KR100788789B1 - Bio-marker proteins for diagnosing exposure to formaldehyde - Google Patents

Abstract

A bio-marker protein in plasma is provided to diagnose exposure to formaldehyde and the degree of the exposure effectively. And an agent for diagnosing exposure to formaldehyde comprising an antibody against the same is provided. A bio-marker for diagnosing exposure to formaldehyde comprises a protein of which expression is changed in accordance to the formaldehyde exposure and which is selected from the group consisting of SNAP-23 having an amino acid sequence of SEQ ID : NO. 1, the molecular weight of 22-24kD and the pI of 4.3-5.3; apolipoprotein A-1 having an amino acid sequence of SEQ ID : NO. 2, the molecular weight of 29-31kD and the pI of 5.0-6.0; apolipoprotein E having an amino acid sequence of SEQ ID : NO. 3, the molecular weight of 34-36kD and the pI of 4.7-5.7; clusterin having an amino acid sequence of SEQ ID : NO. 4, the molecular weight of 24-26kD and the pI of 4.4-5.4; Fibrinogen gamma having an amino acid sequence of SEQ ID : NO. 5, the molecular weight of 49-51kD and the pI of 4.9-5.9; and kinesin having an amino acid sequence of SEQ ID : NO. 6, the molecular weight of 29-31kD, and the pI of 4.4-5.4. An agent for diagnosing exposure to formaldehyde comprises an antibody specifically binding to the protein as an effective ingredient.

Description

Bio-marker proteins for diagnosing exposure to Formaldehyde}

1A is a 2-DE pattern of plasma proteins obtained using 3-5.6 pI strips, and FIG. 1B is a graph comparing the relative intensities of the spots that change with exposure of FA.

FIG. 2A is a 2-DE pattern of plasma proteins obtained using 5.3-6.5 pi strips, and FIG. 2B is a graph comparing the relative intensity of spots that change with exposure of FA.

3A is a 2-DE pattern of plasma proteins obtained using 6-9 pI strips, and FIG. 3B is a graph comparing the relative intensities of spots that change with exposure of FA.

4A is a Western blot photograph of (1) SNAP-23, (2) Apo A-1, (3) Apo E, (4) clusterin, (5) fibrinogen γ, (6) kinesin and (7) tubulin, 4B is a graph comparing the relative intensities of (1) SNAP-23, (2) Apo A-1, (3) Apo E, (4) clusterin, (5) fibrinogen γ, and (6) kinesin to tubulin .

FIG. 5A is a Western blot image of (1) IL-4, (2) IFN-γ and (3) tubulin, and FIG. 5B shows the relative intensities of (1) IL-4 and (2) IFN-γ in tubulin It is a graph comparing.

6 is a 2-DE immunoblot pattern of Apo E in (A) 0 ppm (control), (B) 5 ppm and (C) 10 ppm FA exposed mice.

The present invention relates to the study of toxic protein biomarkers, and specifically, biomarker proteins, antibodies, and the like, which can diagnose and toxicologically monitor the exposure of formaldehyde, known as a carcinogenic substance, to environmental pollutants. It relates to a kit that can be used to diagnose the extent and extent of formaldehyde exposure.

Formaldehyde (FA) is a low molecular weight organic compound and is one of the main causes of SBS (Nakazawa et al., Industrial Health. 2005, 43 (2), 341-5). FA is widely used in workplaces and homes and is commonly used in construction, cloth, paper, resin, wood, insulation, paints, plastics, textiles, adhesives, and cosmetics. Exposure to low levels of FA is suspected of causing or exacerbating airway inflammation mediated by immunological and neurological responses (Fujimaki et al., Toxicology. 2004, 197, 1-13).

FA has been classified as a potential human carcinogen based on animal testing with neoplastic lesions in the contact area and nasal cavity, but there is limited evidence of human respiratory carcinogenicity (Liteplo et al., J. Toxicol. Environ. Health). 2003, Part B 85-114). FA is also a well-known cross-linking agent that can react with many polymers, such as proteins and nucleic acids, or with small molecule materials such as amino acids (Cheng et al., Chem. Res. Toxicol. 2003, 16, 145). -52). Thus, FA-induced levels of DNA adducts in nasal cells can be used as biomarkers and effects of FA exposure (Zhong W .; Que Hee S. S. Mutat. Res. 2004, 563, 13-24).

Genotoxicological studies have shown that low concentrations of FA in environmentally induced DNA damage, growth inhibition and delayed DNA repair after UV irradiation in various human cell types, primary cultures of mouse organ endothelial cells, and mouse cells. (Emri et al., Experimental Dermatology. 2004, 13, (5), 305-15). Previous studies have shown that toxicological effects such as DNA single chain cleavage and the formation of DNA adducts alter gene regulation and protein expression in living organisms (Oh et al., Proteomics. 2004, 4, 3514-3526).

Recently, proteome assays have been introduced as a means of analyzing differential gene expression and identifying biomarkers at the protein level. However, proteomics research has been developed mainly for disease-related studies, and protein studies on toxic substances have been insufficient. Toxicological studies have only recently been published on the discovery of toxicity protein indicators for dioxins and polycyclic aromatic hydrocarbons. In particular, although formaldehyde has recently attracted attention as a representative source of Sick Building Sydrome (SBS), the biological exposure indicators that have been researched and developed are extremely insignificant. In particular, since plasma proteins are composed of a large number of high molecular weight molecules, 2-DE analysis has limitations in terms of plasma protein analysis.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, and as a result, mice exposed to FA using a large 2-DE system and three different pI strips to increase the resolution of proteome analysis Through the 2-DE analysis of the six plasma proteins that specifically react to formaldehyde (plasma) to identify and present as a formaldehyde toxic protein biomarker.

Therefore, the main object of the present invention is to provide a new biomarker protein in plasma that can effectively diagnose the extent and extent of formaldehyde exposure.

Another object of the present invention to provide an antibody and a diagnostic kit using the biomarker protein.

According to an aspect of the present invention, the present invention provides a biomarker for diagnosing formaldehyde exposure, comprising as an active ingredient a protein selected from the following group whose expression changes according to formaldehyde exposure:

Synaptozomal-associated protein 23 (SNAP-23) having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 22-24 kD and a pi of 4.3-5.3;

Apolipopreotein A-1 having the amino acid sequence of SEQ ID NO: 2 and having a molecular weight of 29-31 kD and a pi of 5.0-6.0;

Apolipopreotein E having an amino acid sequence of SEQ ID NO: 3 and having a molecular weight of 34-36 kD and a pi of 4.7-5.7;

Clustererin having the amino acid sequence of SEQ ID NO: 4 and having a molecular weight of 24-26 kD and a pi of 4.4-5.4;

Fibrinogen γ having an amino acid sequence of SEQ ID NO: 5 and having a molecular weight of 49-51 kD and a pi of 4.9-5.9; And,

Kinesin having the amino acid sequence of SEQ ID NO: 6 and a molecular weight of 29-31 kD and a pi of 4.4-5.4.

In the present invention, the expression is changed according to formaldehyde exposure means that the expression is up (down) or down (down). In addition, the molecular weight and pI are the values identified on two-dimensional electrophoresis and include generally accepted experimental error ranges.

According to another aspect of the present invention, the present invention provides a diagnostic agent for formaldehyde exposure comprising an antibody that specifically binds to the biomarker protein of the present invention as an active ingredient.

The antibody of the present invention may be a polyclonal antibody, but is preferably a monoclonal antibody. Polyclonal antibodies can be prepared by injecting a biomarker protein or fragment thereof as an immunogen into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection and are usually administered with an adjuvant to increase antigenicity. Blood is collected periodically from external hosts to collect serum that shows improved titers and specificity for the antigen or to separate and purify antibodies therefrom.

Monoclonal antibodies can be prepared by immortalized cell line generator technology (Koeher and Milstein (1975) Nature, 256: 495) by fusions known to those skilled in the art. The manufacturing method is briefly described as follows. First, 20 μg of pure protein is immunized to Balb / C mice or a peptide is synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and only hybridoma cells that produce the desired monoclonal antibody using ELISA method. After selection and propagation, monoclonal antibodies are isolated and purified from the culture.

According to another aspect of the present invention, the present invention provides a method for diagnosing formaldehyde in a subject, comprising detecting the presence of the biomarker protein of the present invention or an immunogenic fragment thereof in a body fluid of the subject.

In the diagnostic method of the present invention, the bodily fluid is plasma or serum, and the detecting step directly detects the presence of a biomarker protein or immunogenic fragment thereof by two-dimensional electrophoresis (2-DE) from the bodily fluid of the subject, Contacting the bodily fluid with the antibody of the invention to indirectly confirm the presence of the biomarker protein or immunogenic fragment thereof through an antigenic antibody reaction. Currently known immunoassay as an antigen antibody reaction, enzyme immunoassay (ELISA, Coated tube), antibody-bound magnetic particles are bound to a tube, and then antigen-tracer and refractory contaminants are reacted competitively to induce enzymatic reaction. Magnetic particle method, latex particle method using antibody bound latex particle.

According to another aspect of the present invention, the present invention provides a formaldehyde exposure diagnostic kit comprising an antibody that specifically binds to the biomarker protein of the present invention as an active ingredient.

Diagnostic kits of the present invention are prepared by conventional methods known to those skilled in the art, and typically include lyophilized antibodies, buffers, stabilizers, inactive proteins and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes and the like.

The monoclonal antibody of the present invention can be used in a variety of immunoassay kits (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), as well as various environmental toxicity through the development of antibodies showing higher specificity and sensitivity It can also be used to develop protein chips with a detection spectrum.

Hereinafter, the present invention will be described in more detail.

In the present invention, the procedure for analyzing proteomics in plasma of FA exposed animals is as follows.

 ① After collecting blood from exposed rat, plasma is separated and used as analytical sample

 ② Conduct electrophoresis for primary protein separation: IEF of various detailed ranges (pH3 ~ 5.6, 5.3 ~ 6.5, 6 ~ 9)

 ③ Electrophoresis for secondary separation of protein: Large size (35x45cm) SDS gel

 ④ After silver staining the protein of separated SDS gel, identify the spot of protein that is changed according to formaldehyde concentration by image analyzer.

 ⑤ Check peptide information of spot changed by MALDI-TOF / MS

 ⑥ Confirm the protein name by searching the peptide information obtained by MS-FIT

 ⑦ Western blot using monoclonal antibody to verify the accuracy of the changed protein

 ⑧ Proteomics 2-DE Result Confirmation of protein consistent with westernblot result as toxic protein indicator.

In the present invention, by applying two new techniques for protein analysis, the protein resolution was improved to improve the reliability of the results.

① Improved electrophoretic performance for protein primary separation using Immobiline Dry Strip Gel of various detail ranges (pH3 ~ 5.6, 5.3 ~ 6.5, 6 ~ 9).

② The electrophoresis performance for protein secondary separation was improved by using a large size (35x45cm) SDS gel that was arbitrarily manufactured than the 24x20cm that was previously sold.

As described above, a total of 3757 protein spots of plasma protein analysis were isolated by applying a new protein analysis technique. Expression of a total of 32 spots was changed by FA exposure, including 19 spots and 13 spots. The results of the changed proteins by pH range of the primary electrophoretic strip are as follows: (pH 3 ~ 5.6: 7 increase, 8 decreases), (pH 5.3 ~ 6.5: 6 increase, 4 decreases), ( pH 6-9: 6 increases, 1 decrease).

Results of protein name identification through MALDI-TOF / MS and MS-FIT of the proteins whose expression is changed according to the exposure of the formaldehyde are shown in Tables 1 and 2. Among the identified protein names, we purchased a monoclonal antibody that is commercially available and sold, and performed westerblot to find and verify the protein that matches the results. A total of six proteins (SNAP-23, Apolipopreotein A-1, Apolipopreotein E, Clusterin, Fibrinogen γ, and Kinesin) have been shown to specifically change in relation to formaldehyde exposure and have been suggested as indicators of toxic proteins in rat plasma.

In the proteomic analysis of the present invention, lipoxygenase expression was found to be up-regulated in exposed rat plasma in a dose dependent manner. Lipoxygenases are a family of enzymes that dioxygenate unsaturated fatty acids and initiate the synthesis of lipid peroxidation and signaling molecules in the membrane. As a result, they induce structural and metabolic changes in cells under various pathological conditions. Recently, lipoxygenase and hydroperoxides have been reported to have pro-apoptotic effects leading to cell death in different cells and masons (Maccarrone et al., Cell Death & Differentiation. 2001, 8 (8), 776-84).

In the present invention, FA exposure induced Crk expression in rat plasma. Smith et al. Reported that adapter protein C is required for apoptosis signaling of egg extracts, including the regulation of this cell growth and cell motility (Kornbluth S. Journal of Cell Biology. 2000, 151 (7), 1391- 400).

In the present invention, sulfotransferases and glutathione S-transferases (GSTs) were significantly up-regulated by FA. Sulfotransferases catalyze sulfonation, and cytoplasmic sulfotransferases are known to be involved in detoxification, hormone regulation and drug metabolism (Smith et al., Molecular & Cellular Biology. 2002, 22 (5), 1412- 23). GSTs are multifunctional and multigene products. GST enzyme has traditionally been known as a detoxifying agent of electrophiles by glutathione conjugation. Meanwhile, another antioxidant molecule, heme oxygenases (HO), was significantly down-regulated in plasma. HO is a stress-responsive enzyme involved in the catabolism of ham and is known as a novel defense factor with potent anti-inflammatory, antioxidant and anti-proliferative effects (Exner et al., Free Radical Biology & Medicine. 2004, 37 ( 8), 1097-104)

In the present invention, it has been found that some apoproteins, including apo A, E, and J, are down or up regulated. Clusterin / Apolipoprotein J (Apo J) is a multifunctional glycoprotein of heterodimers, expressed in various tissues and found in all human fluids (Ogborne et al., Biochemical Society Transactions. 2004, 32 (Pt 6), 1003-5). Its demonstrated and suggested functions include the transport of lipoproteins, complement-mediated cytolysis and regulation of cell-cell interactions. In light of previous studies, clusterin exerts a protective function on surviving bystander cells as cell viability genes (Trougakos et al., International Journal of Biochemistry & Cell Biology. 2002, 34 (11), 1430-48). In cells undergoing apoptosis, clusterin expression is not enhanced but rather down-regulated. In the present invention, clusterin expression in plasma has been dose-dependently down-regulated and may be a useful biomarker of FA exposure.

In the present invention, Apo A was significantly down-regulated in the high acid range (pI 3.0-5.6). The formation of methionine sulfoxide in apolipoproteins AI and AII is an early event accompanying lipid peroxidation (Koch-Brandt C .; Morgans C. Progress in Molecular & Subcellular Biology. 1996, 16, 130-49). Apolipoprotein E (Apo E) is a 34-kDa lipid-associated protein present in the plasma and central nervous system. Previous studies have demonstrated that Apo E has multiple functions including the ability to transport lipids, regulate cell homeostasis and inhibit lipid oxidation. Moreover, the receptor binding domain of Apo E is responsible for its antioxidant activity (Garner et al., Journal of Biological Chemistry. 1998, 273 (11), 6080-7). In the present invention, Western blotting showed that Apo E expression was significantly up-regulated in plasma with increasing FA concentrations, which was due to an increase in blood cell levels of DNA damage, lipid peroxidation and protein oxidation in rats exposed. It plays a role in homeostasis and antioxidant activity. Moreover, the 2-DE immunoblot pattern showed that the expression of several isoforms of Apo E is down or up regulated. Thus, they can be specific biomarkers of FA in plasma.

In the present invention, Fructose 1, 6-biphosphatase, inositol-1-monophosphatase, myosin heavy chain, calcium binding protein p22 was significantly up-regulated, while kinesin and guanine nucleotide binding proteins were significantly down-regulated. Kinesins are a superfamily of mechanochemical enzymes and are motor proteins that transport membrane organelles and macromolecules required for cellular function along microtubules (Pham et al., Biochemistry. 2005, 44 (20), 7577-82). Moreover, recent studies have shown that kinesins play an important role in the dynamics of mitotic spindle assembly, chromosome division, and the formation of neuronal connections (Hirokawa et al., Experimental Cell Research. 2004, 301 (1), 50-9).

fibrinogens are a major group of plasma coagulation proteins that bind to cell surface receptors, growth factors and coagulation factors using gamma chains and play an important role in fibrin clot formation, platelet aggregation and wound healing (Wordeman L. Current Opinion in Cell Biology. 2005, 17 (1), 82-8). Fibrinogen has been suggested to be a useful biomarker of vascular disease and inflammation (Albert MA .; Ridker PM. Source Reviews in Cardiovascular Medicine. 2004, Suppl 3, S22-7), and low levels of FA exposure are immunological and neurological It has been suspected of causing or exacerbating airway inflammation mediated by the response (Fujimaki et al., Toxicology. 2004, 197, 1-13). Moreover, some cytokines play an important role as inducers and regulators of fibrinogen biosynthesis. For example, interleukins 4, 10, and 13 down-regulate the biosynthesis of fibrinogen (Vasse et al., Br. J. Haematol. 1996, 93, 955-961). In the present invention, similar results have been obtained. IL-4 was up-regulated while FA concentration was up-regulated, while fibrinogen and interferon gamma were down-regulated

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Chemicals and Animals

Formaldehyde (FA) was purchased from Sigma Chemical (St Louis, MO). Urea, thiourea, 3-[(cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), dithiothreitol (DTT), acrylamide, NN'-methylene-bisacrylamide, iodoacetamide, acetonitrile, sodium thiosulfate, trifluoroactic acid, 2-thiobarbituric acid (TBA ), butylated hydroxytoluene (BHT) and 1,1,3,3-tetramethoxypropane (TMP) were purchased from Sigma Chemical (St Louis, MO). 2,4-dintrophenylhydrazine (DNPH) was purchased from Fluka (Buchs, Switzerland) and protease inhibitor cocktail was purchased from Roche (Mannheim, Germany).

Male Sprague-Dawley rats without specific pathogens were purchased from Samtaco Animal Breeding Company (Osan, Korea) and standard laboratory conditions (Tm; 24 ± 2 ° C, humidity; 50 ± 10% and 12-hour day / night cycles) ). Animals were allowed to acclimate for 1-2 weeks and observed for abnormal behavior. They were free to eat the Samtaco Animal chow diet (PMI Nutritional Int. LLC, MO) and drinks and were first exposed at 6-8 weeks of age.

Example 2: Experimental Design and Formaldehyde Exposure

Two formaldehyde exposed groups, 5 ppm and 10 ppm, overexposed control, were used in the experiment. Animals were exposed in an inhalation exposure chamber for 6 hours / day and 5 days / week. To determine the formaldehyde concentration, air samples were collected and the FA concentration was determined using HPLC. Ten rats were exposed for each experiment (total: 30). After the rats were finally sacrificed, plasma samples were collected for proteomic analysis.

FA exposure was performed for 2 weeks at target concentrations of 5 ppm and 10 ppm in 1-m ³ stainless steel and glass suction chambers. FA concentration was generated with Permeater PD-1B (Gastec, Japan) and controlled by changing the humidified air flow in the second mixing chamber. The air in the exposure chamber was controlled at 22 ± 2 ° C. and 50 ± 5% humidity. Inhalation FA concentrations were monitored using HPLC.

Exposure of FA to rats was performed after FA concentrations equilibrated in the mixing chamber. FA concentration was monitored using HPLC. The actual mean FA concentrations obtained after two weeks of study were 5.10 ± 0.01 and 10.08 ± 0.01 ppm.

Example 3: 2-DE PAGE

1. Sample preparation

Plasma samples for 2-DE were prepared by Oh et al., Proteomics. Prepared according to the method described in 2004, 4, 3514-352. Lipids and salts were removed from the samples using a molecular weight cutoff column (3 kDa; Amicon, Millipore, Bradford, Mass.). For this step, plasma and 7 M urea, 2 M thiourea, 40 mM Tris (0.5 M, pH 8.5), 4% CHAPS, 65 mM DTT, 1% IPG buffer (pH 4-7L) and 1% protease inhibitors The containing sample buffer was mixed with eastern blood. The molecular weight cutoff column was centrifuged four times at 12 ° C. for 1 h at 3500 rpm, then each supernatant was separated and stored at −70 ° C. Protein concentration was measured by a modified Bradford assay method (Bradford M, Anal. Biochem. 1976, 72, 248-25).

 2. Isoelectric focusing (IEF) and SDS-PAGE

For one-dimensional electrophoresis of 2-DE, proteins were separated according to their isoelectric point. 450 μl total volume per sample by mixing protein sample solution (150 μg) with rehydration buffer containing 8 M urea, 2% CHAPS, 0.5% IPG buffer, 65 mM DTT and trace bromophenol blue (BPB) Was made. IEF is performed with a commercially available immobilized pH gradient (pH 3-11 nonlinear, 3-5.6 nonlinear, 5.3-6.5, 6-9, 24 cm) using an IPGphor (Amersham Biotech, Amersham, UK) device It was. The gel was rehydrated in the presence of sample for 12 h and focused at 60, 85, and 130 kVh. After IEF, the IPG gel strip was prepared in a solution containing 1% DTT (equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 1% w / v SDS) and containing 2.5% iodoacetamide. Equilibrate twice in 15 minutes under gentle shaking at room temperature in equilibration buffer. In SDS-PAGE, two-dimensional electrophoresis, proteins were used only on their molecular weight in 11.5% gradient polyacrylamide gels (size 35 cm × 45 cm) using an Owl separation system runner (Owl Separation System Co, Portamouth, NH, USA). On the basis of decomposition. IPG strips were embedded in 0.5% w / v molten agarose before running on SDS-PAGE slabs. The agarose contained 0.001% w / v BPB as the tracking dye. Running conditions were 1 w / gel for 30 min and 20 w / gel for 14-16 h until the BPB reached the gel end.

3. Visualization and image analysis

After separation on SDS-PAGE gels, proteins were visualized using a silver staining kit (Amersham Biotech, Amersham, UK) according to the manufacturer's instructions. The negatively stained gel was scanned using a 3600 × 4900 dpi instrument (Epson Expression ™ 10000XL, Epson, Japan) and the image file was converted into TIF format with linear gray scale values. Computer analysis of 2D-images was performed using Progenesis Discovery, 2-DE image analysis software (Nonlinear Dynamics, Newcastle upon Tyne, UK) according to the manufacturer's protocol. Intensity levels were normalized between gels as a percentage of the total protein intensity detected in the entire gel.

Proteomic analysis was performed using three different pI ranges (i.e., 3.0-5.6, 5.3-6.5, 6-9) and large 2-DE systems (Figures 1-3). Mice were exposed to 0, 5, or 10 ppm FA. 1496, 1362 and 633 protein spots were observed in each gel. Thus, a total of 3491 protein spots were isolated.

A total of 32 proteins were found to be up- and down-regulated in exposed cells (Figures 1-3), which are summarized in Tables 1 and 2. Nine spots were upregulated in a dose-dependent manner using strips of pi 3.0-5.6, 5.3-6.5, and 6-9 in exposed rat plasma. Eight spots, four spots and one spot were dose-dependently downregulated in pi 3.0-5.6, 5.3-6.5, and 6-9 strips, respectively.

Example 4: MALDI-TOF / MS analysis and protein identification

Protein spots were obtained from gels stained with 0.12% Coomassie Brilliant Blue G (Amersham Biotech, Amersham, UK) and, with slight modification, described above (Quadroni M. and James P. Electrophoresis. 1999, 20, 664-67) Decomposed according to. In summary, gel spots were cut with a scalpel, crushed and bleached with 25 mM ammonium bicarbonate, 50% acetonitrile. The gel was then dehydrated with the addition of acetonitrile, rehydrated on ice with 1020 μl of 25 mM ammonium bicarbonate with 10 μg / ml sequencing trypsin (Promega) and incubated at 37 ° C. for 12-15 h. . Peptides were extracted three times by adding 25 μl of solution containing 50% acetonitrile, 0.1% trifluoroacetic acid and 20 μl of acetonitrile. The extracted solution was pooled and evaporated to dryness in a Speed Vac centrifuge. Samples were reconstituted in 10 μl of 0.1% trifluoroacetic acid and treated with ZipTips ™ (Millipore Co. Bedford, MA) containing C18 resin according to the manufacturer's instructions. The washed peptide was eluted using saturated matrix solution (-cyano-4-hydroxycinnamic acid in 60% acetonitrile, 0.1% trifluoroacetic acid). Monoisotopic masses (M + 1) of trypsin fragments were determined using a Perspective Biosystem MALDI-TOF / MS voyager DE-STR Mass Spectrometer (Framingham, Mass., USA). The spectrum obtained was internally calibrated for trypsin peak. Tandem mass spectra of the interpreted peptides were searched for NCBInr (NCBInr.1.6.2005) using the MS-Fit (http://prospector.ucsf.edu) program. Known keratin mass and trypsin autolysis products were excluded from the search. The parameters were set to one of missed cleavage and acrylamide modification. Protein identification was given only if at least five peptide masses were matched within a maximum error of 50 ppm and the candidates matched the pI and Mw values measured in the 2-DE gel.

Identification of these differentially expressed proteins was performed by MALDI-TOF / MS, which is involved in apoptosis, transport, signaling, energy metabolism and cell structure and cell movement. Thirty-two proteins including proteins were identified (Tables 1 and 2).

TABLE 1 Up-regulated protein spots in rat plasma exposed to formaldehyde

TABLE 2 Down-regulated protein spots in rat plasma exposed to formaldehyde

The amino acid sequence of the proteins identified using MALDI-TOF / MS as described above was searched in the NCBInr database (http://www.ncbi.nlm.nih.gov/) for SNAP-23 (SEQ ID NO: 1), Apolipopreotein A-1 (SEQ ID NO: 2), Apolipopreotein E (SEQ ID NO: 3), Clusterin (SEQ ID NO: 4), Fibrinogen γ (SEQ ID NO: 5), and Kinesin (SEQ ID NO: 6) were identified.

Example 5: Western Blotting

Murine plasma proteins were isolated by 12% SDS-PAGE. Proteins were electroblotted onto PVDF membranes and the membranes were blocked with 5% dried skimmed milk in TPBS (0.05% Tween 20 in PBS) overnight at 4 ° C., then diluted 1: 500 for 1 h at room temperature. Secondary antibodies (fibrinogen γ cat.no.sc-18032, apo A-1 cat.no. 612331, apo E cat.no.sc-6385, kinesin cat.no.MAB1613, SNAP 23 cat.no.MAB331, clustein cat sc-8354, interleukin 4 cat.no.sc-1260, INF-γ cat.no.551506 and tubulin cat.no.sc-12462, suppuied by Snata Cruz Biotechnology, Inc and BD Biosciences). The membrane was then washed with TPBS and incubated in anti-rabbit, mouse, goat IgG (Snata Cruz Biotechnology, Inc) conjugated to 1: 2000 dilution of peroxidase for 45 min at room temperature. Bands containing murine plasma proteins were visualized by chemiluminescence (Amersham ECL kit), scanned with a flat-bed scanner and digitized with Scion image analysis software (Scion Co, Frederick, MD). For 2-DE immunoblot analysis of Apo E, rat plasma proteins were separated by 2-DE and 11.5% SDS-PAGE using pH 4-7 NL strips, followed by electroblotting on PVDF membrane (20 × 14 cm). It was.

Of a total of 3491 isolated protein spots, 32 were found to be dose-dependently up or down regulated using different pi strips. Western blot assays were performed to confirm the identification of the protein (FIG. 4). Commercially, SNAP 23, apolipoprotein A-1 (Apo A-1) and E, clusterin, kinesin, and fibrinogen γ monoclonal antibodies were purchased and used to confirm their identification as useful candidates for toxicological biomarkers of FA. Expression of five proteins, SNAP-23, Apo A-1, clusterin, fibrinogen γ, and kinesin, was significantly downregulated with increasing FA concentration, but Apo E was upregulated at 5 ppm FA and at 10 ppm FA. Down regulated (FIG. 4).

In order to examine the expression of inflammatory cytokines in the plasma of rats exposed to FA, Western blot assays were performed (FIG. 5). Th2 type cytokine, IL-4, was up-regulated while Th1 cytokine, IFN-γ, was down-regulated in a dose dependent manner in plasma of FA exposed mice. These data suggest that FA has an inflammatory effect.

A 2-DE immunoblot assay was performed to determine the expression pattern of Apo E isoforms (FIG. 6). The two isoforms of Apo E (D1 and D2) were significantly down-regulated and the nine isoforms of Apo E (UI-U9) were significantly up-regulated (p <0.05, n = 10). PI and MW values measured in 2-DE of the Apo E isoforms are shown in Table 3 below.

TABLE 3

Spot No. Regulation pI range Mass range (kDa) U1 Up 5.05 ~ 5.11 44-46 U2 Up 5.03-5.10 38-41 U3 Up 5.28-5.33 38-41 U4 Up 5.33-5.42 37-41 U5 Up 5.40-5.44 38-41 U6 Up 5.42-5.37 38-41 U7 Up 5.48-5.57 35-38 U8 Up 5.48-5.57 34-36 D1 Down 5.10 ~ 5.16 44-46 D2 Down 5.07 ~ 5.12 41-43

As described above, according to the present invention, six plasma proteins that specifically react to formaldehyde in rats using proteomics techniques were identified and presented as formaldehyde toxic protein biomarkers. . The association between the proteins identified in the present invention and formaldehyde, a toxic substance, can be applied to the study of biotoxicity mechanisms of formaldehyde and humans as well as a new toxic protein indicator that can detect the exposure of formaldehyde in animals. It will serve as an important basis for toxicity research.

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Claims (4)

Biomarker for diagnosing whether or not formaldehyde is exposed as an active ingredient comprising a protein selected from the following group whose expression changes according to formaldehyde exposure: Synaptozomal-associated protein 23 (SNAP-23) having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 22-24 kD and a pi of 4.3-5.3; Apolipopreotein A-1 having the amino acid sequence of SEQ ID NO: 2 and having a molecular weight of 29-31 kD and a pi of 5.0-6.0; Apolipopreotein E having an amino acid sequence of SEQ ID NO: 3 and having a molecular weight of 34-36 kD and a pi of 4.7-5.7; Clustererin having the amino acid sequence of SEQ ID NO: 4 and having a molecular weight of 24-26 kD and a pi of 4.4-5.4; Fibrinogen γ having an amino acid sequence of SEQ ID NO: 5 and having a molecular weight of 49-51 kD and a pi of 4.9-5.9; And, Kinesin having the amino acid sequence of SEQ ID NO: 6 and a molecular weight of 29-31 kD and a pi of 4.4-5.4. Formaldehyde exposure diagnostic agent comprising an antibody that specifically binds to the protein of claim 1 as an active ingredient. The agent for exposing formaldehyde according to claim 2, which is a monoclonal antibody. Formaldehyde exposure diagnostic kit comprising an antibody that specifically binds to the protein of claim 1 as an active ingredient.

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