KR20110040158A

KR20110040158A - Marker and kit for detecting contamination of benzoanthracene

Info

Publication number: KR20110040158A
Application number: KR1020090097321A
Authority: KR
Inventors: 이미영; 박슬기; 류재천; 남석우
Original assignee: 순천향대학교 산학협력단
Priority date: 2009-10-13
Filing date: 2009-10-13
Publication date: 2011-04-20

Abstract

A benzoanthracene contamination marker, a kit for diagnosing benzoanthracene contamination using the marker, and a method for detecting a benzoanthracene contamination marker through the formation of an antigen-antibody complex are disclosed. By using this, benzoanthracene contamination can be effectively detected.

Description

Marker and kit for detecting contamination of benzoanthracene

The present invention relates to markers for detecting exposure or contamination to benzoanthracene. The invention also relates to kits for detecting exposure or contamination to benzoanthracene. The present invention also relates to a method for detecting exposure or contamination of benzoanthracene.

Benzo (a) anthracene (benzoanthracene) is a polycyclic aromatic hydrocarbons (PAH) that consists of four aromatic rings and has one or more rings that share two carbons. Soluble in alcohol, ether and benzene but not in water. Also called Benzo (a) phenanthrene, Tetraphene, Benzanthrene, chemical formula is C ₁₈ H ₁₂ , molecular weight is 228.28, colorless, yellow-green to ultraviolet fluorescence, melting point 159-161 ℃, boiling point 400 ℃, vapor pressure 1.10x10 ^-7 mmHg (25 ° C).

Benzoanthracene itself cannot act as a carcinogen, but it can also work with promoters such as phorbol esters. It is known to be destroyed by photoacidification in the atmosphere and slowly degraded by bacteria in the soil. Benzoanthracene is a common environmental pollutant and is not known for industrial or industrial use. It is a common pollutant from incomplete combustion of organics and smoke and flue gas, tobacco smoke, car exhaust, roasted coffee and charcoal meat, barbecue or It is found in many different kinds, including smoked meat. It can also be found in a variety of foods, including cleats, coal tar, petroleum asphalt, and yeast from vegetable oils and bread.

PAHs of the same kind, such as benzoanthracene, have a lethal effect on cells involved in cell proliferation. In animal studies, exposure to other chemical structures with PAHs induces an effect on the blood production system, exacerbating anemia and developing granulocytopenia. In addition, the lymphocyte system was adversely affected, resulting in lymphocyte reduction. Toxic effects are also observed in the rapid cell division of the intestinal epithelium and spermatogonia and in the non-proliferating testicular and ovarian oocytes. Most of these effects are caused by oral and parenteral exposure. Respiratory epithelial cell proliferation and cell hyperplasia are reported to be due to subchronic inhalation exposure.

Benzoanthracene is easily absorbed through the skin, exposed through intravenous, intraperitoneal injections, ingestion, and inhalation, and is known to exhibit neoplastic, papilloma, skin cancer, and mutagenicity upon skin contact. Benzoanthracene has mild carcinogenicity in animal experiments, and liver and lung adenoma and gastric papilloma occurred in animals after repeated oral administration. Local tumors developed when applied to rat skin, but not in rats and hamsters. In general, the oxidative product of benzoanthracene is more active against it than benzoanthracene. There is clear evidence of the effects of oral or inhalation of experimental animals, and there is no evidence of any effects on humans. IARC classifies benzoanthracene as group 2B, a human carcinogen.

In addition, not only the gene depends on its function in the cell depending on post-translational modification in the cell, but also the amount of mRNA synthesized by gene transcription actually makes the protein. It varies greatly accordingly. For this reason, it has recently been accepted that analysis at the protein level, the final product, is more important than analysis at the genetic level.

An object of one embodiment of the present invention, to provide a marker for detecting whether benzoanthracene contamination.

It is an object of another embodiment of the present invention to provide a kit for diagnosing benzoanthracene contamination.

Another object of an embodiment of the present invention is to provide a method for detecting whether benzoanthracene contamination.

The benzoanthracene contamination marker according to an embodiment of the present invention is a marker for diagnosing benzoanthracene contamination in a living body, and is characterized by including one or more of the proteins described in Table 1.

Method for detecting a benzoanthracene contamination marker according to an embodiment of the present invention, (a) reacting a biological sample with an antibody that specifically binds to the benzoanthracene contamination marker; And (b) comparing the amount of the antigen-antibody complex formed by the reaction of step (a) with the control.

The method for diagnosing benzoanthracene contamination according to an embodiment of the present invention includes determining positive benzoanthracene contamination when the amount of the benzoanthracene contamination marker present in the biological sample is more or less than the normal range of the control group. It features.

By using the present invention, it is possible to effectively detect whether or not it is contaminated with benzoanthracene.

Benzoanthracene contamination marker according to an embodiment of the present invention, a marker for diagnosing benzoanthracene (Benzoanthracene) of the living body,

Transketolase; Aldehyde dehydrogenase family 7, member A1; Phenylalanine hydroxylase; Chain A, New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver; Glutathione S-transferase, mitochondrial; Chain A, crystal structure of perchloric acid soluble protein-A translational inhibitor (A); Beta 1 globin; Hemoglobin subunit beta-2 (hemoglobin beta-2 chain) (beta-2-globin) hemoglobin beta chain, minor-form) (Hemoglobin subunit beta-2 (Hemoglobin beta-2 chain) (Beta-2-globin) (Hemoglobin beta chain, minor-form)); Hemoglobin beta-2 subunit analogue (hemoglobin beta-2 chain) (beta-2-globin) hemoglobin beta chain, minor-form) (similar to Hemoglobin beta-2 subunit (Hemoglobin beta-2 chain) (Beta-2-globin ) (Hemoglobin beta chain, minor-form)); And diazepam binding inhibitor.

Looking at the benzoanthracene marker according to an embodiment in detail.

The transketolase catalyzes another nodular reaction of the pentose phosphate pathway, and one of the most important intermediate products of this pathway is ribose-5-phosphate, a phosphorylated pentose sugar. This is necessary for the synthesis of ATP, GTP, nucleic acid, DNA, RNA, NADP and the like. In addition, since the transketolase decreases from the beginning of thiamin binding, it is used to measure its activity in erythrocytes as a method for assessing thiamin nutritional status. In FIG. 1, transketolase is indicated as spot number 1.

Similar to aldehyde dehydrogenase family 7, member A1 increases dependent on benzoanthracene concentration and catalyzes the conversion to carboxylic acids (formic acid) by oxidizing aliphatic or aromatic aldehydes. It is an enzyme that plays an important role in not only liver detoxification but also metabolism of neurotransmitters. In FIG. 1 aldehyde dehydrogenase family 7 member A1 analogues are designated as spot 2.

Phenylalanine hydroxylase is a biological catalyst that converts excess phenylalanine into another amino acid, tyrosine, to finely control the amount of phenylalanine. The disease Phenylketonuria is inherently degraded by the activity of phenylalanine hydrolase, causing phenylalanine and its metabolites to accumulate in the blood, leading to intellectual disability, light brown hair, and skin pigment deficiency. The phenylalanine hydroxylase is 1/10 LD ₅₀ (20 mg / kg) benzoanthracene treated group increased compared to normal group, but 1/2 LD ₅₀ (100 mg / kg) in the benzoanthracene treated group, indicated as spot 3 in FIG.

Chain A, New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver, Glutathione S-transferase, mitochondrial GSTs catalyze the binding between reduced glutathione and several electrophilic substances to protect cells from damage caused by oxidative cytotoxic or carcinogenic substances. GSTs also have a broad spectrum of substrate-specific properties that protect cells from a wide range of toxic chemicals. In Figure 1, the novel crystalline form of chain A, Lett liver-derived Mu class glutathione S-transferase, glutathione S-transferrate and mitochondria are shown as spots 4 and 5, respectively.

Chain A, the crystal structure of perchloric acid soluble protein-A translational inhibitors, is a protein that inhibits cell free protein synthesis, but the mechanism of action is unknown. The 14 kDa translational inhibitor protein is very similar to rat liver perchloric acid-soluble protein (L-PSP) in human monocytes and rat livers. The crystal structure of the chain A, perchloric acid soluble protein-A metastasis inhibitor is reduced in the benzoanthracene treatment group of 1/10 LD _{50 and} the benzoanthracene treatment group of 1/2 LD ₅₀ , and is composed of 136 proteins as protein synthesis inhibitors. In FIG. 1, spot 6 is indicated.

Beta 1 globin, hemoglobin subunit beta-2, and hemoglobin beta-2 subunit analogues (similar to Hemoglobin beta-2 subunit) have heme in hemoglobin and gas exchange in the blood Known as a protein involved in Beta 1 globin, hemoglobin subunit beta-2 and hemoglobin beta-2 subunit analogues are shown in Figure 1 as spans 6, 7, and 8, respectively.

Diazepam binding inhibitor, 1/10 LD ₅₀ (20 mg / kg) benzoanthracene treated group increased compared to normal group, but 1/2 LD ₅₀ (100 mg / kg) in the benzoanthracene treated group. It is also regulated by hormones and is involved in lipid metabolism, regulates the response of pancreatic secretions, and also acts as a synthetic mediator by the release of cholecystokinin and the corticosteroids of steroids in the adrenal cortex after meals. Diazepam binding inhibitor is shown as spot 10 in FIG.

In one embodiment, the benzoanthracene contamination marker is a transketolase of spot number 1, an aldehyde dehydrogenase family of 7 member A1 analogue of spot number 2, a phenylalanine hydroxylase of spot number 3 among proteins having a change in two-dimensional electrophoresis. And chain A, a crystal structure of a perchloric acid soluble protein-A metastatic inhibitor.

In another embodiment, the present invention provides a kit for diagnosing benzoanthracene contamination in a living body including an antibody specifically binding to the benzoanthracene contamination marker.

The present invention also provides a detection method using the benzoanthracene contamination marker. Specifically, the benzoanthracene contamination marker detection method is

(a) reacting a biological sample with an antibody that specifically binds to the benzoanthracene contamination marker; And

(b) comparing the amount of the antigen-antibody complex formed by the reaction of step (a) with a control.

In one embodiment, step (b) is a positive determination of benzoanthracene contamination when the protein expression in the biological sample to be detected is increased or decreased compared to the normal range of the control.

In one embodiment, the present invention provides a method for diagnosing contamination using a benzoanthracene contamination marker. Specifically, if the amount of the benzoanthracene contamination marker present in the biological sample is more or less than the normal range of the control, it will be determined as positive for benzoanthracene contamination.

The benzoanthracene contamination marker according to an embodiment of the present invention may be modified in various modified forms by replacing, deleting and / or adding amino acid residues of a natural amino acid sequence as long as it can be used to detect benzoanthracene contamination. Can be. It may also be in a form in which modifications such as phosphorylation, saccharification, methylation, and pansylation have occurred.

In the method for detecting a benzoanthracene contamination marker according to an embodiment of the present invention, "antibody" means a protein that specifically binds to a benzoanthracene contamination marker, and includes both a polyclonal antibody and a monoclonal antibody. Since the production of antibodies is well known in the art, the antibody may be produced by selecting any one of the antibody production methods well known in the art.

As a method for detecting a benzoanthracene contamination marker according to an embodiment of the present invention, any one of methods well known in the art may be used. For example, various methods can be used, such as western blot, ELISA, or immunoprecipitation techniques.

Polyclonal antibodies can be produced by methods well known in the art for injecting the benzoanthracene contamination marker protein antigens described above into an animal and collecting blood from the animal to obtain serum comprising the antibody. Such polyclonal antibodies can be prepared from any animal species host such as goat, rabbit, sheep, monkey, horse, pig, bovine dog.

Monoclonal antibodies can be made by fusion methods well known in the art (see Kohler and Milstein (1976) European Jounral of Immunology 6: 511-519). One of two populations of cells fused to make a "hybridoma" secreting monoclonal antibodies utilizes cells from an immunologically suitable host animal, such as a mouse injected with a benzoanthracene contaminating marker protein antigen, and the other Cancer or myeloma cell lines are used as the population of. These two populations 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 benzoanthracene contaminating marker protein are either in vitro or in vivo according to standard techniques. Incubate in bulk in. The monoclonal antibodies produced by the hybridomas may be used without purification, but in order to obtain the best results, the monoclonal antibodies are preferably purified and used according to methods well known in the art.

Monoclonal antibodies can be identified and separated by searching for recombinant combinatorial immunoglobulin libraries (eg, antibody phage display libraries) using benzoanthracene contaminating marker proteins, and then separating members of the immunoglobulin library that bind to benzoanthracene contaminating marker proteins. have. Phage display library preparation and screening kits are commercially available (eg, Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurβAPS Phage Display Kit, Catalog No. 240612). In addition, examples of methods and reagents particularly useful for antibody display library preparation and screening are described, for example, in US Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; And 90/02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-1281; Griffiths et al. (1993) EMBO J. 12: 725-734.

In one embodiment of the invention, two antibodies or “sandwich” ELISAs are used to detect overexpression of a benzoanthracene contamination marker in a patient sample. Such "sandwich" or "two site" immunoassays are known in the art. For example, Current Protocols in Immunology. See Indirect Antibody Sandwich ELISA to Detect Soluble Antigens, John Wiley & Sons, 1991. In this aspect, the present invention uses two antibodies specific for two different antigen forming sites on one benzoanthracene contamination marker. "Different antigen forming sites" means that the antibodies are specific to different sites on the desired benzoanthracene contaminating marker protein such that binding of one antibody does not significantly interfere with binding of another antibody to the benzoanthracene contaminating marker protein. . The first antibody, the so-called "capture antibody", is fixed or bound to a solid support. For example, capture antibodies against the desired benzoanthracene contamination markers can be covalently or non-covalently attached to microtiter plate wells, beads, cuvettes or other reaction vessels. In a preferred embodiment, the capture antibody is bound to the microtiter plate wells. Methods of attaching antibodies to solid supports are known in the art. Body samples, in particular serum samples, are contacted with a solid support to allow complexation with the bound capture antibody. Unbound samples are removed and a secondary antibody, the so-called "detection antibody", is added to the solid matrix. The detection antibody is coupled to or labeled with a substance that is specific for the individual antigen forming site on the benzoanthracene contamination marker of interest and provides a detectable signal. Such antibody labels are well known in the art and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. After incubation with the detection antibody, the unbound antibody is removed and the labeled detection antibody bound to the solid support is quantified to determine the expression level of the benzoanthracene contamination marker. Those skilled in the art will appreciate that capture and detection antibodies can be contacted with a body sample continuously or simultaneously as described above. Furthermore, the body sample may first be incubated with the detection antibody prior to contacting the sample with the immobilized capture antibody.

In the benzoanthracene contamination marker detection method according to an embodiment of the present invention, the amount of antigen-antibody complex formed can be quantitatively measured through the size of a signal of a detection label. Such detection labels may be selected from enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but are not limited thereto. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase, GDPase, RNase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase, β -Latamase and the like, but is not limited thereto. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include, but are not limited to, ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, and the like.

Colorimetric method, electrochemical method, fluorimetric method, luminometry, particle counting method, visual assessment and the like to detect antigen-antibody complex formation. A method selected from scintillation counting method may be used, but is not limited thereto.

Referring to the kit for diagnosing benzoanthracene contamination according to an embodiment of the present invention is as follows. A "kit" is intended to be any preparation (eg, package or container) that includes one or more reagents such as antibodies, nucleic acid probes, and the like for specifically detecting a marker of the present invention. Kits may be formulated, dispensed, or sold as units for carrying out the methods of the present invention. Additionally, the kit can include a package insert that describes the kit and how to use it. Any or all kit reagents may be included in a container such as a closed container or pouch that protects it from the external environment.

In certain embodiments, the immunocytochemistry kit of the present invention further comprises two or more reagents, such as antibodies, for specifically detecting the expression of at least two or more individual protein markers. Each antibody can be provided in the kit as an individual reagent or as an antibody cocktail including all antibodies to the different protein markers of interest.

In a preferred embodiment, kits are provided for performing immunochemical methods, in particular “sandwich” ELISAs. Such kits can be used with manual or automated immunochemical techniques. Such kits include one or more first capture antibodies for the protein marker of interest, labeled second detection antibodies specific for other antigenic sites of the protein marker, and compounds for detecting antibody binding to the protein marker. The first capture antibody can then be provided in solution for attachment to the solid support. Alternatively, the capture antibody may be provided as a kit already bound to a solid support, such as a bead or microtiter plate well. Any compound that detects an antigen-antibody complex can be used in the practice of the present invention. In some instances, the second detection antibody is conjugated with an enzyme that catalyzes colorimetric conversion of the substrate. Techniques for their use to detect such enzyme and antibody binding are well known in the art. In a preferred embodiment, the kit comprises a second detection antibody conjugated with HRP. Substrates, in particular chromophores, can be provided which can be used with conjugated enzymes (e.g., tetramethylbenzidine for HRP-labeled second detection antibodies) and solutions for stopping the enzymatic reaction, such as sulfuric acid. In certain instances, compounds for detecting antibody binding include commercially available reagents and kits. In another embodiment, the “sandwich” ELISA kits of the invention comprise antibodies for detecting two or more different biomarkers of interest. Such kits include two or more first capture antibodies and a secondary detection antibody for each biomarker. The capture antibody may be provided as a separate reagent or alternatively in a mixture of all antibodies against the various biomarkers of interest. To verify the correct use and activity of the reagents employed in accordance with the present invention, the kit may contain positive and / or negative controls. Controls may include samples such as tissue fragments known to be with or without the desired biomarker, cells immobilized on glass slides, and the like. In certain embodiments, the positive control is a solution comprising the desired biomarker protein. Construction and use of the control group is standard and self-explanatory within the general ability of those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example

1. Protein Extraction

Normal oil group treated with corn oil for proteome analysis, 1/10 LD ₅₀ (20 mg / kg) and 1/2 LD ₅₀ Liver tissues were isolated from rats treated with (100 mg / kg) benzoanthracene for 72 hours and homogenized, followed by 40 mM Tris containing 7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE. Disintegrate once more in -HCl buffer (pH 8.8). The supernatant obtained after centrifuging the crushed sample at 15 ℃, 12,000xg for 50 minutes was used. The extracted protein was used while stored at -70 ℃.

2. Isoelectric point Electrophoresis

The total protein isolated from rat liver tissues of normal and experimental groups was 1 mg and protein was prepared using 450 ul of rehydration solution (7 M urea, 2 M thiourea, 4.5% CHAPS, 0.25% ampholytes, 100 mM DTE). Melted. The protein in each condition dissolved in 450 ul of rehydration solution was injected into the IPG holder, and a 24 cm immobilized dry strip (pH 3-10 NL, GE Healthcare Bio-) made to move according to the pH of the protein for isoelectric electrophoresis. Science) was used for rehydration 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.

3. Equalization

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

4. SDS - polyacrylamide gel Electrophoresis

After making 9%-16% acrylamide gel, the strip with the isoelectric point electrophoresis was placed on the gel, fixed with 0.5% agarose gel, and then subjected to two-dimensional electrophoresis. The current was first developed with 5 mA per gel for 2 hours and then 18 mA per gel for 10 hours to move proteins according to molecular weight.

5. Gel Dyeing and Bleaching

The developed gel was stained for at least 12 hours using Coomassie Blue G-250 staining solution (34% methanol, 0.1% Coomassie G-250, 17% ammonium sulfate, 3% phosphoric acid) to identify proteins isolated from the gel. And decolorized using 1% acetic acid.

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 were analyzed per condition for accurate analysis.

7. Nanolc - MS Of 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 ㅧ 0.075 mm) used for LC-MS./MS analysis was purchased from Proxeon (Odense M, Denmark) and the slurry was 5 μm, 100 Å pore size Magic C18 stationary phase (Michrom Bioresources, Auburn, CA). Mobile phase A for LC separation was distilled water (0.1% formic acid in deionized water) containing 0.1% formic acid and mobile phase B was acetonitrile (0.1% formic acid in acetonitrile) containing 0.1% formic acid. The chromatographic gradient was set up with a linear increase of 35% D at 5% B for 50 minutes, a linear increase of 60% B at 40% B for 20 minutes, and a 80% B linear increase at 60% 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 (400-1800 m / z) after MS / MS scan. Each MS / MS scan obtained was an average of 1 microscans 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, delatCn ≥ 0.1 and Rsp ≤ 4 were applied, Xcorr ≥ 1.5 for Xcorr, Xcorr ≥ 2.0 for charge state 2+, and Xcorr ≥ 2.0 for charge state 2+. Xcorr ≥ 2.5 was applied, and peptide probability> 0.1 was used to identify proteins as cutoffs. Peptides were oxidized variously at methionine residues and carboxyamidomethylation and carboxymethylation at various cysteines.

7. Results

Two-dimensional electrophoresis of the livers of rats treated with benzoanthracene revealed a total of 1170 spots in the normal group and 1/10 LD _50. When treated with (20 mg / kg) benzoanthracene, 1222 in total, 1/2 LD ₅₀ When treated with (100 mg / kg) benzoanthracene, 1348 spots were identified. Among them, a total of 10 protein spots showing differences in expression between the normal group and the benzoanthracene treatment group were shown in FIG. 1 and Table 1.

In addition, in FIG. 2, the expression amount of each spot protein was shown with respect to the protein shown in FIG. In Figure 2 Example 1 is 1/10 LD ₅₀ (20 mg / kg) benzoanthracene treatment group, Example 2 is 1/2 LD ₅₀ (100 mg / kg) benzoanthracene treated group.

Referring to FIG. 2, a spot 1 transketolase, a spot 2 aldehyde dehydrogenase family 7 member A1 analogue, a spot 4 chain A, Rett liver origin New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver (Chain A), Hemoglobin subunit beta-2 (spot 8), is a normal group 1/10 LD ₅₀ (20 mg / kg) benzoanthracene treatment group and 1/2 LD ₅₀ (100 mg / kg) benzoanthracene treatment group increased more than 1.5 times (Fig. 2. (A)). While chain 6, spot 6, crystal structure of perchloric acid soluble protein-A translational inhibitor, spot 7, beta 1 globin and hemoglobin subunit beta -2 analogue (similar to Hemoglobin beta-2 subunit) was reduced by more than 1.5-fold (Figure 2. (B)). Thus, it can be seen that spots 1, 2, 4 and 8, and spots 6, 7 and 9 are proteins that vary in concentration depending on benzoanthracene.

Spot 3 phenylalanine hydroxylase and Spot 10 diazepam binding inhibitor are 1/10 LD ₅₀ (20 mg / kg) benzoanthracene treated group increased compared to the normal group but 1/2 LD ₅₀ (100 mg / kg) benzoanthracene treated group decreased (Fig. 3. (C)). In addition, glutathione S-transferase (mitochondrial), glutathione S-transferase with spot 5, was 1/10 LD _50. It was decreased in the phenanthrene treated group of (20 mg / kg), but increased in the phenanthrene treated group of 1/2 LD ₅₀ (100 mg / kg) (Fig. 3. (D)).

1 is an electrophoretic photograph showing the expression of benzoanthracene contamination markers according to an embodiment of the present invention;

Figure 2 is an electrophoresis picture of measuring the expression amount of each spot protein according to benzoanthracene treatment according to an embodiment of the present invention.

Claims

As a marker for diagnosing benzoanthracene of a living body,

Transketolase; Aldehyde dehydrogenase family 7, member A1; Phenylalanine hydroxylase; Chain A, New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver; Glutathione S-transferase, mitochondrial; Chain A, crystal structure of perchloric acid soluble protein-A translational inhibitor; Beta 1 globin; Hemoglobin subunit beta-2 (hemoglobin beta-2 chain) (beta-2-globin) hemoglobin beta chain, minor-form) (Hemoglobin subunit beta-2 (Hemoglobin beta-2 chain) (Beta-2-globin) (Hemoglobin beta chain, minor-form)); Hemoglobin beta-2 subunit analogue (hemoglobin beta-2 chain) (beta-2-globin) hemoglobin beta chain, minor-form) (similar to Hemoglobin beta-2 subunit (Hemoglobin beta-2 chain) (Beta-2-globin ) (Hemoglobin beta chain, minor-form)); And at least one selected from the group consisting of diazepam binding inhibitors.

The method of claim 1,

The benzoanthracene contamination marker comprises at least one selected from the group consisting of transketolase, aldehyde dehydrogenase family 7 member A1 analogue, phenylalanine hydroxylase and chain A, crystal structure of perchloric acid soluble protein-A metastasis inhibitor. Benzoanthracene Contamination Marker.

A kit for diagnosing benzoanthracene contamination in a living body comprising an antibody specifically binding to the benzoanthracene contamination marker according to claim 1.

(a) reacting a biological sample with an antibody that specifically binds to the benzoanthracene contamination marker according to claim 1; And

The method of claim 4, wherein

In the step (b), benzoanthracene contamination marker detection method, characterized in that the positive determination of benzoanthracene contamination when the protein expression in the biological sample to be detected increased or decreased compared to the normal range of the control.

A method for diagnosing benzoanthracene contamination comprising determining positive benzoanthracene contamination when the amount of the benzoanthracene contamination marker according to claim 1 or 2 present in the biological sample is greater or less than the normal range of the control group.