KR102349848B1 - Monoclonal antibody specific to Ara h 1 and Ara h 3 from peanut and uses thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody specific to peanut-derived Ara h 1 and Ara h 3 and uses thereof, and is produced by a hybridoma cell line having an accession number of KCTC 14185BP, and Ara h 1, a heat-stable water-soluble protein derived from peanuts. (Arachis hypogaea 1) and to a monoclonal antibody that specifically binds to Ara h 3.

Description

Peanut-derived monoclonal antibody specific to Ara h 1 and Ara h 3 and uses thereof {Monoclonal antibody specific to Ara h 1 and Ara h 3 from peanut and uses thereof}

The present invention relates to peanut-derived monoclonal antibodies specific for Ara h 1 and Ara h 3 and uses thereof.

Food allergy is caused by an immunoglobulin E (IgE)-mediated or non-immunoglobulin-mediated hypersensitivity reaction to dietary protein. It is a relatively common allergic disease that exhibits various symptoms, and although it is a harmless food to normal people, it refers to an excessive immune response of the human body to a specific protein in the food when a specific person ingests it. The incidence of food allergy is reported to be about 8% of children under 3 years of age and about 2% of adults. Although the amount of food that a person consumes in a lifetime amounts to about 2-3 tons and is exposed to a wide variety of foods, the human gastrointestinal immune system has evolved to develop immune tolerance to most dietary proteins, In very limited types of food, sensitization to dietary protein occurs and ultimately allergic symptoms.

Food allergen is defined as a food ingredient that induces an immediate hypersensitivity reaction by inducing the production of immunoglobulin E. In addition to food taken by mouth, inhalation and contact food allergens are also included. Most of the major allergens in food are water-soluble glycoproteins with a molecular weight of about 10,000-60,000 Da. Unlike inhaled allergens, they are likely to be transformed by digestive enzymes, bacteria, and toxins while passing through the gastrointestinal tract, and stay in the intestinal mucosa for a long time. As well as anaphylaxis reactions, there are many cases that cause a delayed type reaction. Common foods that cause allergic reactions due to immunoglobulin E include eggs, milk, soybeans, peanuts, nuts, buckwheat, wheat, fish, and seafood. In addition, adverse reactions may occur due to food preservatives, spices, coloring, etc., but these cases are not due to an immunological mechanism and should be treated separately. In the case of children, milk, egg, peanut, soybean, and wheat are the most common food allergens, and more than 90% of patients are sensitized. do. There is no specific prevention and treatment method for food allergy, and the surest and only way to prevent it is to eliminate the food that causes food allergy from the diet and not consume it.

Therefore, it is very important to analyze the food that causes food allergy, and through this, it is important to check in advance not only the raw material of the food but also the allergen-inducing ingredient in the manufactured/processed processed food accurately.

Accordingly, in the present invention, a hybridoma cell capable of producing a monoclonal antibody capable of simultaneously specifically binding Ara h1 and Ara h3, which are representative allergens of peanut, was prepared, and the monoclonal antibody produced from the hybridoma cell was prepared. It was confirmed that it was possible to quickly analyze whether peanuts were mixed in a mixture of raw and sub-materials of food heat-treated and processed food.

On the other hand, Japanese Patent No. 3853692 discloses 'antibody, test method, and test kit for testing peanut ingredients', and Korean Patent No. 1223256 discloses 'a method for reducing peanut allergy and peanut allergy' Although this is disclosed, peanut-derived Ara h 1 and Ara h 3 specific monoclonal antibodies and uses thereof of the present invention are not described.

The present invention was derived from the above needs, and the present inventors used a heat-stable water-soluble protein of peanut as an antigen, and a hybridoma strain that produces a monoclonal antibody that specifically binds to heat-stable water-soluble protein of peanut was prepared. Among the prepared hybridoma cell lines, a cell line producing a monoclonal antibody capable of simultaneously detecting Ara h 1 and Ara h 3 could be selected, and immunoassay was performed using the monoclonal antibody produced from the hybridoma cell line. As a result, compared to the monoclonal antibody capable of detecting Ara h 1 or Ara h 3 alone, the monoclonal antibody of the present invention can quickly and accurately determine the presence or absence of peanut incorporation up to a lower concentration sample By confirming, the present invention was completed.

In order to solve the above problems, the present invention provides a monoclonal antibody that is produced by a hybridoma cell line with accession number KCTC 14185BP and specifically binds to peanut-derived Ara h 1 and Ara h 3 .

In addition, the present invention provides a hybridoma cell line having an accession number of KCTC 14185BP.

In addition, the present invention provides a composition for determining whether peanuts are mixed in food comprising a monoclonal antibody that specifically binds to Ara h 1 and Ara h 3 derived from peanuts.

In addition, the present invention provides a method for determining whether peanuts are incorporated in food, comprising the step of detecting an antigen-antibody complex formed by contacting a monoclonal antibody that specifically binds to peanut-derived Ara h 1 and Ara h 3 with a specimen sample. to provide.

In addition, the present invention provides a kit for determining whether peanuts in food containing a monoclonal antibody that specifically binds to Ara h 1 and Ara h 3 derived from peanuts.

The monoclonal antibody of the present invention can simultaneously specifically detect Ara h 1 and Ara h 3 among heat-stable water-soluble proteins in peanuts, and effectively detects whether peanuts mixed in intentionally unlabeled food manufactured and processed by heat treatment can be analyzed. Therefore, the monoclonal antibody of the present invention can prevent food allergy induction by providing information on whether or not peanuts are mixed in food to consumers, and can be used to improve food manufacturing and processing processes, thereby contributing to food stability. It is expected that there will be

1 is a SDS-PAGE result of heat-stable protein extracted from non-heat-treated (Non-Heat) and heat-treated (Heat) peanuts. Lane 1: Size marker, Lane 2: Raw peanut sample extracted by non-heat treatment, Lane 3: Raw peanut sample extracted by heat treatment, Lane 4: Boiled peanut sample extracted by non-heat treatment, Lane 5: Boiled peanut sample extracted by heat treatment, Lane 6: Roasted peanut sample extracted by non-heat treatment, Lane 7: Roasted peanut sample extracted by heat treatment, Lane 8: Peanut jam sample extracted by non-heat treatment, Lane 9: Peanut jam sample extracted by heat treatment.
Figure 2 is a Western blot analysis of the reaction specificity of the RP Mab GNU-10, RP Mab GNU-23 and RP Mab GNU-30 antibodies to the peanut extract sample. M: size marker, peanut: roasted peanut sample extracted by non-heat treatment method.
3 is an indirect ELISA result of analyzing the sensitivity of RP Mab GNU-10, RP Mab GNU-23 and RP Mab GNU-30 using a plate coated with various concentrations of raw peanut and roasted peanut extract.
Figure 4 is an indirect ELISA result of analyzing the sensitivity (sensitivity) of RP Mab GNU-10, RP Mab GNU-23 and RP Mab GNU-30 using a plate coated with boiled peanuts and peanut butter extracts of various concentrations.
5 is an indirect ELISA result confirming the detection limit of the monoclonal antibody RP Mab GNU-10.
6 is an indirect ELISA result of analyzing the specificity of the monoclonal antibody RP Mab GNU-10.

In order to achieve the object of the present invention, the present invention provides a monoclonal antibody that is produced by a hybridoma cell line having an accession number of KCTC 14185BP and specifically binds to peanut-derived Ara h 1 and Ara h 3 .

Peanut ( Arachis hypogaea ) is one of the food allergens that can cause severe systemic symptoms and fatal anaphylaxis. Eight protein antigens from peanuts are known from Ara h 1 to Ara h 8, and the most dominant major antigens are known as Ara h 1, Ara h 2 and Ara h 3. These allergens are vicilin, conglutin, and glycinin, respectively, which are seed storage proteins. In 90% of peanut allergy patients, Ara h 1 and Ara h 2 specific IgE was detected, and Ara h 3 was found to be positive in about 45% of patients. According to the study results of Yapo-Crezoit et al. (IOSR Journal of Pharmacy and Biological Sciences (2017) 12(1):31-36), in raw peanut extract, Ara h 1 was about 60-75 kDa and Ara h 2 was 17, 20 kDa, and Ara h 3 are known to correspond to 25, 36, 40, 44 kD.

As used herein, the term "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Monoclonal antibodies are also called monoclonal antibodies, monoclonal antibodies, or monoclonal antibodies. Unlike polyclonal antibodies, which typically include different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are produced by hybridoma culture.

The monoclonal antibody produced by the hybridoma can be used without purification, but in order to obtain the best results, it is purified with high purity (eg, 95% or more) according to a method well known in the art to which the present invention belongs. It is preferable to do As such a purification technique, for example, gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, etc. may be used to separate from the culture medium or ascites fluid.

The monoclonal antibody of the present invention is produced from the KCTC 14185BP hybridoma cell line RP MAb GNU 10 deposited with the Center for Biological Resources, Korea Research Institute of Bioscience and Biotechnology on May 12, 2020.

The antibody of the present invention comprises the steps of (1) mixing peanuts with 0.1 M carbonate buffer in a weight ratio of 1:10; (2) reacting the peanut mixed with the carbonate buffer in boiling water for 15 minutes to extract heat-stable water-soluble protein from the peanut; (3) dialyzing the extracted heat-stable water-soluble protein in a phosphate buffer and then injecting it into the mouse; (4) forming an antibody in the mouse against the injected heat-stable water-soluble protein; And (5) using the antibody-formed mouse splenocytes to develop hybridoma cells for monoclonal antibody production; monoclonal antibodies specific to heat-stable water-soluble proteins Ara h 1 and Ara h 3 of peanuts comprising; Characterized by the manufacturing method.

The present invention also provides a hybridoma cell line having an accession number of KCTC 14185BP that produces monoclonal antibodies specific for the heat-stable water-soluble proteins Ara h 1 and Ara h 3 of the peanut.

The present invention also provides a composition for determining whether peanuts in food containing the monoclonal antibody. The composition includes a monoclonal antibody produced by a hybridoma cell line having an accession number KCTC 14185BP, which specifically binds to peanut-derived Ara h 1 and Ara h 3 at the same time as an active ingredient, and food using the antibody It is possible to determine whether or not my peanuts are mixed.

The food may include all foods that contain or are expected to contain peanuts, but are not limited thereto, but may be processed products such as raw peanuts, roasted peanuts, boiled peanuts, fried peanuts or buttered peanuts. The buttered peanuts refer to products processed like jam generally marketed in the United States.

The present invention also includes the step of detecting the antigen-antibody complex formed by contacting a monoclonal antibody specific for the heat-stable water-soluble proteins Ara h 1 and Ara h 3 of the peanut with a sample sample, Determination of whether peanuts are incorporated in food provide a way

As used herein, the term "antigen-antibody complex" refers to a combination of the peanut heat-stable water-soluble proteins Ara h 1 and Ara h 3 in the sample and the monoclonal antibody according to the present invention recognizing them, and such antigen-antibody complex is a colormetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual assessment and a scintillation counting method. It can be detected by any method selected from the group consisting of. However, it is not necessarily limited to these, and various applications and applications are possible.

In the discrimination method of the present invention, various markers can be used to detect antigen-antibody complexes. Specific examples of the monoclonal antibody label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes, but are not necessarily limited thereto. Preferred enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase or β-lactamase, and preferred fluorescent substances include quantum dots, fluoro Rescein, Eu ³⁺ , Eu ³⁺ chelates or cryptates, preferred ligands are biotin derivatives, preferred phosphors are acridinium esters or isoluminol derivatives, preferred microparticles are colloidal gold or There is a colored latex, and preferred radioactive isotopes include ^{, but are not limited to, 7} Co, ³ H, ¹²⁵ I or ¹²⁵ I-Bolton Hunter's reagent.

The method for detecting the antigen-antibody complex is preferably, but not limited to, enzyme-linked immunosorbent assay (ELISA). The enzyme immunosorbent method is a direct ELISA using an antibody recognizing an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody recognizing a capture antibody in a complex of an antibody recognizing an antigen attached to a solid support, and attached to a solid support Direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen, a label that recognizes the antibody after reacting with another antibody that recognizes an antigen in a complex of an antibody attached to a solid support and antigen It includes various ELISA methods such as indirect sandwich ELISA using a secondary antibody. The antibody may have a detection label, and when it does not have a detection label, these antibodies can be captured, and can be identified by treating another antibody having a detection label.

The present invention also provides a kit for determining whether peanuts in food containing the monoclonal antibody according to the present invention.

In the kit of the present invention, the monoclonal antibody may be a monoclonal antibody produced by a hybridoma cell line having an accession number of KCTC 14185BP that specifically binds peanut-derived Ara h 1 and Ara h 3 at the same time.

Kit systems of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatographic and radiation division immunoassay devices, flow-through devices, and the like.

The kit of the present invention is not limited thereto, as long as it is commonly used in kits, for example, a solid-phase support if using ELISA; monoclonal antibodies of the present invention; and an enzyme-associated immunoassay (ELISA) reaction solution containing an enzyme-labeled antibody solution for reaction with an antigen and a chromogenic solution showing an enzyme reaction.

In the present invention, 'sensitivity' is the probability that the test result is actually positive among positive samples (samples mixed with peanuts) in the discriminant test, and is an index indicating how well the discriminant test selects positive samples.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Immunogen Preparation

A thermal stable-soluble protein (TSSP) extracted from peanuts was used as an immunogen. Specifically, 0.1 M carbonate buffer was added to raw peanuts to finely ground raw peanuts. The mixture was mixed 10 times by weight, put in a glass beaker, and extracted by boiling in boiling water for 15 minutes. After cooling at room temperature, the supernatant was centrifuged at 3,220 x g at 4°C for 15 minutes. 1 was filtered with a filter paper, and the protein was extracted by dialyzing for 3 days while changing the phosphate buffered saline (PBS, pH 7.4) solution every day. The dialyzed crude protein extract was used as an immunogen. The protein concentration of the crude extract was measured using Quick start ^TM Bradford protein assay dye (Bio-Rad Laboratories Inc., USA).

2. Production of monoclonal antibodies against peanut protein

The prepared crude protein extract (100 μg/100 μl PBS) was emulsified with the same amount (100 μl) of Freund's complete adjuvant, and the emulsified immunogen (200 μl/mouse) was added to 7-week-old BALB/c female mice. Injected intraperitoneally. After that, the crude protein extract was emulsified with Freund's incomplete adjuvant at intervals of 2 weeks, and the second and third injections were performed, and the crude protein extract (200 μg/200 μl PBS) was injected 4 days before cell fusion. . After the fourth injection, serum was collected from the caudal vein and the antibody titer was confirmed by ELISA.

Spleen cells of immunized mice exhibiting high antibody titers were fused with SP2/0 myeloma cells, a type of cancer cell. The general procedure of cell fusion was performed with a slight modification from the Kohler and Milsten method. After fusion of splenocytes (2×10 ⁸ ) and SP2/0 myeloma cells (2×10 ⁷ ) using 1 ml of 50% polyethylene glycol (PEG) 1500 solution, hypoxanthine, ami Using HAT medium containing nopterin (aminopterin) and thymidine (thymidine), only the fused hybridoma cells were selectively cultured and proliferated. The absorbance of the culture supernatant of the hybridoma was measured by an indirect enzyme linked immunosorbent assay (ID-ELISA) method using a peanut antigen-coated plate, and a monoclonal antibody specific to the heat-stable water-soluble protein in peanuts. , MAb) was analyzed, and ELISA-positive hybridoma cells were separated by an unlimited dilution method to select a hybridoma cell line that produced a peanut protein-specific MAb.

In order to produce a monoclonal antibody from the hybridoma cell line, 0.5 ml of pristine was intraperitoneally pretreated in BALB/c mice for 7 to 10 days, and then hybridoma cells (1×10 ⁷ ) were injected into the mouse. Ascites was produced by injection into the abdominal cavity. The produced ascites was collected and purified by ammonium sulfate precipitation, dialyzed with phosphate-buffered saline for 3 days, freeze-dried, and stored at -20°C for use in the experiment.

3. Preparation of Western Blot Samples

Using commercially available peanuts, walnuts, almonds, cashew nuts, red beans, soybeans, skim milk and peanut butter The specificity of the monoclonal antibody of the present invention was analyzed. Crude protein from peanuts and other types of nuts was added to a finely ground powder of each ingredient (raw peanut, roasated peanut, boiled peanut, walnut, almond, cashew, red bean and soybean) in 0.1 M carbonate buffer. was mixed at 10 times the amount of each powder sample, put in a glass beaker, extracted by boiling in boiling water for 15 minutes, cooled at room temperature, and centrifuged at 3,220 x g for 15 minutes at 4°C. 1 was filtered with filter paper, and the filtrate was stored frozen until the experiment. Peanut butter was purchased and used commercially, and the extraction method was performed in the same manner as above.

4. Characterization of monoclonal antibodies

4-1. Indirect ELISA analysis

100 μl of each sample containing the protein in 0.1 M carbonate buffer was put into a microplate well (Nunc International, Denmark), and then coated at 37° C. for 1 hour. Plates were washed 3 times with PBST (PBS with 0.05% Tween 20). After adding 200 μl of blocking buffer (1% skim milk in PBS) and blocking at 37° C. for 1 hour, it was washed 4 times with PBST, and the monoclonal antibody purified by ammonium sulfate precipitation method was diluted at a ratio of 1:10,000. Then, 100 μl was added to each well and reacted at 37° C. for 1 hour. After removing the reaction solution from the wells, after washing 5 times with PBST, 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Thermo Fisher Scientific Inc., USA) diluted 1:10,000 in each well was added and reacted at 37°C for 1 hour. After washing 6 times with PBST, 100 μl of a substrate solution (ABTS solution) (Sigma, USA) was added to each well, reacted at 37° C. for 30 minutes, and absorbance was measured at 405 nm.

4-2. western blot

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed by modifying the Laemmli method. Samples (3 μg protein per well) and protein markers (5 μl per well) (prestained broad range protein maker, Bio-Rad, USA) were loaded on a 5% stacking gel (pH 6.8) to a 12% separating gel (pH 8.8) was separated from Electrophoresis was performed at 150V for 1 hour and 30 minutes using a Mini-PROTEAN ^® tetra electrophoresis cell (Bio-Rad) and PowerPac ^{TM Basic (Bio-Rad).}

After electrophoresis, the gel was stained with a staining solution (EZblue ^TM gel staining reagent, Sigma) and decolorized with water. Another gel was used for Western blot analysis. Proteins separated on a 12% separating gel were transferred to a nitrocellulose membrane (Bio-Rad) at 100V for 1 hour using a MiniTrans-Blot unit (Bio-Rad). The membrane was blocked with a blocking solution (3% skim milk) at 37° C. for 1 hour, and then washed 3 times with PBST. The membrane was reacted with an antibody-producing hybridoma cell culture medium (1:4 in 3% skim milk) at 37° C. for 1 hour, washed 4 times with PBST, and then diluted at 1:6,000 with goat anti-mouse IgG alkali. It was reacted with a phosphatase conjugate (goat anti-mouse IgG alkaline phosphatase conjugate, Bio-Rad). The membrane was luminescent by adding ECL (PerkinElmer, USA), and then confirmed through an image analysis device.

Example 1. Preparation of thermal stable-soluble protein in peanut as an immunogen

As a result of analyzing the expression pattern of protein in peanuts extracted by non-heat treatment and heat treatment by SDS-PAGE, the protein (lane 2) expression of the non-heat-treated peanut sample and the protein (lane 3) expression of the heat-treated peanut sample (lane 3) were significantly different was not observed, and it was confirmed that the main protein band appeared in the range of 15 to 100 kDa in all other samples. Through this, it was confirmed that the heat-stable water-soluble protein was present in the peanut extracted by the heat treatment method (FIG. 1).

Example 2. Monoclonal antibody against heat-stable water-soluble protein in peanut

A 0.1 M carbonate buffer was mixed 10 times with respect to the weight of the sample, put in a glass beaker, and three mice were immunized with peanut extract extracted by boiling in boiling water for 15 minutes to select mice exhibiting high antibody titer. Western blot was performed to analyze the specificity of the selected hybridoma clones with respect to the heat-stable water-soluble protein in peanuts by fusing splenocytes and cancer cells extracted from the selected mice.

As a result of Western blot analysis, it was possible to confirm the antibodies reacting with the heat-stable protein of peanuts, and the location of the protein reacting with the antibody was previously reported (Yapo-Crezoit et al., (2017) IOSR Journal of Pharmacy and Biological Sciences 12(1) ):31-36), the RP Mab GNU-10 antibody responds to Ara h 1 and Ara h 3, the RP Mab GNU-23 antibody to Ara h 1, and the RP Mab GNU-30 antibody to Ara h 1 It was confirmed to respond specifically to Ara h 3 (FIG. 2). In particular, whereas the RP Mab GNU-30 antibody showed a specific response only to Ara h 3 of the size of 36 kDa, the RP Mab GNU-10 antibody responded to Ara h3 of 25, 36, and 40 kDa to Ara h 3 It was predicted that the sensitivity would be excellent.

Example 3. Sensitivity analysis of monoclonal antibodies

Various peanut extract contents (100% (3 mg/ml), 1%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002% 0.001%, 0.0005%, 0.0002%, 0% v/v) Accordingly, the sensitivity of RP Mab GNU-10, RP Mab GNU-23 and RP Mab GNU-30 of the present invention was measured through indirect ELISA. Each of the monoclonal antibodies was purified by ammonium sulfate precipitation method, and the antibody (1 mg/ml) was diluted with PBS (pH 7.4) at a ratio of 1:10,000 and then used.

As a result, the RP Mab GNU-10 antibody capable of simultaneously detecting Ara h 1 and Ara h 3 in both raw peanut, roasted peanut, boiled peanut, and peanut butter extract samples could detect Ara h 1 or Ara h 3 alone. It showed significantly superior sensitivity compared to the Mab GNU-23 and RP Mab GNU-30 antibodies ( FIGS. 3 and 4 ). As a result of comparison with the absorbance of the untreated control group (0%), the RP Mab GNU-10 antibody was able to detect raw peanut, roasted peanut and boiled peanut extract up to 0.001% content, and peanut butter extract was detectable up to 0.0002% content. confirmed to be possible. Through this, it was found that the monoclonal antibody produced from the RP Mab GNU-10 hybridoma cells developed in the present invention reacted very specifically to the heat-stable protein of peanuts.

Example 4. Confirmation of detection limit of monoclonal antibody RP Mab GNU-10

The limit of detection (LOD) was analyzed using RP Mab GNU-10, which was confirmed to have excellent specificity to the heat-stable protein of peanuts through Example 3. The analysis method is indirect ELISA after preparing peanut extract (3 mg/ml) with 0.01%, 0.003%, 0.001%, 0.0003%, 0.0001%, 0.00003%, 0.00001%, 0.000003%, 0.000001%, 0% (v/v) was performed. The RP Mab GNU-10 antibody was used by dilution at a ratio of 1:100, 1:200, 1:500 and 1:1,000. As a result, it was confirmed that the LOD of the monoclonal antibody RP Mab GNU-10 was 0.000001%, and the LOD value was the same regardless of the dilution factor of the antibody, and the heat stable protein in the peanut extract of RP Mab GNU-10 of the present invention. It was found that it reacts very sensitively to even a very small amount of (FIG. 5).

In addition, as a result of confirming the reactivity of the monoclonal antibody RP Mab GNU-10 to nuts other than peanuts by indirect ELISA, walnut, almond, cashew nut, red bean, soybean ) and skim milk extract samples, the reaction results were not confirmed, indicating that the specificity of the monoclonal antibody RP Mab GNU-10 was very good (FIG. 6). Through this, it can be seen that the monoclonal antibody produced from the RP Mab GNU-10 hybridoma cells developed in the present invention reacts with high specificity and sensitivity to the heat-stable protein of peanuts. It was predicted to be usefully used in the development of immune sensors or biosensors.

Korea Institute of Biotechnology and Biotechnology KCTC14185BP 20200512

Claims (7)

A monoclonal antibody that is produced by a hybridoma cell line with an accession number of KCTC 14185BP and specifically binds to peanut-derived Ara h 1 (Arachis hypogaea 1) and Ara h 3. A hybridoma cell line whose accession number is KCTC 14185BP for producing the monoclonal antibody of claim 1. A composition for determining whether peanuts are mixed in food comprising the monoclonal antibody of claim 1. A method for determining whether peanuts are mixed in food, comprising the step of detecting an antigen-antibody complex formed by contacting the monoclonal antibody of claim 1 with a sample sample. 5. The method of claim 4, wherein the detection of the antigen-antibody complex is a colormetric method, an electrochemical method, a fluorimetric method, a luminometry, a particle counting method, a visual measurement method. (visual assessment) and scintillation counting method (scintillation counting method), characterized in that carried out by any one method selected from the group consisting of peanuts in the food, characterized in that the determination method. A kit for determining whether peanuts are mixed in food containing the monoclonal antibody of claim 1. According to claim 6, wherein the monoclonal antibody is an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle and any one of a label selected from the group consisting of radioactive isotopes, characterized in that the labeled with peanuts in the food to determine whether the incorporation of the monoclonal antibody for kit.

Images