KR20110115742A - Marker proteins for identifying hempseed and an identification kit for hempseed - Google Patents

Abstract

The present invention relates to a marker that can distinguish blue ginseng seed and complementary seed among hemp seeds, and more particularly, a simple method of blue ginseng seed having low tetrahydrocanabinol content and high tetrahydrocanabinol content as an anesthetic component. It relates to a marker that can be divided into a kit using the same.

Description

Marker protein for identifying hemp seeds and kit for distinguishing hemp seeds using the same

The present invention relates to a marker that can distinguish blue ginseng seed and boseong seed among hemp seeds, and more specifically, the hemp seed of blue ginseng, which is low in anhydrous tetrahydrocanabinol, and high in hemp bovine hemp seed. The present invention relates to a marker that can distinguish seeds by a simple method and a kit using the same.

Hemp is the scientific name Cannabis sativa , also called hemp, and the bast fibers of the stem are used for canvas, linen, tea bags, bills, rolling papers, and clothing (Callaway, JC (2004) Hempseed as A nutritional resource: An overview.Euphytica, 140, 65-72), seeds are used for seasoning or squeezing oil and have been used in some medicines to separate bioactive substances from flowers.

Hemp stems, leaves and flowers are known to contain an anesthetic substance called Tetrahydrocannabinol (THC), reducing the use of hemp. However, the roots, seeds and fibers of hemp contain little tetrahydrocannabinol, and research on active ingredients of hemp and the development of products based on these are being actively conducted in some developed countries.

Hemp fiber is a natural fiber that has been used for a long time. It is an excellent fiber material with high tensile strength, long fiber length, very high cellulose content and very low lignin content. In addition, hemp fiber has excellent antibacterial action, deodorization, insect repellent effect, absorbs ultraviolet rays and has good ventilation, so it can be developed as various clothing fibers, industrial filter materials and food packaging materials. In addition, hemp fibers are also used in the production of interior decorative items, curtains, bedding products, carpets, functional special papers, and insulation for construction, and are also used as interior and exterior materials for automobiles.

In addition to the many benefits of hemp fiber, hemp seed, Cheongsam, contains more than 80% of the high quality unsaturated fatty acids linoleic and linolenic acid, and contains saturated fatty acid but gamma linolenic acid (GLA), which is not commonly found in other plants. I never do that. Such unsaturated fatty acids are known to be effective in the prevention and treatment of cardiovascular diseases and type 2 diabetes. Cheongsam also contains high amounts of arginine, methionine, histidine and cysteine, which are essential for growth. It also does not contain trypsin inhibitors and oligosaccharides and contains large amounts of essential fatty acids and contains tocopherols and various vitamins A, B, C, D and E (Hullar, I., Meleg, I., Fekete, S. and Romvari, R. (1999) Studies on the energy content of pigeon feeds I. Determination of digestibility and metabolizable energy content.Poult. Sci., 78, 1757-1762).

In addition, hemp seed oil has been developed as a quick and effective absorber for skin and capillaries, and has been developed as a treatment for diseases of the ears, nose and throat, skin diseases, atopic dermatitis and functional cosmetics. , for the prevention and treatment of cardiovascular disease and diabetes therapy shows excellent effects (Grigoriev, OV (2002) Application of hempseed (Cannabis sativa L.) oil in the treatment of ear, nose and throat (ENT) disorders. J. Ind. Hemp , 7, 5-15, Callaway, J., Schwab, U., Harvima, I., Halonen, P., Mykkanen, O., Hyvonen, P. and Jarvinen, T. (2005) Efficacy of dietary hempseed oil in patients with atopic dermatitis.J. Dermatolog. Treat . 16, 87-94).

However, hemp containing tetrahydrocannabinol is strictly prohibited according to related laws because of its drug content. Thus, recently, a new low-dose type of ginseng with low tetrahydrocannabinol content has been developed.

However, the blue ginseng seed and the complementary seed containing the conventional tetrahydrocannabinol have the same size and color in appearance and are very difficult to distinguish them.

Accordingly, an object of the present invention is to provide a biomarker that can easily distinguish between blue ginseng seed and complementary seed among hemp seeds.

Another object of the present invention is to provide a kit comprising a biomarker capable of distinguishing blue ginseng seed and complementary seed from hemp seeds, and a method for distinguishing hemp seeds using the same.

In order to achieve the above object, the present inventors confirmed the total protein of two seeds of Cheongsam and Boseong using two-dimensional electrophoresis technology, and the total of 695 proteins in Cheongsam seed and 722 protein spots in Bosung seed can be separated. (FIG. 1, 2). In addition, the identified protein spots are clearly shown in the blue ginseng seed, but not identified in the complementary seed was selected to identify this can be used for the purpose of identifying the blue ginseng seed to invent the hemp seed classification kit using the same.

Two-dimensional electrophoresis gels were divided into 12 sections and the proteins expressed in each section were compared. A total of 26 proteins were expressed in both seeds.

In addition, it was possible to identify five specific proteins expressed only in the 8th and 12th regions of the blue ginseng seed protein among the proteins expressed only in the blue ginseng seed and not in the complementary seed (FIG. 3).

The blue ginseng seed identification marker protein according to the present invention is a transmembrane trafficking protein (molecular weight: 23 kDa, spot 127) and a phosphate-N-acetylmuramoyl-pentapeptide, an enzyme involved in peptidoglycan biosynthesis. Phospho-N-acetylmuramoyl-pentapeptide-transferase (molecular weight: 38 kDa, spot 132), peptidyl-prolyl cis-trans isomerase (molecular weight: 18 kDa, spot 133) ), A group consisting of chloroplast phosphoglycerate kinase (molecular weight: 32 kDa, spot 146) and glyceraldehyde-3-phosphate dehydrogenase (molecular weight: 21 kDa, spot 147) It is one or more selected. These marker proteins are expressed at pH 8-10 and could not be found in two-dimensional electrophoresis of complementary seeds. Therefore, it is possible to easily distinguish the blue ginseng seed and the complementary seed through the location and the presence of spots on the two-dimensional electrophoretic gel of these proteins. That is, when the five protein spots are identified in the region of pH 8 to 10 and a molecular weight of 40 kDa or less, the blue ginseng seed may be seen, and the seed may be complementary seed.

The characteristics of the five marker proteins are as follows.

Transmembrane trafficking protein is a protein involved in protein modification due to secretion and transport of protein after protein synthesis and is one of the essential elements of cell membrane composition.

Phospho-N-acetylmuramoyl-pentapeptide-transferase is a phosphate-N-acetylmuramoyl-pentapeptide-transferase. UDP-Mur ₂ Ac (oyl-L-ala-γ-D-Glu-L-Lys-D-Ala) -D-Ala) + undecaprenyl phosphate UMP + Mur ₂ Ac (oyl-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala) -diphosphate undecapre An enzyme that catalyzes the reaction of diphosphoundecaprenol, the two substrates of which are UDP-Mur ₂ Ac (oyl-L-ala-γ-D-Glu-L-Lys-D-Ala-D-Ala) and Undeka Prenyl phosphate (undecaprenyl phosphate), the product is UMP, Mur ₂ Ac (oyl-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala)-and diphosphoundecaprenol diphosphate )to be. This enzyme is included in the transferase family and is involved in peptidoglycan biosynthesis as an essential material for membrane composition (The initial stage in peptidoglycan synthesis. IV.Solubilization of phospho-N-acetylmuranyl-pentapeptide translocase. Heydanek, MG Jr. & Neuhaus, FC Biochemistry, 8: 1474-1481 (1969)).

Peptidyl-prolyl cis-trans isomerase accelerates the process of converting unfolded proteins into cis-trans isomers of proline peptides (Xaa-Pro) in oligopeptides. It is an enzyme. Cis-type peptide bonds are unstable compared to trans-type bonds. It is a group of four groups of PIPA, FK506-binding proteins (FKBPs), cyclophilines, parvulins, and Ser / Thr phosphatase 2A activator. (Engineering of monomeric FK506-binding protein 22 with peptidyl prolyl cis-trans isomerase.Importance of a V-shaped dimeric structure for binding to protein substrate.Budiman, C., Bando, K., Angkawidjaja, C., Koga, Y , Takano, K. and Kanaya, S. FEBS J., 276: 4091-4101 (2009)).

Chloroplast phosphoglycerate kinase (Chloroplast phosphoglycerate kinase) is an enzyme contained in the substrate of chloroplast, which catalyzes the reversible reaction of the Calvin cycle. diphosphoglycerate). Glycerate phosphate kinase is an important enzyme involved in glycolysis and your life cycle as well as photosynthetic carbon metabolism.In plants, the phosphate kinase activity is mainly in the chloroplast. purification and cDNA isolation of chloroplastic phosphoglycerate kinase from chlamydomonas reinhardtii, kitayama, M. & Togasaki, RK Plant Physiol., 107: 393-400 (1995)).

Glyceraldehyde-3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate dehydrogenase) is an oxidoreductase and glycerin-3-phosphate in the process, 1,3-diphosphate ( It phosphorylates bisphosphoglycerate and catalyzes the only oxidation reaction, NAD ⁺ to NADH, which plays a pivotal role in the production of energy in cells. It is distributed evenly in the cytoplasm, nucleus and membrane within the cell, plays an important role in energy production, and in addition to its glycolytic function, membrane fusion, phosphate transfer activity, microtubule bundling, and nuclear RNA transfer to the outside ( nuclear RNA export, DNA replication and DNA repair (repair), and a variety of functions.

The present invention

Transmembrane trafficking protein (molecular weight: 23 kDa);

Phospho-N-acetylmuramoyl-pentapeptide-transferase (molecular weight: 38 kDa);

Peptidyl-prolyl cis-trans isomerase (molecular weight: 18 kDa);

Chloroplast phosphoglycerate kinase (molecular weight: 32 kDa); And

For distinguishing blue ginseng seed from complementary seed comprising antibodies that specifically bind to one or more marker proteins selected from glyceraldehyde-3-phosphate dehydrogenase (molecular weight: 21 kDa) or immunogenic fragments thereof Provide the kit.

In addition, the present invention is characterized in that the kit is a kit for ELISA (Enzyme linked immunosorbent assay).

In addition,

Transmembrane trafficking protein (molecular weight: 23 kDa);

Phospho-N-acetylmuramoyl-pentapeptide-transferase (molecular weight: 38 kDa);

Peptidyl-prolyl cis-trans isomerase (molecular weight: 18 kDa);

Chloroplast phosphoglycerate kinase (molecular weight: 32 kDa); And

Contacting an unidentified seed sample with an antibody that specifically binds one or more marker proteins or immunogenic fragments thereof selected from glyceraldehyde-3-phosphate dehydrogenase (molecular weight: 21 kDa) It provides a way to distinguish between Cheongsam seeds and Boseong seeds.

The method

a) providing an unidentified seed sample to be analyzed;

b) contacting said sample with an antibody that specifically binds said marker protein;

c) quantitatively detecting the antigen-antibody complex; And

d) comparing the detection result of step c) with the blue ginseng seed control group.

The immunogenic fragment refers to some of the proteins that can produce reactive antibodies when injected into an external host.

The present invention provides an antibody that specifically binds to the marker protein or immunogenic fragment thereof. The antibody of the present invention can be prepared according to methods well known to those skilled in the art to which the present invention pertains.

The present invention provides a kit for distinguishing hemp seeds, characterized in that it distinguishes between the green ginseng seed and the complementary seed by comparing the expression of the marker protein or the amount of expression compared to the blue ginseng seed control group. The kit includes a kit for an Enzyme linked immunosorbent assay (ELISA).

In the present invention, the "marker" is a substance that can distinguish the hemp seed, Cheongsam seed, from other hemp seeds, the complementary seed, and may be a polypeptide marker for the marker protein, and these markers may be expressed in the blue ginseng seed as compared to the seed. Increasing or two-dimensional electrophoresis indicates that the protein spot can be clearly identified.

Therefore, by checking the presence or expression of the marker protein in the seed sample, it is possible to distinguish whether it is a blue ginseng seed or a complementary seed. Separation of the protein from the seed sample can be carried out using a known process, the amount of protein can be measured by various methods known to those skilled in the art.

In the present invention, "protein level measurement" is a process of confirming the expression level or expression level of the marker protein expressed or much expressed in blue ginseng seed in a sample, preferably by using an antibody that specifically binds to the marker protein. Check the amount of protein.

"Antibody" means a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the marker protein, and includes all polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Assays for measuring protein levels using antibodies include Western blot, Enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, Tissue immunostaining, immunoprecipitation, complement fixation assay, FACS, protein chips, and the like, are not limited to the described methods.

Through the above analysis method, it is possible to compare the amount of antigen-antibody complex formation of Chongsam seed and the amount of antigen-antibody complex formation of unidentified hemp seed, and to determine whether significant expression or increased expression level of marker protein is identified. It can be determined whether the seed is a blue ginseng seed or Boseong seed.

"Antigen-antibody complex" means a combination of the marker protein and an antibody specific thereto, and the amount of antigen-antibody complex formed can be quantitatively determined through the signal size of the detection label. The detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not limited to the materials described above. When enzymes are used as detection labels, the available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase and the like, and are not limited to the ranges described above. Fluorescent materials include fluorescein, phycocyanin, fluorescarmine and the like, and are not limited to the above-described materials. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include luciferin and the like, but are not limited thereto. The microparticles include colloidal gold and the like, but are not limited thereto. Redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, hydroquinone, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, and the like.

The protein expression level is preferably measured using ELISA (Enzyme-Linked ImmunoSorbent Assay). In ELISA, a direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, and reacted with another antibody that recognizes the antigen in the complex of the antibody and antigen attached to the solid support Various ELISA methods include an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody. More preferably, the antibody is attached to a solid support and reacted with a sample, followed by enzymatic color development by attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by the sandwich ELISA method which attaches a labeled secondary antibody and enzymatically develops. By confirming the degree of complex formation of the marker protein and the antibody will be able to determine whether the blue ginseng seed.

Alternatively, one or more antibodies against the marker may be arranged at a predetermined position on the substrate to use a protein chip immobilized at a high density. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. You can check it.

Another method is western blotting using antibodies to markers. The whole protein is isolated from the sample, which is electrophoresed to separate the protein according to size and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed by using the labeled antibody to determine whether or not the blue ginseng seed.

Such detection methods include comparing the expression levels of marker proteins in unidentified seeds with the marker protein expression levels of blue ginseng seed. The level of protein can be expressed in terms of absolute or relative differences in marker proteins.

The present invention relates to a hemp seed classification kit comprising an antibody that specifically binds to the marker protein.

Since the five marker proteins have been identified as the blue ginseng seed identification marker in the present invention, the production of antibodies using the same can be easily prepared using techniques well known in the art. As is well known in the art, polyclonal antibodies are injected into an animal and collected from the animal to obtain a serum comprising the antibody. Animals can be prepared using any animal host such as goat, rabbit, pig. Monoclonal antibodies are known in the art to which the present invention pertains, such as the hybridoma method (see Kohler & Milstein (1976) European J. Immunology 6 : 511-519) or phage antibody libraries (Clackson et al., Nature , 352 : 624-628, 1991; Marks et al, J. Mol. Biol. , 222 : 58, 1-597, 1991).

Hybridoma methods utilize cells of an immunologically suitable host animal, such as a mouse injected with a marker protein antigen, and the other uses a cancer or myeloma cell line. These two kinds of cells are fused by methods well known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. After obtaining a uniform cell population by subcloning by the limited dilution technique, hybridomas capable of producing antibodies specific for the marker protein are mass cultured in vitro or in vivo according to standard techniques.

The phage antibody library method obtains an antibody gene for a marker protein and expresses it in the form of a fusion protein on the surface of the phage to prepare an antibody library in vitro, and isolates the monoclonal antibody binding to the marker protein from the library. , How to make.

Antibodies prepared by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.

Antibodies included in the kits of the present invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means a fragment which retains at least antigen binding function, and includes Fab, F (ab '), F (ab') ₂ , F (ab) ₂ and Fv.

The hemp seed separation kit of the present invention comprising an antibody that specifically binds to a marker protein is preferably a diagnostic kit for ELISA, and may include an antibody specific for a control protein, in addition to detecting an antigen-antibody complex. Reagents, such as labeled secondary antibodies, chromophores, enzymes conjugated with the antibody and other substrates or other substances capable of binding to the antibody, and the like.

The marker for identifying ginseng seed according to the present invention can easily and specifically detect whether an unidentified hemp seed is a blue ginseng seed by detecting the expression or change in the expression amount of one or more proteins among the five proteins.

Figure 1 is a photograph showing the results of two-dimensional electrophoresis of the whole protein ginseng seed.
Figure 2 is a photograph showing the results of two-dimensional electrophoresis of the whole seed seed protein.
3 is a result of confirming the specific protein expressed only in the blue ginseng seed compared to FIG. (A): blue ginseng seed protein, (B): complementary seed protein

Hereinafter, the configuration of the present invention will be described in detail by examples. However, the scope of the present invention is not limited by the description of the examples.

Example 1: Protein Extraction

1 g of each ginseng seed and complementary seed, completely peeled, were frozen with liquid nitrogen and crushed with a homogenizer, and then 0.01M sodium phosphate buffer solution containing 0.01M sodium phosphate (pH 7.5) and 0.6M sucrose ( crushed once more at pH 7.5). The crushed sample was centrifuged at 4 ° C., 16,000 rpm for 30 minutes, and the obtained supernatant was treated with cold 10% (v / v) trichloroacetic acid to extract protein. Washing was repeated five times to remove each substance remaining in the extracted protein, the extracted protein was used while stored at -70 ℃.

Example 2: Isoelectric Point Electrophoresis

The total amount of protein extracted from blue ginseng seed and complementary seed was 1 mg, and 450 μl of rehydration solution (7M urea, 2M thiourea, 4.5% CHAPS, 0.25% ampholytes, 100mM DTE) was used. The protein was dissolved. A protein of each condition dissolved in 450 µl of rehydration solution was injected into an IPG holder, and a 24 cm immobilized dry strip (pH 3-10 NL) made to move according to the pH of the protein for isoelectric electrophoresis. , GE Healthcare Bio-Science) was rehydrated for 18 hours. In the field of 200 V / 200 Vhrs, 500 V / 500 Vhrs, 1,000 V / 1,000 Vhrs, 8,000 V / 13,500 Vhrs, 8,000 V / 100,000 Vhrs conditions were moved in the electric field according to the pH of the protein.

Example 3: Equilibration

Strips subjected to isoelectric point electrophoresis were subjected to primary equilibration in primary equilibration buffer (6M urea, 0.375M Tris-HCl, pH 8.8), 20% glycerol, 2% SDS, 25% acrylamide, 2mM TBP for 20 minutes each. After the second equilibration buffer (6M urea, 0.375M Tris-HCl (pH 8.8), 20% glycerol, 2% SDS, 25% acrylamide, 2mM TBP) was subjected to the second equilibration for 20 minutes.

Example 4: SDS-polyacrylamide gel electrophoresis

After the 8% ~ 16% acrylamide gel was made, a strip of which isoelectric point electrophoresis was completed was placed on the gel, fixed with 0.5% agarose gel, and then subjected to two-dimensional electrophoresis. The current was first developed by giving a current of 5 mA per gel for 2 hours, and then the protein was moved according to the molecular weight by giving a current of 18 mA per gel for 10 hours.

Example 5: Gel Dyeing and Decolorization

The developed gel was run for 12 hours using a Coomassie Blue G-250 staining solution (34% methanol, 0.1% Coomassie Blue G-250, 17% Ammonium Sulfate, 3% Phosphoric Acid) to identify proteins isolated from the gel. After the abnormal staining, it was decolorized using 1% acetic acid.

Example 6: Gel Image Analysis

For gel image analysis, gels stained with UMAX powerLook 1100 were scanned and image analyzed with ImageMaster 2D Platinum 6.0 (2D software, GE Healthcare Bio-Science). Three gels per condition were analyzed for accurate analysis.

Example 7: NanoLC-MS / MS Analysis

After in-gel digestion of each spot with trypsin for NanoLC-MS / MS analysis, the peptides of the spots were agilent 110 Series nano-LC and LTQ-mass spectrometer (Thermo Electron, San Jose, CA). The capillary column (150 mm × 0.075 mm) used for LC-MS / MS analysis was purchased from Proxeon (Odense M, Denmark), and the slurry was a Magic C18 stationary phase (5 µm, 100 mm pore size) (Michrom Bioresources, Auburn, CA). Mobile phase A for LC separation was distilled water containing 0.1% formic acid and mobile phase B was acetonitrile containing 0.1% formic acid. The chromatographic gradient was set up as 35% D linear increase at 5% B for 50 minutes, 61% B linear increase at 541% B for 0 minutes, and 81% B linear increase at 561% B for 5 minutes. The flow rate was maintained at 300 ml per minute splitting. Mass spectra were obtained with data-dependent acquisition with full mass scan (5410-1810 m / z) after MS / MS scan. The obtained several MS / MS scans were the average of rmmicroscans in LTQ. The temperature of the ion transfer tube was set at 200 ° C. and the spray was 1.5.0-2.0 kV. Normalized collision energy was set at 35% for MS / MS. Sequest software was used to identify peptide sequences. In order to obtain reliable results, the conditions of delatCn = 0.1 and Rsp = 4 were applied, Xcorr = 1.5 in Charge state 1+, Xcorr = 2.0, Charge in charge state 2+ In state 3+, Xcorr = 2.5 was applied, and peptide probability> 0.1 was used to identify proteins as cutoffs.

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Claims (4)

Transmembrane trafficking protein (molecular weight: 23 kDa);
Phospho-N-acetylmuramoyl-pentapeptide-transferase (molecular weight: 38 kDa);
Peptidyl-prolyl cis-trans isomerase (molecular weight: 18 kDa);
Chloroplast phosphoglycerate kinase (molecular weight: 32 kDa); And
For distinguishing blue ginseng seed from complementary seed comprising antibodies that specifically bind to one or more marker proteins selected from glyceraldehyde-3-phosphate dehydrogenase (molecular weight: 21 kDa) or immunogenic fragments thereof Kit.
The method of claim 1,
The kit is a diagnostic kit, characterized in that the kit for ELISA (Enzyme linked immunosorbent assay).
Transmembrane trafficking protein (molecular weight: 23 kDa);
Phospho-N-acetylmuramoyl-pentapeptide-transferase (molecular weight: 38 kDa);
Peptidyl-prolyl cis-trans isomerase (molecular weight: 18 kDa);
Chloroplast phosphoglycerate kinase (molecular weight: 32 kDa); And
Contacting an unidentified seed sample with an antibody that specifically binds one or more marker proteins or immunogenic fragments thereof selected from glyceraldehyde-3-phosphate dehydrogenase (molecular weight: 21 kDa) How to distinguish between Cheongsam seeds and Boseong seeds.
The method of claim 3,
a) providing an unidentified seed sample to be analyzed;
b) contacting said sample with an antibody that specifically binds said marker protein;
c) quantitatively detecting the antigen-antibody complex; And
d) comparing the detection result of step c) with the blue ginseng seed control group.

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