KR101395918B1 - Biomarkers for diagnosing the addiction of dichloromethylene - Google Patents

Abstract

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

Methylene chloride, biomarker, toxicity, poisoning

Description

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

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

Dichloromethylene is a kind of volatile organic compounds, which is also called methylene chloride, methylene dichloride, methylene dichloride, and has the formula CH ₂ Cl ₂ and a molecular weight of 84.93. It is a volatile liquid. It is toxic when inhaled and soluble in most organic solvents and is a solvent used in the chemical process of organic compounds. It has a specific gravity of 1.336 (20 ℃), a melting point of -96 ℃, a boiling point of 40.2 ℃ and a refractive index of 1.4216. It is miscible with benzene, ethanol, ether, chloroform and carbon disulfide. Chlorinated methylene chloride or chloroform with zinc and acetic acid gives a similar odor to chloroform. It was once used as a raw material and solvent to make ethylene glycol and polysulfide rubber. Today, it is mainly used for the manufacture of vinyl chloride and gasoline Is used as a component of cyethylpentane which is an antioxidant.

Methylene chloride can be used as a solvent for paints, printed circuit board cleaners, metal degreasing cleaners, urethane foaming aids, aerosol sprays, low boiling point organic solvents (nonflammable films, fats, alkaloids, resins, rubbers, waxes, It is used for reaction solvent of polycarbonate, refrigerant, racquet, fabric and leather, analysis, linoleum, ink, extinguishing agent and so on. It is also used in the food industry to remove caffeine from coffee and extracts from hops and other plants to produce seasonings.

Methylene chloride, which has high volatility, contains health hazards that cause acute inhalation and cause carbon monoxide poisoning due to the metabolism of carbon monoxide. Prolonged contact with skin can cause skin irritation or chemical laceration due to the dissolution of methylene chloride in some adipose tissue. It has been suggested that methylene chloride may be absorbed into the placenta and affect the fetus when exposed during pregnancy, but it has not been proven yet. Animal studies showed fetal toxicity but did not cause anomalies. In the International Agency for Research on Cancer (IARC), methylene chloride has been identified as a potential group 2B for human carcinogenicity.

It is an object of the present invention to provide a biomarker for methylene chloride poisoning diagnosis with high specificity.

In order to achieve the above object, the inventors of the present invention conducted a two-dimensional electrophoresis technique in which a protein significantly increased in expression or markedly lowered in the methylene chloride treatment test group as compared with a normal control cell was selected and diagnosed as methylene chloride poisoning And to develop a diagnostic kit using the same.

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

The present invention provides a protein biomarker or immunogenic fragment thereof whose expression is markedly increased or decreased in the methylene chloride exposure test group as compared to normal cells.

The protein biomarker has an A chain (glutathione transferase mutant Y115f) of a glutathione transferase mutant Y115f having a molecular weight of about 26 kD and a pI of 8.6, a glutathione S-transferase having a molecular weight of about 25 kD and a pI of 9.1, mitochondrial), a molecular weight of about 26 kD, a glutathione S-transferase alpha-2 of pI 8.9, a molecular weight of about 16 kD, a beta 1 globin of pI 8, a molecular weight of about 16 kD, a pI 9 hemoglobin beta-2 subunit, and alpha-2u globulin having a molecular weight of about 17 kD and a pI of 5.4.

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 methylene chloride poisoning by an increase or decrease in the amount of the biomarker protein as compared to a normal control. The kit includes a kit for ELISA (Enzyme linked immunosorbent assay).

The present invention provides a method for detecting methylene chloride 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" may be a polypeptide marker for the biomarker protein, which can be diagnosed by distinguishing methylene chloride intoxication cells from normal cells. These markers are expressed in methylene chloride- Or < / RTI >

In the present invention, "diagnosis" means confirming the poisoning or characteristic of the methylene chloride 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 for confirming the expression level of the biomarker protein expressed or increased in methylene chloride-intoxicated cells in a biological sample, preferably 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 and the amount of antigen-antibody complex formation in the suspect patients with methylene chloride poisoning could be compared with each other and the increase of the biomarker protein expression level was judged by 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 confirmed to confirm methylene chloride poisoning.

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 amount of biomarker protein expression in a normal control with the expression level of a marker protein in methylene chloride-intoxicated cells. Protein levels can be expressed as absolute or relative differences in biomarker protein.

The present invention relates to a methylene chloride poisoning diagnostic kit comprising an antibody that specifically binds to the biomarker protein.

In the present invention, since the seven types of biomarker proteins have been identified as methylene chloride poisoning markers, the antibody can be easily produced using techniques well known in the art. As is well known in the art to which this invention pertains, a polyclonal antibody is obtained by injecting an animal with the biomarker protein antigen and collecting the antibody-containing serum from the animal. 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

<Protein Extraction>

For proteomic analysis, the normal group treated with corn oil, 1/10 LD ₅₀ (160 mg / kg) and The liver tissues were separated from the rats treated with 1/2 LD ₅₀ (800 mg / kg) of methylene chloride for 72 hours, disrupted with a homogenizer, and then treated with 7M urea, 2M thiourea, 4.5% CHAPS {3 [(3-Cholamidopropyl) dimethylammonio] -propanesulfonic acid}, and 100 mM DTE (dithioerythritol) at pH 8.8. 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.

<Isoelectric point electrophoresis>

Total protein isolated from normal liver and rat liver tissues in the experimental and control groups was 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 a 24 cm fixed drying strip (pH 3-10 NL, GE Healthcare Bio- Science) for 18 hours. 200V / 200Vhrs, 500V / 500Vhrs, 1,000V / 1,000Vhrs, 8,000V / 13,500Vhrs and 8,000V / 100,000Vhrs depending on the pH of the protein.

<Equilibrium>

The isoelectric point electrophoresis strips were subjected to primary equilibration for 20 minutes in primary equilibration buffer (6 M 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.

&Lt; 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% Agrose gel, and then subjected to two dimensional electrophoresis. The cells were first developed with a current of 5 mA per gel for 2 hours, and then subjected to a current of 18 mA per gel for 10 hours to transfer the protein according to the molecular weight.

&Lt; Gel dyeing and bleaching &

The developed gel was incubated with a Coomassie blue G-250 staining solution (34% methanol, 0.1% Coomassie G-250, 17% ammonium sulfate, 3% phosphoric acid) for more than 12 hours After staining, it was decolorized with 1% acetic acid.

<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.

<Nano LC-MS / MS analysis>

After in-gel digestion of each spot with trypsin for nano LC-MS / MS analysis, the peptides of the spots were analyzed using agilent 110 Series Nano LC and LTQ-mass spectrometer (Thermo Electron, San Jose, CA). Capillary columns (150 mm x 0.075 mm) used in the LC-MS / MS analyzes were purchased from Proxeon (Odense M, Denmark) and slurries were prepared in 5 탆, 100 Å pore size Magic C18 stationary phase (Michrom Bioresources, Auburn, CA) Lt; / RTI > 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 (400-1800m / 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 1.5.0-2.0 kV. The normalized collision energy was adjusted to 35% for MS / MS. Sequest software has been used to identify peptide sequences. To obtain reliable results, delatCn ≥ 0.1 and Rsp ≤ 4 were applied. In the case of Xcorr, Xcorr ≥ 1.5 was applied in charge state 1+, Xcorr ≥ 2.0 in charge state 2+, Xcorr ≥ 2.5, and protein identity> 0.1 was used to identify the protein as the cutoff. Peptides have been oxidized at various methionine residues, and carboxyamidomethylation and carboxymethylation have been variously carried out in cysteine.

result

The liver of rats treated with methylene chloride 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 (160 mg / kg) of methylene chloride was treated, When methylene chloride of LD ₅₀ (800 mg / kg) was treated, 1376 spots were observed. Among them, there were 7 protein spots showing a difference in expression between the normal control group and methylene chloride treatment group (Fig. 2).

Spot No. Identified protein Accession No. Cov% pI M.W Change 1/10 LD ₅₀ 1/2 LD ₅₀ One Chaine, glutathione transferase mutant Y115f 29726512 47 8.6 25920 ↘ ↘ 2 glutathione S-transferase, mitochondrial 310077128 69 9.13 25493 ↘ ↘ 3 Glutathion S-transferase alpha-2
(Glutathione S-transferase Ya-2) (GST Ya2) (GST 1b-1b) (GST A2-2) 121713 50 8.89 25559 ↘ ↘ 4 beta 1 globin 546056 82 7.98 15834 ↘ ↘ 5 Similar to Hemoglobin beta-2 subunit (Beta-2-globin) (Hemoglobin beta chain, minor-form) 109459168 87 8.91 15982 ↘ ↘ 6 Alpha-2u globulin 204264 46 5.4 17340 ↘ ↘

(Chain A, glutathione transferase mutant Y115f) of glutathione transferase mutant Y115f with a molecular weight of about 26 kD, pI 8.6, spot number 1, a Glutathione S-transferase having a molecular weight of about 25 kD, pI 9.1, (Glutathione S-transferase alpha-2) having a pI of 8.9, a beta 1 globin having a molecular weight of about 16 kD and a pI of 8, a spot number 4, a molecular weight of about 26 kD, , A spot-5 hemoglobin beta-2 subunit with a molecular weight of about 16 kD, pI 9 and an alpha-2u globulin with a molecular weight of about 17 kD and a pI of 5.4, It was confirmed that protein expression was decreased in dependence on methylene chloride concentration (FIG. 3A).

A chain of glutathione transferase mutation Y115f (Chain A, glutathione transferase mutant Y115f), spot 2 mitochondrial glutathione S-transferase, mitochondrial, spot 3 glutathione S - Glutathione S-transferase alpha-2 catalyzes the linkage between reduced glutathione and a number of electrophilic substances to protect cells from damage caused by oxidative cytotoxic or carcinogenic substances . Glutathione S transferases also have broad substrate specificity to protect cells from a wide range of toxic chemicals, including carcinogens, therapeutic drugs, environmental toxins and oxidative stress.

A spot 1 globin, a spot 5 hemoglobin beta-2 subunit, a spot 6 hemoglobin beta-2 subunit-like protein (similar to Hemoglobin beta-2 subunit) -2 subunit has heme in hemoglobin and is known to play a role of oxygen transport and binding with sickle hemoglobin.

Spot-7 alpha-2u globulin is a small protein of 20 kDa that can be found in the liver, mandible, lacrimal gland, foreskin, and mammary gland of rats. It is also known as the major urinary protein and belongs to the lipocalin protein group. It is complexly regulated with hormones and is expressed in a specific way depending on tissue, age, and sex. This protein is mainly expressed in the liver and is removed from the plasma through kidneys and excretion. It is reported that male rats are more likely to have bladder cancer than humans because of the high concentration of alpha-2u globulin and albumin contained in the urine.

It has been found for the first time in the present invention that these six proteins are useful as specific and sensitive biomarkers for methylene chloride poisoning.

FIG. 1 is a diagram showing protein change by methylene chloride. FIG.

(A): control _{group, (B): 1/10 LD 50} (160 mg / kg) in methylene chloride-treated _{group, (C): 1/2 LD 50} (800 mg / kg) in methylene chloride-treated group

2 is a graph showing changes in protein expression of each spot by methylene chloride.

Example 1: 1/10 LD ₅₀ (160 mg / kg) methylene chloride treated group, Example 2: 1/2 LD ₅₀ (800 mg / kg) methylene chloride treatment group

Claims (4)

Chain A of glutathione transferase mutant Y115f (Chain A, glutathione transferase mutant Y115f) having a molecular weight of 26 kD and a pI of 8.6; Mitochondrial glutathione S-transferase (mitochondrial) with a molecular weight of 25 kD, pI 9.1; Glutathione S-transferase alpha-2 with a molecular weight of 26 kD, pI 8.9; Beta 1 globin with a molecular weight of 16 kD, pI 8; A similar to Hemoglobin beta-2 subunit with a molecular weight of 16 kD, pI 9; And Alpha-2u globulin with a molecular weight of 17 kD, pI 5.4; Wherein the antibody specifically binds to at least one biomarker protein or immunogenic fragment thereof selected from the group consisting of: The method according to claim 1, Wherein the kit is an ELISA (Enzyme Linked Immunosorbent Assay) kit. Chain A of glutathione transferase mutant Y115f (Chain A, glutathione transferase mutant Y115f) having a molecular weight of 26 kD and a pI of 8.6; Mitochondrial glutathione S-transferase (mitochondrial) with a molecular weight of 25 kD, pI 9.1; Glutathione S-transferase alpha-2 with a molecular weight of 26 kD, pI 8.9; Beta 1 globin with a molecular weight of 16 kD, pI 8; A similar to Hemoglobin beta-2 subunit with a molecular weight of 16 kD, pI 9; And Alpha-2u globulin with a molecular weight of 17 kD, pI 5.4; Contacting an antibody that specifically binds to at least one biomarker protein or immunogenic fragment thereof selected from the group consisting of 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.

Images