KR101396300B1 - Biomarkers for diagnosing the addiction of ethylbenzene - Google Patents

Abstract

TECHNICAL FIELD The present invention relates to a biomarker protein for diagnosing ethylbenzene poisoning, more specifically, a protein biomarker whose expression is markedly increased or decreased in an ethylbenzene exposure test group as compared with normal cells, and a diagnostic kit using the protein biomarker .

Ethylbenzene, biomarker, toxicity, poisoning

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for ethylbenzene poisoning,

The present invention relates to a biomarker capable of diagnosing ethylbenzene poisoning, which is a toxic substance, and more specifically, a protein biomarker whose expression is significantly increased or significantly decreased in an ethylbenzene exposure test group as compared with a normal cell, And a diagnostic kit using the same.

Ethylbenzene is a kind of Volatile Organic Compounds (VOC), which is also called Ethylbenzol, Phenyl ethane, and Athylbenzol, and its chemical formula is C ₆ H ₅ C ₂ H ₅ , It is a colorless, flammable liquid with a molecular weight of 106.17.

Ethylbenzene is used as an organic synthesis solvent and diluent as an intermediate raw material of styrene monomer. A mixture of ethylbenzene and xylene is used for paint, insecticide spraying, gasoline blending, etc. It is also used for the manufacture of synthetic rubber and fuel for automobiles and airplanes.

The source of ethylbenzene may be released into the atmosphere during the process of ethylbenzene and styrene production process and organic solvent, and waste may leak. The biggest factor in the release of ethylbenzene into the environment is the petroleum industry, which is spread to the atmosphere due to the very high volatility and low solubility of ethylbenzene. In addition to this, it is also possible to reduce the amount of oil in the oil refining process, the volatilization and combustion caused by the use of gasoline, the volatilization and leakage of gasoline and kerosene at the gas station, the volatilization and leakage during storage and transportation at low oil storage, And is released into the atmosphere.

Ethylbenzene causes irritation of the skin, nose, throat and eyes during exposure (Desogus, GF (2007). The environmental monitoring of the exposure to chemical contamination in operating rooms. -418). Liquid ethylbenzenes are absorbed into the body through the skin and intestinal tract, and in the case of gaseous form, they are absorbed into the body through inhalation (US Environmental Protection Agency (1988). Environ. Contam. Toxicol., 106, 189-203), 40-60% remain in the lungs and absorbed ethylbenzene is reported to accumulate in fat.

Animal studies have shown that ethylbenzene in the air can cause central nervous system and liver, kidney, eye and skin inflammation (Futamura, M., Goto, S., Kimura, R., Kimoto, I., Miyake, M., Ito, K. and Sakamoto, T. (2008) Differential effects of topically applied formalin and aromatic compounds on neurogenic-medicated microvascular leakage in rat skin.

In studying the exposure of ethylbenzene in the workplace, it is difficult to define its own effects due to simultaneous exposure with similar substances such as xylene. In 1988, Bardodej and Cirek reported that 200 workers who produced ethylbenzene for 20 years did not find any abnormality in blood or liver (Bardodej, Z. and Cirek, A. (1988 ) Long-term study on workers occupationally exposed to ethylbenzene. J. Hyg. Epidemiol. Microbiol. Immunol. The International Agency for Research on Cancer (IARC) has classified this substance into Group 2B, which has potential for carcinogenicity to humans.

Ethylbenzene is generated by household detergents, paint, oil refineries, automobile exhaust gas, etc., and workers in the petrochemical industry are exposed to benzene to cause poisoning. However, the diagnosis of ethylbenzene poisoning has been based on observation of clinical symptoms such as the amount of ethylbenzene in the blood, the analysis of the cytomegalovirus, the urine test, and the chromosomal abnormality.

Therefore, it is necessary to develop a biomarker capable of early diagnosis of ethylbenzene exposure in order to identify and treat ethylbenzene poisoning.

It is an object of the present invention to provide a biomarker for diagnosing ethylbenzene poisoning with high specificity.

In order to achieve the above object, the inventors of the present invention conducted studies to overcome the problems of the prior art. As a result, it was found that the expression of 2-dimensional electrophoresis in the group treated with ethylbenzene was significantly higher than that of normal control cells, The inventors of the present invention have invented a diagnostic biomarker using a protein which is remarkably lowered.

The biomarker for the detection of ethylbenzene poisoning according to the present invention can sensitively and specifically detect ethylbenzene poisoning by detecting the change in the expression level of at least one protein among the eight proteins.

The present invention provides a protein biomarker or immunogenic fragment thereof whose expression is markedly increased or decreased in the ethylbenzene exposure test group when compared with normal cells.

The protein biomarker is a transketolase having a molecular weight of about 68 kD and a pI of about 7.3, a molecular weight of about 59 kD, an aldehyde dehydrogenase family 7 analogous protein having a pI of about 7.99, a member Al (similar to aldehyde dehydrogenase family 7, member A1) Type I keratin KA17 having a molecular weight of about 48 kD and a pI of about 4.97, aflatoxin B1 aldehyde reductase having a molecular weight of about 37 kD and a pI of about 6.83, a rat having a molecular weight of about 26 kD and a pI of about 8.6 Glutathione S-transferase alpha-2, having a molecular weight of about 26 kD and a pI of about 8.89, a molecular weight of about 15 kD, a pI of about 7.98 (SEQ ID NO: (Β 1 globin), and a similar to hemoglobin β-2 subunit having a molecular weight of about 16 kD and a pI of about 8.91.

The immunogenic fragment refers to a portion of the protein capable of producing a reactive antibody when injected into an external host.

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

The present invention provides a diagnostic kit characterized by diagnosing ethylbenzene poisoning by an increase or decrease in the amount of the biomarker protein as compared with a normal control. The kit includes a kit for ELISA (Enzyme linked immunosorbent assay).

The present invention provides a method for diagnosing ethylbenzene poisoning through biomarker detection comprising contacting an antibody specifically binding to the biomarker protein with a biological sample selected from urine, blood, serum and plasma.

Further, the marker detection method of the present invention

a) providing a biological sample to be analyzed;

b) contacting the sample with an antibody that specifically binds to the one or more biomarker proteins;

c) quantitatively detecting the antigen-antibody complex; And

d) comparing the detection result of step c) with a normal control group.

In the present invention, the term "marker" is a substance capable of diagnosing ethylbenzene intoxication cells differentiated from normal cells, and may be a polypeptide marker for the biomarker protein. These markers are expressed in ethylbenzene- Or < / RTI >

In the present invention, "diagnosis" means identifying the presence or characteristic of an ethylbenzene poisoning state.

The increase or decrease in the amount of the biomarker protein expression in the biological sample can be detected by confirming the amount of the protein. The process of separating the protein from the biological sample can be performed using a known process. The amount of the protein can be measured by various methods known to those skilled in the art .

In the present invention, the "biological sample" is a tissue or cell capable of detecting a difference in the level of the biomarker protein, and is preferably, but not limited to, urine, blood, plasma, serum and the like.

In the present invention, "measurement of protein level" is a process of confirming the expression level of the biomarker protein expressed or decreased in the ethylbenzene intoxicated cell in a biological sample, preferably using an antibody that specifically binds to the biomarker protein To confirm the amount of protein.

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

Analysis methods for measuring protein levels using antibodies include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, Immunoprecipitation, Complement fixation assay, FACS, Protein chip, and the like, and the method is not limited thereto.

Antibody-antibody complex formation in the normal control group was compared with the amount of antigen-antibody complex formation in suspect patients with ethylbenzene poisoning, and the increase in the biomarker protein expression level was assessed through the above analysis method. Can be diagnosed.

The term "antigen-antibody complex" means a combination of the biomarker protein and the antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through the signal label of the detection label. The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule and a radioactive isotope, and is not limited to the substance described above. Examples of enzymes that can be used when the enzyme is used as a detection label include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase, and the like, and is not limited to the range described above. Fluorescent substances include fluorescein, phycocyanin, fluorescamine, and the like, and are not limited to the substances described above. Examples of the ligand include, but are not limited to, biotin derivatives and the like. The luminescent material includes, but is not limited to, luciferin. The fine particles include, but are not limited to, colloidal gold. Examples of redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, and hydroquinone. Radiation isotopes include, but are not limited to, ³ H, ¹⁴ C, and the like.

Determination of protein expression levels preferably uses an ELISA. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, an antibody against an antigen attached to a solid support, and another antibody recognizing the antigen And an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The degree of complex formation between the biomarker protein and the antibody can be checked to confirm whether or not ethylbenzene is poisoned.

Alternatively, one or more antibodies against a biomarker may be arranged at a predetermined position on a substrate to use a protein chip immobilized at a high density. A method for analyzing a sample using a protein chip is to separate the protein from the sample, hybridize the separated protein with the protein chip to form an antigen-antibody complex, and read the protein to confirm presence or expression of the protein .

Alternatively, Western blotting is performed using an antibody against the biomarker. The whole protein is separated from the sample, and the protein is separated according to size by electrophoresis, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the generated antigen-antibody complex can be confirmed using the labeled antibody to confirm the poisoning.

Such detection methods include comparing the biomarker protein expression level of the normal control with the expression level of the marker protein in the ethylbenzene intoxicated cell. Protein levels can be expressed as absolute or relative differences in biomarker protein.

The present invention relates to an ethylbenzene poisoning diagnostic kit comprising an antibody specifically binding to the biomarker protein.

In the present invention, since the eight kinds of biomarker proteins are identified as ethylbenzene poisoning markers, the antibody can be easily produced using techniques well known in the art. As is well known in the art to which the present invention pertains, a polyclonal antibody is obtained from an animal by injecting the biomarker protein antigen into the animal to obtain a serum containing the antibody. The animal can be produced using any animal host such as goat, rabbit, pig, etc. Monoclonal antibodies may be obtained from the hybridoma method (see Kohler & Milstein (1976) European J. Immunology 6 : 511-519) or the phage antibody library (Clackson et al. Nature , 352 : 624-628, 1991; Marks et al . , J. Mol. Biol. , 222 : 58, 1-597, 1991).

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

The phage antibody library method involves obtaining an antibody gene for a biomarker protein and expressing it in the form of a fusion protein on the surface of a phage to produce an antibody library in vitro and to prepare a monoclonal antibody Is a method of separating and manufacturing.

The antibody produced by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography, or the like.

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

The diagnostic kit of the present invention comprising an antibody that specifically binds to a biomarker protein is preferably a diagnostic kit for ELISA and can include an antibody specific for a control protein and can also detect an antigen- A reagent, such as a labeled secondary antibody, a chromophore, an enzyme conjugated to an antibody, and other substances capable of binding to the substrate or antibody thereof.

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited thereto.

Experimental Method

Example 1: Protein extraction

For the proteomic analysis, a normal control group treated with corn oil, 1/10 LD ₅₀ (350 mg / kg) and The liver tissues were separated from the rats treated with 1/2 LD ₅₀ (1750 mg / kg) of ethylbenzene for 72 hours, disrupted with a homogenizer, and resuspended in 7M urea, 2M thiourea, 4.5% CHAPS {3 [ (PH 8.8) containing 100 mM DTE (dithioerythritol) at a concentration of 1 mM. The shredded sample was centrifuged at 12,000 × g for 50 minutes at 15 ° C., and the resulting supernatant was used. The extracted proteins were stored at -70 ° C.

Example 2: Isoelectric point electrophoresis

Total protein isolated from rat liver tissue of normal control and experimental group was made up to 1 mg, and 450 μl of rehydration solution (7M urea, 2M thiourea, 4.5% CHAPS, 0.25% ampholytes, 100 mM DTE) was used to dissolve the protein. Protein of each condition dissolved in 450 μl of rehydrated solution was injected into IPG holder, and immobilized dry strip (pH 3-10 NL , GE Healthcare Bio-Science) for 18 hours. 200V / 200 Vhrs, 500V / 500Vhrs, 1,000V / 1,000Vhrs, 8,000V / 13,500Vhrs and 8,000V / 100,000Vhrs depending on the pH of the protein.

Example 3: Equilibration

The isoelectric point electrophoresis strips were subjected to primary equilibration for 20 minutes in primary equilibration buffer (6M urea, 0.375 M Tris-HCl (pH 8.8), 20% glycerol, 2% SDS, 25% acrylamide, 2 mM TBP) Secondary equilibration was performed for 20 minutes in a secondary 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.

Example 4: SDS-polyacrylamide gel electrophoresis

After making the 9% - 16% acrylamide gel, the strip with the isoelectric transfer was placed on the gel, fixed with 0.5% agarose gel, and then subjected to two dimensional electrophoresis. The protein was transferred according to the molecular weight by applying a current of 5 mA per gel for 2 hours and then applying 18 mA per gel for 10 hours.

Example 5: Gel staining and discoloration

The developed gel was incubated for 12 hours to confirm the protein separated from the gel using Coomassie blue G-250 staining solution (34% methanol, 0.1% Coomassie blue G-250, 17% ammonium sulfate, 3% phosphoric acid) And stained with 1% acetic acid.

Example 6: Gel image analysis

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

Example 7: Nano LC-MS / MS analysis

After in-gel digestion of each spot with trypsin for nano LC-MS / MS analysis, the peptides of the spot were analyzed with agilent 110 Series nano-LC and LTQ-Mass Spectrometer (Thermo Electron, San Jose, Calif.). The capillary column (150 mm x 0.075 mm) used in the LC-MS / MS analysis was purchased from Proxeon (Odense M, Denmark) and the slurry was placed in a 5 urn, 100 Å pore size Magic C18 stationary phase (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 with a straight line increase of 35% B at 5% B for 50 minutes, a linear increase of 60% B at 40% B for 20 minutes and an 80% B linear increase at 60% B for 5 minutes. The flow rate was maintained at 300 ml per minute of 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 adjusted to 200 ° C, and the spray was 1.50-2.0 kV. The normalized collision energy was adjusted to 35% for MS / MS. Sequest software was used to identify the peptide sequence. To obtain reliable results, delatCn ≥ 0.1 and Rsp ≤ 4 conditions were applied. In the case of Xcorr, Xcorr ≥ 1.5 was applied in charge state 1+, Xcorr ≥ 2.0 in charge state 2+, charge state 3+ , Xcorr ≥ 2.5 was applied, and the peptide identification> 0.1 was used to identify the protein as the cutoff. Peptides have been oxidized at various methionine residues and have been carboxyamidomethylation and carboxymethylation in cystein.

Example 8: Western blot analysis of glutathione S-transferase

A normal control group treated with corn oil, 1/10 LD ₅₀ (350 mg / kg) and Liver tissues were separated from rats treated with 1/2 LD ₅₀ (1750 mg / kg) of ethylbenzene for 72 hours and homogenized and then disrupted with 7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE Lt; / RTI > in a 40 mM Tris-HCl buffer (pH 8.8). The shredded sample was centrifuged at 12,000xg for 50 minutes at 15 ° C, and the resulting supernatant was taken to quantitate the protein. Then, the same amount of each protein sample was added to a buffer solution (0.05M Tris-HCl buffer (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) After mixing, the protein was completely denatured by heating at 100 ° C for 5 minutes. Samples and protein markers were separated by electrospray by SDS-PAGE on a running gel of 5% acrylamide stacking gel and 10% acrylamide. The separated proteins were transferred to a blotting kit with PVDF (Polyvinylidene Difluoride) membrane at 400 mA for 1 hour in a state filled with transfer buffer (192 mM glycine, 25 mM Tris, 20% methanol). (1: 3000/5% defatted milk blocking solution) at room temperature for 1 hour and 30 minutes, followed by washing with TBST buffer solution. Lt; / RTI > After washing several times for 15 minutes with TBST buffer solution, the cells were reacted with horseradish peroxidase-conjugated anti-goat IgG (1: 5000/5% skim milk blocking solution) for 30 minutes at room temperature. After washing several times for 15 minutes with TBST buffer solution, the membrane was reacted with ECL solution (Amersham) for 10 seconds, and then the signal was detected by exposure to X-ray film.

The PVDF membrane as above was incubated at 55 ° C for 40 min in a reprobing buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 100 mM β-mercaptoethanol) After the reaction, the cells were washed several times with TBST buffer solution and reacted at 4 ° C for one day in 5% defatting solution. Rat IgG-β-actin (1: 1000/5% skim milk blocking solution) at room temperature for 1 hour and 30 minutes. After washing several times for 15 minutes with TBST buffer solution, the cells were reacted with horseradish peroxidase-conjugated anti-mouse IgG (1: 12,000 / 5% skim milk blocking solution) for 30 minutes at room temperature. After washing several times for 15 minutes with TBST buffer solution, the membrane was reacted with ECL solution (Amersham) for 3 minutes, and the signal was detected by exposure to X-ray film.

result

The liver of rats treated with ethylbenzene was separated and proteome was applied. As a result, a total of 1170 spots were expressed in the normal control group. When 1/10 LD ₅₀ of ethylbenzene was treated, 1242 total, and ethyl of 1/2 LD ₅₀ In the case of benzene treatment, 1299 spots were confirmed. Among them, a total of 11 protein spots showing differences in expression between the normal control group and the ethylbenzene treatment group (Fig. 1). The characteristics of each spot are summarized in Table 1 below. Among these 11 proteins, the 10th and 11th spots are considered to be unsuitable for use as biomarkers because the protein expression increases and then decreases according to the ethylbenzene dose.

Spot No. Identified protein Accession No. Cov% pI M.W Change 1/10 LD ₅₀ 1/2 LD ₅₀ One Transketolase 1729977 25 7.3 68370 ↘ ↘ 2 similar to aldehyde dehydrogenase family 7, member A1 62664437 39 7.99 58748 ↗ ↗ 3 Type I keratin KA17 47087085 51 4.97 48122 ↗ ↗ 4 aflatoxin B1 aldehyde reductase 433611 45 6.83 36742 ↗ ↗ 5 aflatoxin B1 aldehyde reductase 433611 44 6.83 36742 ↗ ↗ 6 Chaina, New Crystal Forms Of A Mu Class Glutathione S-Transferase From Rat Liver 442967 52 8.6 25940 ↗ ↗ 7 Glutathion S-transferase alpha-2
(Glutathione S-transferase Ya-2) (GST Ya2) (GST 1b-1b) (GST A2-2) 121713 50 8.89 25559 ↗ ↗ 8 beta 1 globin 546056 82 7.98 15834 ↘ ↘ 9 Similar to Hemoglobin beta-2 subunit (Beta-2-globin) (Hemoglobin beta chain, minor-form) 109459168 82 8.91 15982 ↗ ↗ 10 디 아페페 항화염 13937379 51 8.8 10010 ↗ ↘ 11 Alpha-2u globulin 204264 46 5.4 17340 ↗ ↘

Spots 2, 6, 7, and 9 showed a 1.5-fold increase in expression in the ethylbenzene-treated group of 1/10 LD _{50 and} the ethylbenzene-treated group of 1/2 LD ₅₀ than the normal control group. On the other hand, spots 1 and 8 decreased expression by more than 1.5 times and spots 10 and 11 were increased in ethylbenzene treated group 1/10 LD ₅₀ compared to the normal control group but decreased in the ethylbenzene treated group of 1/2 LD ₅₀ . In spots 3, 4 and 5, the expression was minimal in the normal control group, but the expression change was more than 2 times in the ethylbenzene-treated group of 1/10 LD _{50 and} the ethylbenzene-treated group of 1/2 LD ₅₀ .

Spot # 1 transketolase catalyzes another critical reaction of the pentose phosphate pathway and one of the most important intermediate products of this pathway is phosphorylated pentose, ribose-5-phosphate, phosphate, which is necessary for the synthesis of ATP, GTP, nucleic acid, DNA, RNA, NADP, etc. Since transketolase is degraded from the early stage of thiamine binding, it is used for the evaluation of thiamine nutritional status and its activity in red blood cells.

Spot No. 3 keratin KA17, Type I keratin KA17, has been shown to be involved in activating protein synthesis machinery necessary for cell growth for regeneration. The keratin protein is a basic component of the cytoskeletal fibrous polymer known as the keratin intermediate filament and has been reported to help cells resist physical damage.

Spots 4 and 5 are aflatoxin B1 aldehyde reductase, and aflatoxin B1 has been reported to cause necrosis around the portal vein in mice and centrilobular necrosis in mice (Goldblatt, LA 1969) "Aflatoxin ", Academic Press, New York, pp. 472). Aflatoxin B1 aldehyde reductase is involved in the metabolism of aflatoxin B1, an environmental carcinogen, as the Aldo-Keto reductase 7 family. It is known that this enzyme gene is regulated through an antioxidant responsive element (ARE) in the promoter region (Elizabeth M. Ellis et al. (2003) Characterization of the rat aflatoxin B1 aldehyde reductase gene, AKR7A1 Structure and chromosomal localization of AKR7A1 as well as identification of antioxidant response elements in the gene promoter, Carcinogenesis, 24 (4), 727-737).

Glutathione S transferase, which is spot 6 and 7, is one of the important defense mechanisms against oxidative damage. It is conjugated with glutathione, a very important antioxidant in the cell, It is known to be involved in the detoxification of tic compounds. The glutathione S-transferase also has broad substrate specificity, protecting cells from a wide range of toxic chemicals (Sherratt, PJ, Manson, MM, Thomson, AM, Hissink, EA, Neal, GE, van Bladcren, and Skov, KA (1984) The contribution of hydroxyl radical to radiosensitization: a study of DNA damage. Radiat Res., 99, 502-510).

Western blotting of the glutathione s-transferase protein, which showed a 1.5-fold change in expression at spot 7, resulted in an increase in protein in the 1/2 LD ₅₀ ethylbenzene-treated group compared to the normal control (Fig. 2).

Similar to aldehyde dehydrogenase family 7 (member A1), a member of the aldehyde dehydrogenase family, SpO2, is increased by ethylbenzene, which oxidizes aliphatic or aromatic aldehydes to form carboxy acids carboxylic acid, and is known to play an important role in the degradation of the neurotransmitter as well as the liver detoxification process.

The spot-8 beta 1 globin and the hemoglobin beta-2 subunit of spot 9 are known to be involved in blood gas exchange.

It was first discovered in the present invention that these eight proteins are useful as specific and sensitive biomarkers for ethylbenzene poisoning.

FIG. 1A is a 2-D gel photograph of a normal control group administered with corn oil. FIG.

1B is a 2-D gel photograph of a group treated with 1/10 LD ₅₀ ethylbenzene.

1C is a 2-D gel photograph of the group treated with 1/2 LD ₅₀ ethylbenzene.

2 is a Western blot photograph of the glutathione S-transferase of the normal control group and the 1/2 LD ₅₀ ethylbenzene treatment group.

Claims (4)

Transketolase having a molecular weight of 68 kD, pI 7.3; Aldehyde dehydrogenase family 7 analogous protein, member A1 (similar to aldehyde dehydrogenase family 7, member A1) with a molecular weight of 59 kD and a pI of 7.99; Type I keratin KA17 with a molecular weight of 48 kD and a pI of 4.97; Aflatoxin B1 aldehyde reductase having a molecular weight of 37 kD and a pI of 6.83; Class Glutathione S-transferase from rat liver with a molecular weight of 26 kD and a pI of 8.6; Glutathione S-transferase alpha-2 with a molecular weight of 26 kD and a pI of 8.89; A beta 1 globin with a molecular weight of 15 kD and a pI of 7.98; And A similar to hemoglobin beta-2 subunit with a molecular weight of 16 kD and a pI of 8.91; Wherein the antibody specifically binds to at least one biomarker protein selected from the group consisting of: The method according to claim 1, Wherein the kit is an ELISA (Enzyme Linked Immunosorbent Assay) kit. Transketolase having a molecular weight of 68 kD, pI 7.3; Aldehyde dehydrogenase family 7 analogous protein, member A1 (similar to aldehyde dehydrogenase family 7, member A1) with a molecular weight of 59 kD and a pI of 7.99; Type I keratin KA17 with a molecular weight of 48 kD and a pI of 4.97; Aflatoxin B1 aldehyde reductase having a molecular weight of 37 kD and a pI of 6.83; Class Glutathione S-transferase from rat liver with a molecular weight of 26 kD and a pI of 8.6; Glutathione S-transferase alpha-2 with a molecular weight of 26 kD and a pI of 8.89; A beta 1 globin with a molecular weight of 15 kD and a pI of 7.98; A similar to hemoglobin beta-2 subunit with a molecular weight of 16 kD and a pI of 8.91; Contacting the antibody that specifically binds to at least one biomarker protein selected from urine, blood, serum, and plasma with a biological sample selected from urine, blood, serum, and plasma. The method of claim 3, a) providing a biological sample to be analyzed; b) contacting the sample with an antibody that specifically binds to the biomarker protein; c) quantitatively detecting the antigen-antibody complex; And and d) comparing the detection result of step c) with a normal control group.

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