specific antibody that is indicated in the art as a single antigenic site. Monoclonal antibodies may also be referred to as monoclonal antibody, monoclonal antibody or monoclonal antibody. Typically, unlike polyclonal antibodies that contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays that utilize antigen-antibody binding and also have other advantages that are not contaminated by other immunoglobulins because they are produced by hybridoma cultures.
상기한 하이브리도마가 생산하는 단일클론항체는 정제하지 않고 사용할 수도 있으나, 최선의 결과를 얻기 위해서는 본 발명이 속하는 기술분야에 잘알려져 있는 방법에 따라 고순도(예컨대, 95% 이상)로 정제하여 사용하는 것이 바람직하다. 이러한 정제 기술로는, 예를 들어 겔 전기영동, 투석, 염 침전, 이온교환 크로마토 그래피, 친화성 크로마토그래피 등의 정제 방법을 이용하여 배양 배지 또는 복수액으로부터 분리될 수 있다.The monoclonal antibody produced by the hybridoma may be used without purification. However, in order to obtain the best results, the monoclonal antibody produced by the hybridoma may be purified to a high purity (for example, 95% or more) according to a method well known in the technical field of the present invention . Such a purification technique can be separated from the culture medium or the plural liquids using a purification method such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography and the like.
본 발명의 단일클론항체는 한국생명공학연구원 생물자원센터에 2017년 07월 04일자로 기탁된 KCTC 13295BP인 하이브리도마 세포주 BP 3A1-12로부터 제조된 것이다.The monoclonal antibody of the present invention was prepared from the hybridoma cell line BP 3A1-12, KCTC 13295BP deposited on Jul. 04, 2017 at the BRC.
본 발명의 항체는 (1) 땅콩에 0.5 M 염화나트륨을 중량비 1:2로 섞는 단계; (2) 상기 염화나트륨과 섞어진 땅콩을 끓는 물에서 15분간 반응시켜 땅콩에서 열 안정성 수용성 단백질을 추출하는 단계; (3) 상기 추출한 열 안정성 수용성 단백질을 인산염 버퍼에 투석한 후 마우스에 주입하는 단계; (4) 상기 주입된 열 안정성 수용성 단백질에 대하여 마우스에서 항체를 형성시키는 단계; (5) 항체가 형성된 마우스의 비장세포를 이용하여 단일클론항체 생산용 하이브리도마 세포를 개발하는 단계를 포함하는 땅콩의 열 안정성 수용성 단백질에 특이적인 단일클론항체의 제조방법을 특징으로 한다.The antibody of the present invention comprises (1) mixing peanuts with 0.5 M sodium chloride in a weight ratio of 1: 2; (2) reacting the peanut mixed with sodium chloride in boiling water for 15 minutes to extract a thermostable water-soluble protein from the peanut; (3) dialyzing the extracted thermostable water-soluble protein into a phosphate buffer, and injecting it into a mouse; (4) forming an antibody in the mouse against the injected thermostable water-soluble protein; (5) developing a hybridoma cell for monoclonal antibody production using spleen cells of a mouse in which an antibody is formed. The method for producing a monoclonal antibody specific to a water-soluble protein of the peanut is disclosed.
본 발명에 따른 면역원으로는 땅콩에 0.5 M 염화나트륨을 중량비 1:2로 섞어 끓는 물에서 15분간 반응시켜 열 안정성 수용성 단백질을 추출하여 사용하였다.As an immunogen according to the present invention, peanut was mixed with 0.5 M sodium chloride at a weight ratio of 1: 2 and boiled for 15 minutes to extract a thermostable water-soluble protein.
상기 면역원을 마우스에 주입한 후 높은 항체가를 나타내는 면역된 마우스의 비장세포(spleen cell)를 암세포 일종인 마우스 유래 SP2/0 골수종 세포(myeloma cell)에 융합하여 하이브리도마 세포주를 제조하였다.The immunogen was injected into a mouse, and a spleen cell of an immunized mouse showing a high antibody titer was fused to a mouse-derived SP2 / 0 myeloma cell, which is a cancer cell, to prepare a hybridoma cell line.
본 발명의 단일클론항체는 하이브리도마 세포주 BP 3A1-12로부터 최종 선정되었으며, 땅콩 유래 Ara h1에 특이적으로 결합하는 단일클론항체임을 확인하였고, 이에 본 발명의 단일클론항체는 BP 3A1-12로 명명하였다.The monoclonal antibody of the present invention was finally selected from the hybridoma cell line BP 3A1-12 and confirmed to be a monoclonal antibody that specifically binds to Ara h1 from peanuts. Thus, the monoclonal antibody of the present invention was identified as BP 3A1-12 Respectively.
또한, 본 발명은 상기 단일클론항체를 생산하는 기탁번호가 KCTC 13295BP인 하이브리도마 세포주를 제공한다.In addition, the present invention provides a hybridoma cell line wherein the accession number for producing said monoclonal antibody is KCTC 13295BP.
본 발명의 하이브리도마 세포주는 한국생명공학연구원 생물자원센터에 2017년 07월 04일자로 기탁번호 KCTC 13295BP로 기탁하였다.The hybridoma cell line of the present invention was deposited at KCTC 13295BP on Jul. 04, 2017 at the BRC, Korea Research Institute of Bioscience and Biotechnology.
상기 하이브리도마 세포주는 항체 생산 세포와 마우스 유래 골수종세포와의 융합에 의해서 형성된 세포이며, 상기 하이브리도마 세포는 본 발명의 항체를 지속적으로 공급할 수 있다.The hybridoma cell line is a cell formed by fusion of an antibody-producing cell and a mouse-derived myeloma cell, and the hybridoma cell can continuously supply the antibody of the present invention.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
1. 면역원 제조1. Manufacture of immunogen
땅콩에서 추출한 열 안정성 수용성 단백질(thermal stable-soluble protein, TSSP)을 면역원으로 사용하였다. 상세하게는, 잘게 분쇄한 생땅콩에 0.5 M 염화나트륨(NaCl) 용액을 생땅콩 중량 대비 2배 혼합하여 유리 비이커에 넣고 끓는 물에서 15분간 중탕하여 추출하였고, 상온에서 식힌 후 4℃에서 15분간 3,220 xg로 원심분리하여 얻은 상층액을 Whatman No. 1 여과지로 여과하였으며, 매일 인산완충식염수(phosphate buffered saline, PBS, pH 7.4) 용액을 바꾸어 가며 3일간 투석하여 단백질을 추출하였다. 상기 투석한 단백질 조추출물을 면역원으로 사용하였다. 조추출물의 단백질 농도는 Quick startTM Bradford protein assay dye(Bio-Rad Laboratories Inc., 미국)를 이용하여 측정하였다. A thermostable soluble protein (TSSP) extracted from peanuts was used as an immunogen. Specifically, a 0.5 M sodium chloride (NaCl) solution was added to finely ground soybeans The mixture was cooled to room temperature and centrifuged at 3,220 x g for 15 min at 4 ° C. Then, the supernatant was collected by using Whatman No. 1. 1 filter paper, and the protein was extracted by dialyzing for 3 days while changing the phosphate buffered saline (PBS, pH 7.4) solution. The dialyzed protein crude extract was used as an immunogen. Protein concentrations of crude extracts were determined using a Quick start ™ Bradford protein assay dye (Bio-Rad Laboratories Inc., USA).
2. 땅콩 단백질에 대한 단일클론항체 생산2. Production of monoclonal antibodies against peanut protein
단백질 조추출물(100 ㎍/100 ㎕ PBS)은 동량(100 ㎕)의 완전 프로인트보강제(Freund's complete adjuvant)로 유화하여 7주령된 BALB/c 암컷 마우스에 유화된 면역원(200 ㎕/마우스)을 복강 내 주사하였다. 이후 2주 간격으로 불완전 프로인트보강제(Freund's incomplete adjuvant)로 단백질 조추출물을 유화하여 2차, 3차 주사하였고, 세포 융합 4일 전에 단백질 조추출물(200 ㎍/200 ㎕ PBS)을 4차 주사하였다. 4차 주사 후, 미정액(caudal vein)에서 혈청(serum)을 수집하고 높은 항체가를 ELISA로 확인하였다.The protein crude extract (100 μg / 100 μl PBS) was emulsified with the same amount (100 μl) of Freund's complete adjuvant, and the emulsified immunoglobulin (200 μl / mouse) was intraperitoneally injected into 7-week old BALB / . Subsequently, the protein crude extract was emulsified with an incomplete Freund ' s incomplete adjuvant at 2-week intervals, followed by second and third injection, and fourth injection of protein crude extract (200 쨉 g / 200 쨉 l PBS) . After the fourth injection, serum was collected from caudal vein and high antibody titers were confirmed by ELISA.
높은 항체가를 나타내는 면역된 마우스의 비장세포(speen cell)와 암세포 일종인 SP2/0 골수종 세포(myeloma cell)를 융합하였다. 세포 융합의 일반적 과정은 Kohler and Milsten 방법에서 약간 변형하여 수행하였다. 비장세포(2×108)와 SP2/0 골수종 세포(2×107)를 1 ㎖의 50% 폴리에틸렌 글리콜(polyethylene glycol, PEG) 1500 용액을 이용하여 융합한 후, 히포잔틴(hypoxanthine), 아미노프테린(aminopterin) 및 티미딘(thymidine)을 포함하는 HAT 배지를 이용하여, 융합된 하이브리도마만을 선택적으로 배양하고 증식시켰다. 상기 하이브리도마의 배양상등액을 땅콩 항원이 코팅된 플레이트를 이용한 간접 ELISA(indirect enzyme linked immunosorbent assay, ID-ELISA)법으로 흡광도를 측정하여 땅콩 내 열 안정성 수용성 단백질에 특이적인 단일클론항체(monoclonal antibody, MAb)의 생성 여부를 분석하였고, ELISA-양성 하이브리도마 세포는 무한대희석법(unlimited dilution method)으로 분리하여 땅콩 단백질에 특이적인 MAb을 생산하는 하이브리도마 세포주를 선별하였다.Spin cells of immunized mice expressing high antibody titers were fused with SP2 / 0 myeloma cells, a kind of cancer cells. The general process of cell fusion was performed with slight modification in the Kohler and Milsten method. After splenocytes (2 × 10 8 ) and SP2 / 0 myeloma cells (2 × 10 7 ) were fused with 1 ml of a solution of 50% polyethylene glycol (PEG) 1500, hypoxanthine, Using a HAT medium containing aminopterin and thymidine, only the fused hybridomas were selectively cultivated and propagated. The culture supernatant of the hybridomas was measured by an indirect enzyme linked immunosorbent assay (ID-ELISA) using a plate coated with peanut antigens to determine the absorbance of the monoclonal antibody , MAb). ELISA-positive hybridoma cells were separated by an unlimited dilution method to select hybridoma cell lines that produce MAb specific to peanut protein.
상기 하이브리도마 세포주로부터 단일클론항체를 생산하기 위해, 7~10일 동안 0.5 ㎖의 프리스틴(pristine)을 BALB/c 마우스의 복강 내에 선처리한 후, 하이브리도마 세포(1×107)를 마우스 복강 내에 주입하여 복수를 생산하였다. 생산된 복수를 채취하여 황산암모늄(ammonium sulfate) 침전법으로 정제한 후, 인산완충식염수로 3일 동안 투석하고 동결건조시켜 -20℃에 보관하면서 실험에 사용하였다. To produce a monoclonal antibody from the hybridoma cell line, 0.5 ml of pristine was pre-treated in the abdominal cavity of BALB / c mice for 7 to 10 days, and then hybridoma cells (1 x 10 7 ) Were injected into the abdominal cavity to produce a plurality. The produced ascites was collected and purified by ammonium sulfate precipitation, dialyzed with phosphate-buffered saline for 3 days, lyophilized and stored at -20 ° C.
3. 웨스턴 블롯 시료의 준비3. Preparation of western blot samples
시판되고 있는 땅콩(peanut), 월넛(walnut), 아몬드(almond), 캐슈넛(cashew nut), 팥(red bean), 대두(soybean), 탈지유(skim milk) 및 땅콩 버터(peanut butter)를 이용하여 본 발명의 단일클론항체의 특이성을 분석하였다. 땅콩 및 다른 종류의 견과류의 조단백질(crude protein)은 각 재료(열처리된 땅콩(raw peanut, roasated peanut, boiled peanut), 월넛, 아몬드, 캐슈넛, 팥 및 대두)의 잘게 분쇄한 분말에 0.5 M 염화나트륨 용액을 중량 대비 2배 혼합하여 유리 비이커에 넣고 끓는 물에서 15분간 중탕하여 추출하였고, 상온에서 식힌 후 4℃에서 15분간 3,220 xg로 원심분리하여 얻은 상층액을 Whatman No. 1 여과지로 여과하고, 상기 여과액을 실험 전까지 냉동 보관하였다.Using commercially available peanut, walnut, almond, cashew nut, red bean, soybean, skim milk, and peanut butter, The specificity of the monoclonal antibodies of the present invention was analyzed. Crude protein of peanuts and other types of nuts is prepared by adding 0.5 M sodium chloride solution to finely crushed powder of each material (raw peanut, roasted peanut, boiled peanut, walnut, almond, cashew nut, Was added to a glass beaker, and the mixture was extracted with boiling water for 15 minutes. After cooling at room temperature, the mixture was centrifuged at 3,220 x g for 15 minutes at 4 ° C. 1 filter paper, and the filtrate was stored frozen until the experiment.
4. 단일클론항체의 특성 분석4. Characterization of monoclonal antibodies
4-1. 간접 ELISA 분석4-1. Indirect ELISA analysis
마이크로플레이트 웰(Nunc International, 덴마크)에 50 mM 인산완충식염수 내 5 ㎍의 단백질을 포함하는 각각의 시료 100 ㎕로 37℃에서 1시간 동안 코팅하였다. 플레이트를 PBST(PBS with 0.05% Tween 20)로 3번 세척하였다. 200 ㎕의 블로킹 버퍼(1% skim milk in PBS)를 첨가하여 37℃에서 1시간 동안 블로킹한 후 PBST로 4번 세척하였고, 정제한 단일클론항체 100 ㎕를 각 웰에 첨가하여 37℃에서 1시간 동안 반응시켰다. 웰 내의 반응액을 제거한 후, PBST로 5번 세척한 후, 1:5,000으로 희석시킨 HRP(horseradish peroxidase) 축합 염소 항-마우스 IgG(Thermo Fisher Scientific Inc., 미국)를 각 웰에 100 ㎕씩 첨가하여 37℃에서 1시간 동안 반응시켰다. PBST로 6회 세척한 다음 기질 용액(ABTS 용액)(Sigma, 미국)을 각 웰에 100 ㎕씩 첨가하여 37℃에서 30분 동안 반응시킨 후 405 nm에서 흡광도를 측정하였다.Were coated with 100 μl of each sample containing 5 μg of protein in 50 mM phosphate buffered saline at 37 ° C for 1 hour in Microplate wells (Nunc International, Denmark). Plates were washed 3 times with PBST (PBS with 0.05% Tween 20). After blocking with PBST for 1 hour, 100 μl of the purified monoclonal antibody was added to each well and incubated for 1 hour at 37 ° C. Lt; / RTI > The reaction solution in the wells was removed, washed 5 times with PBST, and 100 쨉 l of HRP (horseradish peroxidase) -conjugated goat anti-mouse IgG (Thermo Fisher Scientific Inc., USA) diluted 1: 5,000 was added to each well And reacted at 37 DEG C for 1 hour. After washing with PBST 6 times, 100 μl of substrate solution (ABTS solution) (Sigma, USA) was added to each well, reacted at 37 ° C for 30 minutes, and the absorbance was measured at 405 nm.
4-2. 웨스턴 블롯4-2. Western blot
SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)는 Laemmli 방법을 변형하여 수행하였다. 시료(웰 당 3 ㎍ 단백질) 및 단백질 마커(웰 당 5 ㎕)(prestained broad range protein maker, Bio-Rad, 미국)는 5% stacking gel(pH 6.8)에 로딩하여 12% separating gel(pH 8.8)에서 분리되었다. 전기영동은 Mini-PROTEAN® tetra electrophoresis cell(Bio-Rad)과 PowerPacTM Basic(Bio-Rad)을 이용하여 (150V에서 1시간 30분) 동안 수행하였다. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed by modifying the Laemmli method. The samples (3 μg per well) and protein markers (5 μl per well) (Prestained Broad Range Protein Maker, Bio-Rad, USA) were loaded on 5% stacking gel (pH 6.8) . Electrophoresis was performed for the Mini-PROTEAN ® tetra electrophoresis cell ( Bio-Rad) and PowerPac TM Basic using (Bio-Rad) (1 hour and 30 minutes at 150V).
전기영동 후 겔은 염색 용액(EZblueTM gel staining reagent, Sigma)을 사용하여 염색하고, 물로 탈색시켰다. 또 다른 겔은 웨스턴 블롯 분석에 사용되었다. 12% separating gel에서 분리된 단백질은 MiniTrans-Blot unit(Bio-Rad)을 이용하여 100V에서 1시간 동안 나이트로셀룰로스 멤브레인(nitrocellulose membrane, Bio-Rad)으로 이동시켰다. 상기 멤브레인은 블로킹 용액(3% skim milk)으로 37℃에서 1시간 동안 블로킹한 후 PBST로 3번 세척하였다. 항체를 생산하는 하이브리도마 세포 배양액(1:4 in 3% skim milk)에 멤브레인을 37℃에서 1시간 동안 반응시키고 PBST로 4번 세척한 후, 1:6,000으로 희석한 염소 항-마우스 IgG 알칼리포스파타제 콘쥬게이트(goat anti-mouse IgG alkaline phosphatase conjugate, Bio-Rad)에 반응시켰다. 상기 멤브레인은 ECL(PerkinElmer, 미국)을 첨가하여 발광시킨 후 이미지 분석 장치를 통해 확인하였다. 또한, 본 발명의 단일클론항체가 Ara h1에 결합하는지 분석하기 위해, 시판되는 Ara h1 단백질(20 μg/ml, INDOOR Biotechnologies, 미국)을 구매하여 결합력을 분석하였다.After electrophoresis, the gel was stained with a staining solution (EZblue ™ gel staining reagent, Sigma) and discolored with water. Another gel was used for western blot analysis. The proteins separated from the 12% separating gel were transferred to a nitrocellulose membrane (Bio-Rad) for 1 h at 100 V using a MiniTrans-Blot unit (Bio-Rad). The membrane was blocked with blocking solution (3% skim milk) at 37 ° C for 1 hour and then washed 3 times with PBST. The membrane was incubated at 37 ° C for 1 hour in a hybridoma cell culture (1: 4 in 3% skim milk) producing antibody, washed 4 times with PBST, and then incubated with a goat anti-mouse IgG alkali And reacted with a goat anti-mouse IgG alkaline phosphatase conjugate (Bio-Rad). The membrane was illuminated by adding ECL (PerkinElmer, USA) and confirmed by an image analyzer. In order to analyze whether the monoclonal antibody of the present invention binds to Ara h1, commercially available Ara h1 protein (20 μg / ml, INDOOR Biotechnologies, USA) was purchased and the binding force was analyzed.
실시예 1. 면역원으로서의 땅콩 내 열 안정성 수용성 단백질(thermal stable-soluble protein) 준비 Example 1. Preparation of a thermal stable-soluble protein in a peanut as an immunogen
땅콩 내 열 안정성 수용성 단백질의 존재는 SDS-PAGE로 확인하였으며, 항원성은 열 안정성 수용성 단백질의 마우스 면역 후 항-혈청 역가 측정으로 확인하였다. The presence of water-soluble water-soluble proteins in peanuts was confirmed by SDS-PAGE and antigenicity was confirmed by anti-serum titers after mouse immunization of thermostable soluble proteins.
SDS-PAGE로 비열처리 및 열처리 방법으로 추출된 땅콩 내 단백질의 발현 양상을 분석한 결과, 비열처리한 땅콩 시료의 단백질(레인 2) 발현과 열처리한 땅콩 시료의 단백질(레인 3) 발현은 큰 차이를 보이지 않았으며, 기타 모든 시료에서 15~100 kDa 범위에서 주 단백질 밴드가 나타난 것을 확인하였다. 이를 통해, 열처리 방법으로 추출된 땅콩 내에 열 안정성 수용성 단백질이 존재함을 확인하였다(도 1).The protein expression (lane 2) of the non-heat-treated peanut samples and the protein (lane 3) expression of the heat-treated peanut samples were significantly different from those of the non-heat treated peanuts by SDS-PAGE analysis And in all other samples, the main protein band appeared in the range of 15-100 kDa. As a result, it was confirmed that a thermostable water-soluble protein was present in peanuts extracted by the heat treatment method (FIG. 1).
또한, 0.5 M 염화나트륨 용액을 시료의 중량 대비 2배 혼합하여 유리 비이커에 넣고, 끓는 물에서 15분간 중탕하여 추출된 땅콩 추출물을 3마리의 마우스에 면역하여 높은 항체가를 나타내는 마우스를 선별하였다(도 2).In addition, a 0.5 M sodium chloride solution was mixed with 2 times the weight of the sample, put in a glass beaker, and the peanut extract extracted by boiling in boiling water for 15 minutes was immunized with 3 mice to select mice showing high antibody titers 2).
실시예 2. 땅콩 내 열 안정성 수용성 단백질에 대한 단일클론항체Example 2. Thermal stability in peanuts Monoclonal antibodies to soluble proteins
땅콩 내 열 안정성 수용성 단백질에 대한 본 발명의 BP 3A1-12 및 RP 4C12-10 항체 특이성을 분석하기 위해 웨스턴 블롯과 ELISA를 실시하였다.Western blot and ELISA were performed to analyze the BP 3A1-12 and RP 4C12-10 antibody specificities of the present invention against heat stable water soluble proteins in peanuts.
웨스턴 블롯 분석 결과(도 3A, B) 구운 땅콩(레인 2), 삶은 땅콩(레인 3), 땅콩 버터(레인 4) 및 대두(레인 9)에서 20~100 kDa 가량의 단백질 밴드가 관찰되었지만, 땅콩 시료에서 대두 시료에 비해 더 강한 반응을 나타내었으며, ELISA 분석 결과(도 3C)에서는 땅콩 시료를 제외한 월넛(walnut), 아몬드(almond), 캐슈넛(cashew nut), 팥(red bean), 대두(soybean) 및 탈지유(skim milk)에서 전혀 교차반응이 일어나지 않는 것을 확인하였다. 따라서 본 발명의 항체가 시료 내 땅콩의 혼입 여부를 판별하는데 사용 가능하다는 것을 확인하였다.Western blot analysis (Fig. 3A, B) Protein bands of 20-100 kDa were observed in roasted peanuts (lane 2), boiled peanuts (lane 3), peanut butter (lane 4) and soybean The results of the ELISA analysis (Fig. 3C) showed that the walnut, almond, cashew nut, red bean, soybean (soybean) ) And skim milk at the same time. Therefore, it was confirmed that the antibody of the present invention can be used to determine whether or not the sample contains peanuts.
상기 웨스턴 블롯 결과, 대두 시료에서 약한 교차반응성이 나타났으나, ELSIA 결과에서는 교차반응이 전혀 일어나지 않은 이유로는, 웨스턴 블롯의 경우 단백질의 3차 구조를 1차 구조로 변형시켜 목적 단백질의 발현을 확인하는 것이지만, ELISA의 경우 단백질 본연의 3차 구조에 대한 친화성을 확인하는 것이기 때문에 웨스턴 블롯과 ELISA의 분석 결과에 일부 차이가 있는 것으로 유추되었다.As a result of the western blot, weak cross-reactivity was observed in the soybean sample. However, in the case of Western blot, the ELISA results showed that the Western blot transformed the tertiary structure of the protein into the primary structure and confirmed the expression of the target protein However, since ELISA confirms the affinity for the tertiary structure of the protein, it is deduced that there is some difference in the results of Western blotting and ELISA analysis.
또한, 도 4에 개시된 바와 같이 항체가 반응하는 단백질의 위치를 비교해 볼 때 본 발명의 3A1-12 항체는 Ara h1에 반응하는 항체인 것으로 확인되었다. 현재 알려진 Ara h1의 분자량은 66 kDa이므로 본 발명의 항체에 결합하는 타겟 단백질은 Ara h1으로 확인되었다. In addition, when comparing the positions of the proteins to which the antibody reacts, as shown in Fig. 4, the 3A1-12 antibody of the present invention was confirmed to be an antibody reactive with Ara h1. Since the molecular weight of Ara h1 currently known is 66 kDa, the target protein binding to the antibody of the present invention is identified as Ara h1.
실시예 3. 가공식품 내 땅콩 혼입 유무 확인Example 3. Confirmation of peanut contamination in processed foods
본 발명의 BP 3A1-12 및 RP 4C12-10 항체를 이용하여 하기 표 1의 가공식품 내 땅콩의 혼입 유무를 확인하기 위해 간접 ELISA를 실시하였다.Indirect ELISA was performed using the BP 3A1-12 and RP 4C12-10 antibodies of the present invention to determine the presence or absence of peanuts in the processed foods of Table 1 below.
가공식품의 구성 성분Components of processed foods
종류Kinds 주요 성분main ingredient
국희샌드National sand 땅콩 가루, 땅콩 버터, 대두Peanut flour, peanut butter, soybean
마가렛트Margaret 땅콩, 대두Peanut, soybean
버터링Buttering 대두Big head
그레이스Grace 땅콩 페이스트Peanut paste
에이스ace --
엄마손 파이Mommy pie 대두유Soybean oil
땅콩 전병Peanut cracker 땅콩, 대두(마가린)Peanuts, soybeans (margarine)
꼬마포 땅콩구이Grilled chicken egg and peanut 땅콩peanut
피넛 월남쌈 소스Peanut seasoning 땅콩peanut
두유soy milk 땅콩 페이스트, 대두Peanut paste, soybean
그 결과, 도 5에 나타난 바와 같이 본 발명의 BP 3A1-12 및 RP 4C12-10 항체는 열처리된 구운 땅콩(roasted peanut), 삶은 땅콩(boiled peanut) 및 땅콩 버터(peanut butter)에 반응할 뿐만 아니라, 상기 표 1의 땅콩을 주요 성분으로 포함하고 있는 가공식품에서만 특이적인 반응을 나타내었으며, 특히 시료의 농도가 1/10로 희석되어도 검출될 수 있다는 것을 확인하였다. As a result, as shown in Fig. 5, the BP 3A1-12 and RP 4C12-10 antibodies of the present invention not only reacted to roasted peanut, boiled peanut and peanut butter, , Only the processed foods containing the peanut of Table 1 as a main component showed a specific reaction. In particular, it was confirmed that even when the concentration of the sample is diluted to 1/10, it can be detected.
실시예 4. 단일클론항체의 민감도 분석Example 4. Sensitivity analysis of monoclonal antibodies
다양한 땅콩 추출물 함량(0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100%, v/v)에 따라 본 발명의 BP 3A1-12 및 RP 4C12-10 항체의 민감도(sensitivity)가 어떠한지를 간접 ELISA를 통해 측정하였다.The sensitivity of the BP 3A1-12 and RP 4C12-10 antibodies of the present invention was determined according to various peanut extract contents (0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100% Were measured by indirect ELISA.
그 결과, 구운 땅콩, 삶은 땅콩 및 땅콩 버터의 추출물에서 0.0001%(w/v)의 농도까지 본 발명의 항체가 땅콩 단백질을 검출할 수 있음을 확인하였다(도 6). 이를 통해, 본 발명에서 개발된 하이브리도마 세포에서 생산된 단일클론항체는 땅콩 단백질, 특히 열 안정성 단백질에 매우 특이적으로 반응함을 알 수 있었고, 열처리를 비롯 다양한 식품 첨가물이 가미된 가공식품에서 땅콩 혼입 여부를 신속하게 판별할 수 있어, 추후 면역센서 또는 바이오 센서 개발에 유용하게 사용될 것으로 예측되었다.As a result, it was confirmed that the antibody of the present invention can detect the peanut protein at a concentration of 0.0001% (w / v) in the roasted peanut, boiled peanut and peanut butter extract (FIG. 6). As a result, the monoclonal antibody produced in the hybridoma cells developed in the present invention reacted very specifically with the peanut protein, particularly the heat-stable protein, and it was found that the processed food containing various food additives, It is predicted that it will be useful for the future development of immune sensor or biosensor since it can quickly discriminate the presence of peanuts.
[수탁번호][Access number]
기탁기관명 : 한국생명공학연구원Institution name: Korea Biotechnology Research Institute
수탁번호 : KCTC13295BPAccession number: KCTC13295BP
수탁일자 : 20170704Checked on: 20170704

Claims (8)

  1. 땅콩 유래 Ara h1(Arachis hypogaea 1)을 포함하는 식품 내 땅콩 혼입 여부 판별용 마커.Marker for determining the presence of peanuts in food, including peanut-derived Ara h1 (Arachis hypogaea 1).
  2. 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 조성물.A composition for discriminating the presence or absence of peanut in a food comprising an antibody specifically binding to peanut-derived Ara h1.
  3. 제2항에 있어서, 상기 항체는 단일클론항체, 다클론항체, 다중특이적 항체, 항체의 단편, 재조합 항체 및 화학적으로 수식된 항체로 구성된 군에서 선택된 하나 이상인 것을 특징으로 하는 식품 내 땅콩 혼입 여부 판별용 조성물.3. The method according to claim 2, wherein the antibody is at least one selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a fragment of an antibody, a recombinant antibody, and a chemically modified antibody. / RTI >
  4. 제3항에 있어서, 상기 단일클론항체는 기탁번호가 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산되는 것을 특징으로 하는 식품 내 땅콩 혼입 여부 판별용 조성물.The composition according to claim 3, wherein the monoclonal antibody is produced by a hybridoma cell line having a deposit number of KCTC 13295BP.
  5. 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 포함하는 식품 내 땅콩 혼입 여부 판별용 키트.A kit for determining the presence or absence of peanuts in a food comprising an antibody specifically binding to peanut-derived Ara h1.
  6. 땅콩 유래 Ara h1에 특이적으로 결합하는 항체를 검체 시료와 접촉시켜 형성된 항원-항체 복합체를 검출하는 단계를 포함하는 식품 내 땅콩 혼입 여부의 판별방법.And detecting an antigen-antibody complex formed by contacting an antibody specifically binding to a peanut-derived Ara h1 with a test sample to determine whether or not the peanut is present in the food.
  7. 기탁번호가 KCTC 13295BP인 하이브리도마 세포주에 의하여 생산되고, 땅콩 유래 Ara h1에 특이적으로 결합하는 단일클론항체.Monoclonal antibody produced by a hybridoma cell line with accession number KCTC 13295BP and specifically binding to pean-derived Ara h1.
  8. 제7항의 단일클론항체를 생산하는 기탁번호가 KCTC 13295BP인 하이브리도마 세포주.A hybridoma cell line, wherein the accession number for producing the monoclonal antibody of claim 7 is KCTC 13295BP.
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