KR20120060051A - Composition and Kit for Detecting Biomarkers for Obesity in Adipose Tissue - Google Patents

Abstract

PURPOSE: A composition and kit for detecting a biomarker for obesity are provided to determine obesity and progression in obesity. CONSTITUTION: A composition for detecting a biomarker for obesity in a fatty tissue contains an antibody which specifically binds to the biomarker of a laminin receptor, antiquitin(aldehyde dihydrogenase family 7), protein DJ-1, or transglutaminase A chain. The antibody is a monoclonal antibody, polyclonal antibody, multi-specific antibody, antibody fragment, recombinant antibody or chemically formulated antibody. A method for screening a candidate material for preventing and treating obesity comprises: a step of feeding high calorie foods to experimental animals and selecting animals which is susceptible to obesity; a step of feeding a predetermined material with the high calorie foods to the selected animals; a step of measuring the expression level of the biomarker from the selected animals; and a step of comparing the expression level of the selected animals with the expression level of a normal group and determining the material for preventing or treating obesity.

Description

Composition and Kit for Detecting Obesity Biomarkers in Adipose Tissues {Composition and Kit for Detecting Biomarkers for Obesity in Adipose Tissue}

The present invention relates to compositions and kits for detecting obesity biomarkers of adipose tissue.

Recently, the obese population is rapidly increasing due to the improvement of living standards according to economic development, lack of exercise due to busy living environment, and excessive intake of nutrition. The proportion of obese populations in Korea is growing rapidly from 11.7% in 1995 to 29.4% in 2001 and 18.0% in 1995 to 32.6% in 2001 (Ministry of Health and Welfare, 2007).

Obesity is an abnormal increase in adipose tissue due to an imbalance in calorie intake and consumption [Kopelman PG. Neuroendocrine function in obesity. Clinical Endocrinology 28: 675-689, 1998; Clinical Obesity (eds. Kopelman PG, Stock MJ) 248-89 (Blackwell Science, Oxford 1998) .Clinically, the BMI (Body Mass Index) defines 25.0 to 30.0 as overweight and is overweight. define.

Obesity persists as a cause of diseases such as hypertension, arteriosclerosis, diabetes, fatty liver, gallstones, gout, and especially in obese women, endometrial cancer, cervical cancer, ovarian cancer, breast cancer, etc. In addition, in mortality, obesity is reported to be 1.3 times higher than normal weight (American Cancer Society, 2003).

The cause of obesity is that genetic factors are greater than environmental factors, and there is a 70% chance of obesity in a child whose parents are both obese, and a 40% chance of obesity when one of the parents is obese. have.

In addition, studies of non-obesity people who deliberately try to gain weight by overeating have shown that they are resistant to this weight gain and can only maintain high weight by very high calorie intake. People are vulnerable to weight gain and are found to be able to maintain their condition by eating normal or moderately high calories.

Like humans, pigs, cattle and other livestock have different constitutions for obesity, and rats and other experimental animals are known to have a constitution that is vulnerable to obesity and resistant to obesity. [Archer ZA , et al., Obes. Res. 2003, 11: 1376-1383.

Thus, biomarkers for obesity have been developed primarily to identify individuals who are genetically or constitutionally vulnerable to this high calorie intake.

Markers such as CALB2, RARB, and PBX1 genes with genetic polymorphisms (Korean Patent Publication No. 2010-0011719), Wnt10B polymorphic markers (Korean Patent Publication No. 2007-0113586), obR gene (International Publication No. WO 1997/19952) And such are examples.

Such obesity biomarkers can be effective in the early treatment of obesity or prevention of obesity by enabling the identification of genetic or constitutionally vulnerable individuals.

The present invention also discloses a composition and kit for detecting obesity biomarkers useful for the identification of individuals vulnerable to high calorie intake.

Disclosure of Invention An object of the present invention is to provide a composition for detecting obesity biomarker of adipose tissue which is useful for the identification of individuals vulnerable to obesity.

Another object of the present invention is to provide a kit for detecting obesity biomarkers of adipose tissue useful for the identification of individuals vulnerable to obesity.

Other objects of the present invention will be presented below.

In one aspect, the present invention relates to a composition for the detection of obesity biomarkers of adipose tissue.

The composition for detecting obesity biomarker of adipose tissue of the present invention includes a laminin receptor, an antiquitin (aldehyde dehydrogenase family 7), protein DJ-1 and trans It characterized in that it comprises an antibody that specifically binds to the obesity biomarker selected from the group consisting of glutaminenase A chain (transglultaminase A chain).

The obesity biomarker can be used to identify individuals who are vulnerable to obesity or to be obese. In other words, individuals who show high calorie intake and are non-obesity).

If a marker differs from a normal subject in an expression quantity that is different from an obese resistant subject, an obesity-vulnerable subject cannot be distinguished from an obese resistant subject. Obesity-vulnerable individuals cannot be distinguished from normal individuals unless the expression level is different from the individuals.

The inventors performed the experiment in this respect.

Specifically, as confirmed in the following Experimental Examples and Examples, Sprague-Dawley (SD) rats of 5 weeks of age were divided into the control group of the low-calorie pay group and the experimental group of the high-calorie dietary pay group, while measuring body weight every other day. The experimental group, which was a high-calorie diet, was again fed the obesity-prone group (“OP group”), which continuously gained weight on the high-calorie diet, and the obesity-resistant group, which did not gain weight even on the high-calorie diet. (obesity-resistant, hereinafter "OR" group). Then, using a sample of adipose tissue, there was a significant difference in expression level when the OP group was compared with the control group, and a new protein related to obesity, which also showed a significant difference in expression level when the OP group was compared with the OR group. I identified it.

These identified proteins are proteins listed above as obesity biomarkers.

The obesity biomarker proteins are all known proteins, and in Table 1 below, each acession number, isoelectric point, molecular weight, and the like on the Genebank of the marker proteins can be identified.

In the present invention, as shown in Table 1 below, (1) protein DJ-1 and transglutaminase A chain of the obesity biomarker are expressed in OP. High in the group, (2) laminin receptor (laminin receptor) and antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7) its expression is high in the normal (control) and OR group.

Obesity biomarker composition of the adipose tissue of the present invention is provided based on the results of these experiments, the obesity biomarker composition of the present invention, as well as the identification of the subjects vulnerable to the above-mentioned obesity or the presence of obesity, It can be used directly or indirectly to determine the progress of obesity, screening for the prevention or treatment of obesity, the determination of the progress of obesity. Here, the term "directly or indirectly used" means that the biomarker of the present invention is used alone or in combination with other obesity biomarkers or clinical symptoms of obesity, such as weight, and the like. This is because it can be used to determine the presence or absence, to determine the progress of obesity, screening for the prevention or treatment of obesity, and to determine the progress of obesity.

The composition of the present invention may comprise only one antibody against one obesity biomarker or may comprise a mixture of different antibodies against two or more obesity biomarkers, the composition of which is lyophilized, aqueous or buffered solution. And the like.

The composition for detecting obesity biomarker of adipose tissue of the present invention is used to measure the expression level of the obesity biomarker by contacting adipose tissue isolated from a biological sample, in particular a mammal.

The composition for detecting obesity biomarker of adipose tissue of the present invention can be used for the detection of obesity biomarker in mammals, especially humans, rats and the like.

As used herein, "obesity biomarker" refers to a protein in which the amount of expression in its adipose tissue increases or decreases as compared to a normal control as well as an obesity resistant control group when obesity progresses or occurs.

As used herein, the term "specific binding" refers to the case where an antibody forms an antigen-antibody conjugate with the obesity biomarker of the present invention, which is its target protein, and does not form such a conjugate substantially with other proteins. . The meaning of the "specific binding" may be expressed in other words that the binding is determined by a specific structure of the protein, that is, the epitope which is the determining site of the antigen. "Substantially" herein means that a low but nonspecific conjugate can be formed. Such nonspecific binding can be eliminated in the pretreatment for analysis and quantification of specific binding.

As used herein, "epitope" refers to an amino acid region (antigen determinant) of a part having antigenicity or immunogenicity in the obesity biomarker of the present invention. Epitopes will typically include at least 10 amino acids. Such epitopes can be identified by known and commonly used epitope analysis methods in the art, such as phage display, reverse immunogenetics, and the like.

In the present specification, "antibody" is meant to include all forms of the specific binding to the obesity biomarker of the present invention. Thus, in addition to including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (ie, antibodies that recognize two or more antigens or two or more epitopes, such as bispecific antibodies, etc.), the obesity of the invention Fragments of antibodies that possess the ability to specifically bind to biomarkers, recombinant antibodies, chemically modified antibodies. Examples of fragments of the antibody include Fab, F (ab ') ₂ , scFv (an antibody obtained by linking Fv of a heavy or light chain with a suitable linker), Fv, Fab / c (an antibody having one Fab and a complete Fc), and an antibody. It is meant to include antibody fragments obtained by introducing and expressing genes for antibody fragments and fragments obtained by treatment with protein cleavage enzymes such as papain and pepsin into a host cell by genetic recombination as described below. The globulin type of the antibody is not particularly limited as long as it specifically binds to the obesity biomarker of the present invention, and the globulin type may be any one of IgG, IgM, IgA, IgE, and IgD.

Using the antibody, the expression level of the obesity biomarker of a biological sample specifically bound to the antibody is determined by various techniques known in the art, such as enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, and luminescence immunoassay. Quantitative and / or qualitative analysis is possible by measuring methods, and through such quantitative and qualitative analysis, identification of individuals vulnerable to obesity, existence of obesity, progression of obesity, screening of obesity prevention or therapeutic substances In addition, the course of treatment of obesity can be determined.

In the enzyme immunoassay, enzymes such as peroxidase (POD), alkaline phosphatase, β-galactosidase, urease, catalase, glucose oxidase, lactic acid dehydrogenase, amylase or biotin-avidin complex, may be used. In the case of the measurement method, such as fluorescein isothiocyanate, tetramethylodamine isothiocyanate, substituted rhodamine isothiocyanate, dichlorotriazine isothiocyanate, Alexa or Alexa Fluooro, etc. Fluorescent materials or fluorophores may be used, and radioimmunoassays include tritium, iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹²¹ I), phosphorus ( ³² P), sulfur ( ³⁵ S), metals (e.g., Radioactive isotopes such as ⁶⁸ Ga, ⁶⁷ Ga, ⁶⁸ Ge, ⁵⁴ Mn, ⁹⁹ Mo, ⁹⁹ Tc, ¹³³ Xe, etc.) may be used, and the luminescence immunoassay method includes diocetans such as luciferase method and luminol peroxidase POD method. It can be used together with a luminescent material of the compound or the like.

The antibody may be combined with a labeling substance as needed, such as when using the avidin-biotin method, the streptoavidin-biotin method, or the like. The binding of the labeling substance to the antibody may be carried out by methods such as glutaraldehyde method, maleimide method, pyridyl disulfide method, periodic acid method, etc. in the enzyme immunoassay, and in the radioimmunoassay, chloramine T method, Bolton-hunter Methods such as law may be used.

As an immunological measurement method, in addition to the four methods exemplified above, an immunoprecipitation method, an immunobinding method, a Western blotting method, an immunostaining method, an immunodiffusion method, and the like may be further exemplified, but it is preferable to use the above four methods. More preferably enzymatic immunoassay, most preferably an enzyme-linked immunosorbent assay (ELISA).

As a result of the immunological measurement method, the accuracy of determining whether the subject is vulnerable to obesity, obesity, etc. can be used by the receiver operating characteristic (ROC) method and the AUC (Area Under Curve) can be used as a measure of accuracy. . ROC is a representative method for determining sensitivity and specificity (Zweig, M.H., and Campbell, G., Clin. Chem. 39 (1993) 561-577). The AUC of the ROC has a value between 0.5 and 1, and the value of 0.5 means that there is no difference between the patient and the normal person, and 1 means that the evaluation is completely distinct between the patient and the normal person.

On the other hand, polyclonal antibodies can be prepared by immunizing obesity biomarkers of the present invention on animals such as birds (eg chickens), mammals (eg rabbits, goats, horses, sheep, mice, etc.). Can be. Antibodies can be purified from the blood of an immunized animal using methods known in the art such as ion exchange chromatography, affinity-chromatography and the like.

Monoclonal antibodies can be obtained by hybridoma cell lines that produce monoclonal antibodies specific for the obesity biomarkers of the invention. As a method for producing such hybridoma cell lines, for example, an animal (e.g., a mouse) is immunized with the obesity biomarker of the present invention, spleen cells are harvested from the immunized animal, and the spleen cells are fused to a myeloma cell line. Thus, a method of identifying hybridoma cell lines that generate hybridoma cells and produce a monoclonal antibody of interest can be given. Isolation and recovery of monoclonal antibodies from such cell lines is possible by known and conventional techniques in the art.

Looking in more detail for the production method of such a monoclonal antibody, first to obtain a monoclonal antibody, the obesity biomarker of the present invention, an immunogen, need to be administered to mammals, for example, rats, mice, rabbits, monkeys, goats, etc. There is.

Single doses of an immunogen may be appropriately determined by one skilled in the art within the scope of their usual capacity, taking into account the type of immune animal, the route of administration, and the like. Typically, it will be about 50-200 μg per animal. Administration can usually be carried out by diluting and suspending the immunogen in an appropriate amount of phosphate-buffered saline (PBS), physiological saline, or the like, and mixing and emulsifying the usual adjuvant, followed by subcutaneous injection into the abdominal cavity. Such administration will take place 2 to 10 times, preferably 3 to 4 times, at intervals of days to weeks after the initial administration, preferably at intervals of 1 to 4 weeks. After continuing the administration of the immunogen, the antibody titer was measured in the serum of the immunized animal by the ELISA method or the like. When the antibody titer reached the plateau, the immunogen was finally administered intravenously or intraperitoneally. After 5 days antibody producing cells are harvested. The antibody producing cells include spleen cells, lymph node cells, peripheral blood cells and the like, but spleen cells or lymph node cells are preferred.

After harvesting the antibody producing cells, a hybridoma cell line is produced which produces a monoclonal antibody specific for the administered immunogen, i.e., the obesity biomarker of the present invention. Such hybridomas can be produced and identified by known or conventional techniques in the art. Normally, antibody-producing cells, preferably, splenocytes are harvested from immunized animals, the splenocytes are fused to myeloma cell lines to produce hybridoma cells, and hybridomas that produce monoclonal antibodies that bind to immunogens. You will go through the steps of identifying cell lines. Myeloma cell lines used for fusion with antibody-producing cells can be cell lines derived from animals such as mice, which are commercially available. Preferred myeloma cell lines are derived from immune and allogeneic animals, have drug selectivity for antibiotics, etc., and survive in HAT selection medium containing hypoxanthine, aminoputerine and thymine without being fused with spleen cells. It is desirable to have a property that cannot survive and can survive only in the state of fusion with spleen cells. Specific examples of the myeloma cell line include P3X63 (ATCC TIB9), which is a HGPRT (hypoxantine guanine phosporibosyl-transferase) deficient cell line derived from BALB / c mice.

Fusion of splenocytes and myeloma cell lines, which are antibody-producing cells, results in an appropriate ratio (about 1: 1 to 20: 1) of antibody-producing cells and myeloma cell lines in animal cell culture media such as serum-free DMEM and RPMI-1640 medium. Ratio) and by performing a fusion reaction in the presence of a cell fusion promoter. As the cell fusion promoter, polyethylene glycol and the like having an average molecular weight of 1500 to 4000 daltons can be used at a concentration of about 10 to 80%. In addition, in some cases, an adjuvant such as dimethyl sulfoxide may be used in combination to increase the fusion efficiency. In addition, it can also be fused using a commercially available cell fusion device.

After cell fusion treatment, the desired hybridomas should be selected. Normally, the cell suspension is appropriately diluted with fetal calf serum-containing RPMI-1640 medium or the like, and then, the microtiter plate is divided into about 2 million cells per well, selection medium is added to each well, and then appropriately exchanged with the same selection medium. Incubate while supplying fresh medium. The culture temperature is usually 20 to 40 ℃. When the myeloma cell line is an HGPRT-deficient or thymidine kinase-deficient line, selective hybridization of only antibody-producing cells and myeloma cell lines is performed by using a selection medium (HAT medium) containing hypoxanthine-aminopterin-thymidine. Can be cultured and expanded. Then, cells grown about 14 days after the start of the culture in the selection medium can be obtained as hybridomas. The presence of the antibody of interest is then screened in the supernatant culture of the propagated hybridomas. Screening of such hybridomas can be made according to well-known and conventional techniques in the art. Such techniques include, for example, enzyme immunoassay (EIA: Enzyme Immuno Assay and ELISA), radioimmunoassay, and the like. Cloning of fusion cells can be accomplished by a limiting dilution method or the like.

The cloned hybridomas may be cultured in animal cell culture media such as 10% fetal bovine serum-containing RPMI-1640 medium, DMEM medium, and serum-free medium under normal culture conditions (eg, 37 ° C., 5% CO 2 concentration). . Incubation period is about 2 to 10 days. Monoclonal antibodies can be obtained from their upper cultures.

Monoclonal antibodies can be recovered using known and conventional techniques in the art. As a well-known technique of those skilled in the art, methods such as ammonium sulfate salting method, ion exchange chromatography, affinity chromatography, gel filtration chromatography, or a combination thereof are mentioned.

For the production of the monoclonal antibodies of the present invention, genetic recombination techniques obtained by cloning antibody genes from hybridomas, inserting them into appropriate vectors, and introducing and expressing them into appropriate host cells may be used (Vandamme, AM et al., Eur. J. Biochem., 192, 767-775, 1990).

Specifically, mRNA encoding the variable region of the antibody of the present invention is obtained from hybridomas producing the antibody of the present invention. Obtaining such mRNA is known in the art and commonly used methods such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry., Vol 18, 5294-5299, 1979), AGPC method (Chomczynski, P. et. al., Anal. Biochem., 162, 156-159) and the like to obtain total RNA and from the total mRNA to obtain the desired mRNA using the mRNA Purification Kit (Pharmacia) and the like. Alternatively, mRNA may be obtained directly by using the QuickPrep mRNA Purification Kit (manufactured by Pharmacia).

From the obtained mRNA, cDNA of the antibody V region is synthesized using reverse transcriptase. If necessary, RACE PCR may be applied to cDNA synthesis and amplification. The cDNA encoding the variable region thus obtained is inserted into an expression vector containing DNA encoding the constant region (C region) of the antibody. Such expression vectors may include regulatory sequences such as promoters, enhancers, replication initiation points, polyadenylation signals, ribosomal binding sites, and the like, as described below in connection with the method for recombinant DNA production of obesity biomarkers of the present invention. When the expression vector is transformed into a host cell, the production of antibodies is possible. Expression of the antibody gene may be recombined with the DNA encoding the antibody heavy chain (H chain) or the light chain (L chain) into an expression vector, respectively, to co-transform host cells, or the single expression of DNA encoding the heavy and light chains. The host cell may be transformed by inserting it into a vector (WO 94/11523).

On the other hand, the obesity biomarker of the present invention as an immunogen used to obtain the antibody of the present invention can be obtained by well-known and commonly used DNA recombination techniques known in the art. Typically, the cDNA of the obesity biomarker of the present invention is prepared and the cDNA is inserted into an expression vector, the expression vector is transformed into prokaryotic or eukaryotic host cells, and the transformed host cell is cultured in a suitable medium, From that culture or cell. The cDNA can be produced within the range of ordinary skill of those skilled in the art based on the gene sequence provided by the gene / protein database such as GenBank, RefSeq, Entrez Gene, UniProtKB, Swiss-Prot or the like.

In the preparation of such cDNA, a DNA synthesis apparatus using the phosphoramidite method or the like, an RT-PCR method, a hybridization method for obtaining a desired cDNA from a cDNA library, and the like can be used. You can also amplify.

The expression vector is a biotechnology company such as Novagen, Takarashujo, Qiagen, Stratagene, Promega, Roche Diagnositics Commercially available from, Inc., Invitrogen, Genetics Institute.

Such expression vectors facilitate the addition of regulatory elements such as promoters, enhancers, polyadenylation signals, ribosomes, binding sites, initiation points, terminators, selection markers, isolation and purification, in addition to the DNA encoding the obesity biomarker of the present invention. Labeled peptide sequences (eg, nucleotide sequences encoding histidine repeat sequences) and the like.

Host cells include prokaryotic cells such as bacteria (e.g., E. coli, Bacillus subtilis), and yeast (e.g., Saccharomyces cerevisiae )), insect cells (eg Sf cells), mammalian cells (eg COS, CHO, BHK) and the like.

Purification of obesity biomarkers of the present invention from host cells or their cultures includes ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography (when labeled peptide is bound), HPLC, hydrophobic chromatography, isoelectric point chromatography, and the like. The method of or the combination of these can be used.

Production of obesity biomarkers of the present invention by DNA recombination techniques is described in addition to those described herein in "Samrook et al., Molecular Cloning A Laboratory Mannual, Cold Spring Harbor Laboratory Press, US (1989)", "Ausubel et al., Current Protocols in Molecular Biology, Jon Willey & Sons, US (1993), and Sambrook, J. & Russel, D., Molecular Cloning, A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Volume 15, January 15, 2001 7.42 to 7.45, Vol. 2, 8.9 to 8.17 ", and the like. These documents are considered part of this specification.

The obesity biomarker of the present invention as an immunogen for producing the antibody of the present invention may be used as a fragment thereof. This is because antibodies obtained using the fragments will also retain the ability to specifically bind to the obesity biomarker of the present invention.

In another aspect, the present invention relates to a kit for detecting obesity biomarker of adipose tissue.

Obesity biomarker kit for detecting adipose tissue of the present invention is characterized in that it comprises an antibody that specifically binds to the obesity biomarker of the present invention as described above.

In the kit for detecting the obesity biomarker of adipose tissue of the present invention, the antibody specifically binding to the obesity biomarker of the present invention may be in the form of a single substance or a mixture, or may be bound to a solid carrier such as a protein chip or may be free. It may be in the form.

The kit of the present invention is an obese bio-labeled with a secondary antibody (e.g., a fluorescent dye capable of detecting the expression level of an obesity biomarker, etc.) used in an immunological method capable of quantitatively or qualitatively confirming the expression level of an obesity biomarker. Antibodies to markers), carriers, wash buffers, sample dilutions, enzyme substrates, reaction stoppers, and the like.

The kit of the present invention is also preferably vulnerable to obesity from the expression level of quantitatively or qualitatively detected obesity biomarkers, the presence or absence of obesity, the progression of obesity, the screening of obesity prevention or therapeutic substances, and / or obesity Instructions for determining the course of treatment, and the like.

In another aspect, the present invention relates to a composition and kit for diagnosing obesity comprising an antibody that specifically binds to the obesity biomarker of the present invention as described above.

The diagnostic composition or kit for obesity of the present invention can be used for the diagnosis of obesity, including the early diagnosis of obesity, determining the prevalence of obesity by quantifying the expression level of obesity biomarkers.

That is, if the expression level of the obesity biomarker is higher or lower than the cut-off value, it will be determined to be obese or vulnerable to obesity. Here, the reference value may be determined from the expression level of obesity biomarkers measured in adipose tissue samples of various individuals, and may use the ROC (Receiver Ooperating Characteristic) as described above.

In the obesity diagnostic composition and kit of the present invention, the above description of the obesity biomarker detection composition and kit of the present invention is effective as it is.

In another aspect, the present invention relates to a method for screening a candidate substance for preventing or treating obesity.

The screening method of the anti-obesity agent of the present invention comprises the steps of: (a) feeding a high-calorie diet to experimental animals, particularly rats, to screen for obese susceptible experimental animals, (b) high-calorie any substance to the selected obese susceptible experimental animals Feeding with diet, (c) laminin receptor, antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7) from adipose tissue of obese susceptible experimental animals administered with the substance Measuring the expression level of the obesity biomarker of the present invention selected from 1 (protein DJ-1) or the transglultaminase A chain, and (d) expressing the expression level of the obesity biomarker in the normal group. Comparing with the expression level of is configured to determine whether the candidate substance to prevent or treat obesity.

If a substance is administered to an obese susceptible animal, the obesity biomarker expression level of the obese susceptible animal to which the substance is administered is not statistically significant different from that of a normal subject. It will be identified as a candidate for treatment. The expression level of this obesity biomarker will be used alone or in combination with other phenotypes of obesity such as body weight to determine which substance is a candidate for preventing or treating obesity.

As described above, according to the present invention, it is possible to provide a composition and kit for detecting obesity biomarkers of adipose tissue useful for the identification of individuals vulnerable to obesity.

1 is a graph showing the weight of the control group, OP group and OR group (Fig. 1-A) and the weight gain of each group (Fig. 1-B) according to the breeding period.
2 to 13 show representative images of a control group, an OP group, and an OR group among a total of 72 images stained with 2D electrophoresis gels.
Figures 14 and 15 show the serial numbered electrophoretic photographs divided into BAT and WAT, respectively.
16-19 are MS spectra used for the identification of novel obesity biomarkers of the present invention.
20 is a graph showing the spot magnification of the new obesity biomarkers of the present invention and the volume concentration of the spot
21 is a result of re-identification by Immunoblot for some proteins of the spot protein having a difference in the expression amount.

Hereinafter, the present invention will be described with reference to Examples. However, the scope of the present invention is not limited to these examples.

< Example  1> Breeding of experimental animals OP Army OR Judo

Five-week-old Sprague-Dawley rats were purchased from Korean laboratory animals (Korea) and adapted to laboratory animals in the department of biotechnology at Daegu University (Daegu, Korea) for 7-8 days while freely supplying general feed and water. After the experiment was used.

The experimental animals were separated and reared one by one to exclude various effects between the experimental animals.

The experimental animals were randomly divided into 8 control groups and 37 test groups. The control group was fed a low calorie diet (12% calories from fat), and the experimental group was fed a high calorie diet (45% calories from fat).

The animals were weighed every other day for 8 weeks, and according to the measured weight, the experimental groups were divided into the OP group (n = 6) with fast weight gain and the OR group (n = 6) with little weight gain.

Animal testing was conducted with the approval of the Animal Experimental Ethics Committee of Daegu University, and the guidelines for the use and management of experimental animals were followed.

1 shows the weight of the control group, OP group and OR group according to the breeding period (Fig. 1-A) and the weight gain of each group (Fig. 1-B) in a graph.

Significant differences between the OP group and the control / OR group can be seen (* P <0.05, ** P <0.01)).

< Example  2> Preparation of sample

After the breeding of <Example 1>, the rats were sacrificed to immediately separate brown adipose tissue (hereinafter referred to as "BAT") and white adipose tissue (hereinafter referred to as "WAT") and cooled NaCl solution. Washed with. The washed adipose tissue was triturated with liquid nitrogen and stored at −80 ° C. until the experiment. The ground adipose tissue was pulverized with 300 μl of rehydration buffer (7M urea, 2M thiourea, 4% (w / v) 3-[(3-cholamidopropyl) dimethylamonio] -1-propanesulfonate (CHAPS), 20mM dithiothreitol (DTT), It was dissolved in 1 mM phenylmethylsulfonyl fluoride (PMSF), 2% IPG buffer, bromophenol blue) and homogenized using a homogenizer (PT 1200E, Kinematica Ltd., Luzern, Switzerland). Homogenates were centrifuged (14000 × g, 20 min), the supernatant was taken and stored at -80 ℃ until the experiment was used for two-dimensional electrophoresis.

Protein concentrations in adipose tissue samples were determined by Bradford method [Bradford, M. Biochem. 1976, 72: 248-254 (1976).

Samples of adipose tissue were obtained from 6 adipose tissues for each group of <Example 1>.

< Example  3> Conducting 2D Electrophoresis

Immobilized pH gradient (IPG) -isoelectric focusing (IEF) of adipose tissue samples was performed using 18 cm IPG DryStrips at pH 3-10 in PROTEIN IEF cells using the manufacturer's (Bio-Rad) protocol. IPG strips were placed in a strip holder with 350 μl of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 20 mM DTT, 2% IPG buffer (Bio-Rad)) and 150 μg of sample protein. Rehydrate for 26 hours.

IEF was performed for 15 minutes at 250V, 3 hours at 250-10,000V, and 6 hours at 10,000V, after which time 500V was maintained.

After IEF, the gel strip was allowed to 20 minutes in a first equilibration solution (6 M urea, 2% sodium dodecyl sulfate (SDS), 1% DTT, 30% glycerol, 50 mM Tris-HCl, pH 6.8), and a second equilibration solution. (6 M urea, 2% SDS, 2.5% iodoacetamide (Bio-Rad), 30% glycerol) again equilibrated for 20 minutes. Carefully wash the equilibrated IPG strip twice with electrophoresis buffer, arrange the washed gel strips on 20 × 20 cm 8% or 12% (w / v) SDS-PAGE and at a constant voltage of 20 mA per gel. Electrophoresis was performed for 15 hours.

Silver staining was performed as follows to visualize the protein of the gel after 2D electrophoresis: The gel was reacted by shaking for 30 minutes in 50% ethanol (Duksan Chemical, Korea) and 5% acetic acid (Duksan Chemical) solution. After fixing the protein, the reaction was shaken again for 30 minutes in 30% ethanol solution and washed three times for 5 minutes with water. The gel was sensitized by shaking for 10 minutes with a 0.02% sodium thiosulfate (Sigma, USA) solution, and the gel was then washed three times with water for 30 seconds. And 0.3% nitric acid (Kojima, Japan) was shaken for 25 minutes in a solution to stain the protein and washed twice with water for 30 seconds. The protein was then visualized by shaking the reaction solution with a reaction solution (3% sodium carbonate (Duksan Chemical), 0.02% sodium thiosulfate, 0.05% formalin (DC Chemical, Japan)) for 25 minutes. The gel was immersed in 6% acetic acid to stop the reaction and washed with water.

Silver stained gels were scanned with UMAX PowerLook 1120 (Maxium Technologies, Taipei, Taiwan), an image scanner, for a total of 72 (6 BAT 8% electrophoresis photos, 6 BAT 12% electrophoresis photos, WAT per group) 6 images of 8% electrophoresis and 6 images of WAT 12% electrophoresis) are shown in FIGS. 2 to 13.

2 to 5 are BAT 8% electrophoresis picture, BAT 12% electrophoresis picture, WAT 8% electrophoresis picture, and WAT 12% electrophoresis picture of the control group, respectively, and FIGS. 6 to 9 are BAT of the OP group, respectively. 8% electrophoresis picture, BAT 12% electrophoresis picture, WAT 8% electrophoresis picture, and WAT 12% electrophoresis picture, Figures 10-13 are BAT 8% electrophoresis picture, BAT 12% electrophoresis of OR group respectively Electrophoresis photos, WAT 8% electrophoresis photos, and WAT 12% electrophoresis photos.

< Example  4> Image analysis

Quantitative analysis to confirm expression changes of protein spots from the scanned image was performed by modified ImageMaster 2-D software V4.95 (Amersham Biosciences). The reference gels were randomly selected from the control group and the spots of the other gels were compared with the spots of the reference gels, and the volume percentage of each spot was compared.

For BAT, a total of 92 spots showing significant changes in expression levels between the control group, the OP group and the OR group were selected, and 66 spots were selected for the WAT and serial numbers were assigned to each of them.

[Table 1] and [Table 2] below divide the spot serial number and the volume percentage of each group's spot corresponding to the serial number by dividing into BAT and WAT, respectively. For reference, in [Table 1] and [Table 2] below, only the volume percentage of the identified protein spots was shown.

% Volume of spots with significant change in expression levels between control, OP and OR groups in BAT No. in gel
Alternation (Vol%) Normal OP OR 4 0.07 ± 0.03 0.11 ± 0.03 ** 0.03 ± 0.01 ‡‡ †† 6 0.03 ± 0.01 0.07 ± 0.02 ** 0.03 ± 0.01 ‡‡ 7 0.12 ± 0.032 0.07 ± 0.023 ** 0.1 ± 0.04 ‡ 30 0.22 ± 0.03 0.13 ± 0.046 ** 0.22 ± 0.07 ‡‡ 39 0.35 ± 0.08 0.81 ± 0.25 ** 0.46 ± 0.06 ‡‡ †† 49 0.14 ± 0.06 0.07 ± 0.03 ** 0.12 ± 0.06 †† 13 0.24 ± 0.07 0.16 ± 0.05 ** 0.22 ± 0.08 ‡ 10 0.11 ± 0.02 0.13 ± 0.05 0.04 ± 0.01 ‡‡ †† 25 0.05 ± 0.015 0.032 ± 0.008 * 0.04 ± 0.01 ‡‡ 42 0.12 ± 0.17 0.03 ± 0.01 0.14 ± 0.132 ‡ 43 0.18 ± 0.04 0.17 ± 0.07 0.08 ± 0.05 ‡‡ †† 40 0.25 ± 0.13 0.16 ± 0.05 0.34 ± 0.11 ‡‡ 59 0.12 ± 0.04 0.19 ± 0.07 ** 0.17 ± 0.04 † 84 0.67 ± 0.103 1.02 ± 0.36 * 0.44 ± 0.2 ‡‡ †† 85 0.76 ± 0.24 0.89 ± 0.18 0.36 ± 0.2 ‡‡ †† 90 0.21 ± 0.04 0.15 ± 0.05 ** 0.2 ± 0.06 ‡ 44 0.14 ± 0.04 0.32 ± 0.08 ** 0.13 ± 0.07 ‡‡ 21 0.04 ± 0.02 0.02 ± 0.01 ** 0.04 ± 0.02 ‡‡ 91 1.01 ± 0.44 0.86 ± 0.37 1.42 ± 0.65 ‡ † 8 0.06 ± 0.01 0.11 ± 0.03 ** 0.04 ± 0.01 ‡‡ †† 28 0.07 ± 0.03 0.1 ± 0.04 * 0.07 ± 0.03 88 0.53 ± 0.04 0.34 ± 0.13 ** 0.63 ± 0.37 ‡ 89 0.7 ± 0.2 1.04 ± 0.3 * 0.79 ± 0.11 ‡ 25 0.22 ± 0.06 0.13 ± 0.05 ** 0.12 ± 0.04 †† 11 0.06 ± 0.02 0.03 ± 0.01 ** 0.028 ± 0.004 †† 12 0.05 ± 0.03 0.121 ± 0.048 ** 0.064 ± 0.02 ‡‡ 46 0.21 ± 0.08 0.1 ± 0.03 ** 0.08 ± 0.07 †† 52 0.07 ± 0.02 0.05 ± 0.01 * 0.04 ± 0.01 ‡ †† 54 0.04 ± 0.01 0.03 ± 0.01 * 0.03 ± 0.01 †† 56 0.06 ± 0.02 0.04 ± 0.01 ** 0.036 ± 0.01 †† 36 0.23 ± 0.03 0.16 ± 0.04 ** 0.24 ± 0.06 ‡‡ 32 0.15 ± 0.01 0.11 ± 0.04 * 0.17 ± 0.04 ‡‡ 87 1 ± 0.315 1.49 ± 0.911 0.74 ± 0.33 ‡ 60 0.07 ± 0.02 0.09 ± 0.03 * 0.06 ± 0.03 ‡ 13 0.11 ± 0.04 0.17 ± 0.09 ** 0.07 ± 0.01 ‡‡ †† 57 0.06 ± 0.02 0.04 ± 0.01 ** 0.06 ± 0.03 ‡ 53 0.08 ± 0.02 0.04 ± 0.01 ** 0.04 ± 0.01 †† 17 0.4 ± 0.08 0.29 ± 0.09 ** 0.27 ± 0.04 ‡‡ 22 0.04 ± 0.01 0.01 ± 0.01 ** 0.03 ± 0.003 ‡‡ 35 0.17 ± 0.02 0.14 ± 0.03 0.21 ± 0.07 ‡‡ 37 0.12 ± 0.02 0.15 ± 0.04 * 0.1 ± 0.016 ‡‡ † 45 0.04 ± 0.01 0.02 ± 0.002 ** 0.02 ± 0.01 †† 55 0.14 ± 0.04 0.08 ± 0.03 ** 0.08 ± 0.02 †† 86 0.24 ± 0.07 0.41 ± 0.11 ** 0.27 ± 0.11 ‡ 65 0.15 ± 0.03 0.11 ± 0.03 0.11 ± 0.04 ‡ † 18 0.04 ± 0.013 0.02 ± 0.003 ** 0.03 ± 0.01 ‡‡ 3 0.04 ± 0.01 0.07 ± 0.02 ** 0.02 ± 0.01 ‡‡ †† 83 1.82 ± 0.57 1.01 ± 0.18 ** 1.62 ± 0.01 ‡‡ 5 0.04 ± 0.01 0.091 ± 0.017 ** 0.05 ± 0.02 ‡‡ 33 0.26 ± 0.07 0.15 ± 0.04 ** 0.26 ± 0.05 ‡‡ 34 0.16 ± 0.03 0.23 ± 0.06 ** 0.11 ± 0.04 ‡‡ 51 0.09 ± 0.03 0.05 ± 0.01 ** 0.05 ± 0.02 †† 90 0.39 ± 0.18 0.35 ± 0.20 0.59 ± 0.25 ‡ Statistical significance was determined by a t-test, where p-value is * p <0.05 and ** p <0.01: Nor vs. OP or † p <0.05 and †† p <0.01: Nor vs. OR or ‡ p <0.05 and ‡‡ p <0.01: OP vs. OR. For each protein, the relative intensity was averaged and expressed as a mean ± SE of six separate experiments.

% Volume of Spots with Significant Changes in Expression in WAT Between Control, OP and OR Groups No. in gels
Alteration (Vol%) Normal OP OR 4 0.19 ± 0.08 0.31 ± 0.06 ** 0.21 ± 0.04 ‡‡ 16 0.2 ± 0.06 0.33 ± 0.09 ** 0.18 ± 0.04 ‡‡ 17 0.15 ± 0.04 0.15 ± 0.04 0.09 ± 0.04 ‡‡ 27 0.07 ± 0.02 0.11 ± 0.06 0.07 ± 0.02 61 0.33 ± 0.04 0.407 ± 0.05 ** 0.27 ± 0.09 ‡‡ One 0.26 ± 0.15 0.42 ± 0.13 ** 0.32 ± 0.13 26 0.1 ± 0.03 0.08 ± 0.03 * 0.09 ± 0.03 36 0.04 ± 0.01 0.06 ± 0.02 * 0.04 ± 0.02 ‡ 18 0.11 ± 0.06 0.07 ± 0.02 * 0.1 ± 0.03 ‡ 21 0.442 ± 0.126 0.602 ± 0.179 * 0.521 ± 0.168 38 1.33 ± 0.43 0.92 ± 0.21 * 0.79 ± 0.11 † 45 0.18 ± 0.05 0.259 ± 0.06 * 0.25 ± 0.05 3 0.38 ± 0.09 0.55 ± 0.13 ** 0.37 ± 0.13 ‡ 5 0.23 ± 0.11 0.16 ± 0.09 0.29 ± 0.13 ‡‡ 7 0.22 ± 0.09 0.14 ± 0.05 * 0.2 ± 0.06 ‡ 32 0.07 ± 0.03 0.05 ± 0.01 0.08 ± 0.03 ‡ 8 0.2 ± 0.07 0.374 ± 0.17 * 0.31 ± 0.11 † 12 0.14 ± 0.04 0.205 ± 0.05 * 0.139 ± 0.08 ‡ 22 0.07 ± 0.02 0.11 ± 0.03 ** 0.1 ± 0.01 ‡‡ 28 0.3 ± 0.06 0.44 ± 0.07 ** 0.295 ± 0.09 ‡‡ 29 0.06 ± 0.04 0.13 ± 0.02 ** 0.08 ± 0.03 ‡‡ 2 0.52 ± 0.15 0.18 ± 0.35 ** 0.46 ± 0.19 ‡‡ 6 0.09 ± 0.03 0.17 ± 0.07 ** 0.13 ± 0.03 51 0.42 ± 0.07 0.57 ± 0.15 * 0.45 ± 0.13 9 0.27 ± 0.09 0.403 ± 0.09 ** 0.24 ± 0.06 ‡‡ 11 0.17 ± 0.06 0.27 ± 0.07 ** 0.15 ± 0.06 ‡‡ 40 0.19 ± 0.05 0.13 ± 0.05 * 0.08 ± 0.05 ‡ † 56 0.17 ± 0.05 0.28 ± 0.08 ** 0.15 ± 0.029 ‡‡ 35 0.18 ± 0.03 0.255 ± 0.04 ** 0.16 ± 0.04 ‡‡ 13 0.19 ± 0.03 0.315 ± 0.13 * 0.24 ± 0.08 64 0.3 ± 0.14 0.17 ± 0.04 * 0.32 ± 0.13 ‡‡ 19 0.08 ± 0.04 0.14 ± 0.04 ** 0.12 ± 0.04 33 0.08 ± 0.023 0.13 ± 0.02 ** 0.01 ± 0.02 ‡ One 0.09 ± 0.04 0.12 ± 0.02 ** 0.25 ± 0.05 50 0.17 ± 0.04 0.181 ± 0.059 0.099 ± 0.029 ‡‡ †† 57 0.22 ± 0.08 0.42 ± 0.12 ** 0.22 ± 0.07 ‡‡ 20 0.63 ± 0.1 0.83 ± 0.24 * 0.56 ± 0.13 ‡ 14 0.1 ± 0.03 0.203 ± 0.03 ** 0.13 ± 0.05 ‡‡ 43 0.09 ± 0.03 0.14 ± 0.04 * 0.09 ± 0.02 ‡‡ 59 0.17 ± 0.1 0.41 ± 0.18 ** 0.22 ± 0.12 ‡‡ 15 0.06 ± 0.02 0.1 ± 0.02 ** 0.06 ± 0.03 ‡‡ 62 0.32 ± 0.1 0.51 ± 0.14 ** 0.34 ± 0.2 63 0.09 ± 0.03 0.17 ± 0.07 * 0.13 ± 0.04 10 0.12 ± 0.05 0.21 ± 0.08 * 0.16 ± 0.07 Statistical significance was determined by a t-test, where p-value is * p <0.05 and ** p <0.01: Nor vs. OP or † p <0.05 and †† p <0.01: Nor vs. OR or ‡ p <0.05 and ‡‡ p <0.01: OP vs. OR. For each protein, the relative intensity was averaged and expressed as a mean ± SE of six separate experiments.

Figures 14 and 15 show the electrophoresis pictures serially numbered as BAT and WAT, respectively.

< Example -5> Protein for Mass Spectrometry Sectioning

Protein spots are described by Shevchenko et al. [Anal. chem. 1996, 68: 850-858] were enzymatically digested into small fragments using modified porcine trypsin.

First, the gel of the spot showing the significant expression change was cut out. It was washed with 50% acetonitrile to remove impurities such as SDS, organic solvents, staining reagents from the cut pieces. After drying the gel pieces, trypsin (8-10 ng / μl) solution was added to the gel operation to decompose the protein, followed by proteolysis by rehydration at 37 ° C for 8-10 hours. After the proteolytic reaction was terminated by the addition of 5 μl of 0.5% trifluoroacetic acid, the protein fragments cut by trypsin were recovered in an aqueous solution, and desalted and concentrated to a volume of 1-5 μl using C18ZipTips (Millipore). This concentrate was mixed with α-cyano-4-hydroxycinnamic acid saturated in an equivalent 50% aqueous acetonitrile and loaded onto the target plate for mass spectrometry.

< Example  6> Identification of Proteins Using Mass Spectrometry

The mass spectrometer used Ettan MALDI-TOF (Amersham Biosciences). Protein fragments loaded on the target plate were vaporized by 337nm N2 laser irradiation and accelerated by 20Kv injection pulse. The mass spectrum of each protein spot was determined by cumulative peaks of 300 laser shots. For the analysis of the mass spectrum, the ion peak m / z (842.510, 2211.1046) of the peptide generated by autolysis of trypsin was used as standard peaks. The MASCOT Program search engine (http://www.matrixscience.com/) operated by Matrixscience was used to identify proteins from the mass spectrum after analysis.

As a result, out of a total of 158 spot proteins (92 BAT spots, 66 WAT spots) showing significant changes in expression levels between the control, OP and OR groups, a total of new obese biomarkers not reported in relation to obesity Four proteins: laminin receptor at spot 32 in BAT, antiquitin at spot 18 in WAT (aldehyde dehydrogenase family 7), protein DJ at spot 56 in WAT- 1 (protein DJ-1) and the transglutaminase A chain of the 33 spot of the WAT was confirmed (transglultaminase A chain).

In Table 3 and Table 4 below, the results of identification of the spot protein including the four novel obesity biomarkers are shown separately by BAT and WAT (the remaining spots were not identified).

<Results of Identification of BAT Spot Proteins>

<Result of Identification of WAT Spot Protein>

For reference, FIGS. 16 to 19 below show MS spectra used for identification of novel obesity biomarkers shown in [Table 1]. FIG. 16 is a laminin receptor, FIG. 17 is an antiquitin (aldehyde dehydrogenase family 7), FIG. 18 is a protein DJ-1, and finally FIG. 19 is a trans MS spectrum of the glutaminenase A chain, and FIG. 20 is a graph showing the volume concentration of each test subject and the average volume concentration of each test group in each experimental group of the new obesity biomarkers. .

< Example  7> Immunoblot  analysis

Among the proteins showing significant changes in expression levels between the control, OP and OR groups, 14-3-3 (spot 12, 14-3-3 protein zeta / delta), FABP4 (spot 85, Fatty) of BAT. acid-binding protein 4), EHD1 (spot 13, Ehd1 protein) and gelsolin (spot 52), and WAT's CES3 (spot 1, Carboxylesterase 3), PON2 (spot 35, Paraoxonase 2) and osteonectin (13). Burn spot) expression was reconfirmed using the immunoblot method.

Tissue lysates were prepared using RIPA buffer (Sigma-Aldrich, St. Louis, MO, USA), homogenized and centrifuged (12000 × g, 20 min). The supernatant was diluted in 5X sample buffer (50 mM Tris of pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue, 5% β-mercaptoethanol) and heated at 95 ° C. for 10 minutes. After electrophoresis using SDS-PAGE, the protein was transferred to polyvinylidene difluoride (PVDF, Santa Cruz Biotechnology, Santa Cruz, CA, USA) membrane and TBS-T buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1). % Tween 20 containing 5% skim milk) was blocked for 1 hour. After blocking, the membrane was washed five times with TBS-T buffer, and primary polyclonal antibodies (14-3-3, FABP4, EHD1, CES3 and osteonectin were purchased from Santa Cruz Biotechnology (USA), and gelsolin and PON2 were Ab Frontier). (Purchased from Korea) was incubated for 1 hour by diluting 1: 1000 in TBS-T buffer containing 1% skim milk. After washing the membrane five times, horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology (USA)) was incubated for 2 hours by diluting 1: 1000 in TBS-T buffer containing 1% skim milk. And developed using enhanced chemiluminescence (ECL; Amersham Biosciences). Immunoblot analysis was scanned using UMAX PowerLook 1120 (Maxium Technologies, Akron, OH, USA) and digitized using image analysis software (KODAK 1D, Eastman Kodak, Rochester, NY, USA).

The results are shown in FIG. 21, and the results that are consistent with the results shown in [Table 2] and [Table 3] were obtained.

Statistical processing

All experimental results were compared by one-way analysis of variance (ANOVA) using the Statistical Package of Social Science (SPSS) program, and measurements were expressed as mean ± standard deviation. It was considered significant when the mean difference between groups was less than or equal to p value of 0.05. Correlation analysis was performed using Pearson's linear correlation test.

Claims (9)

Select from the group consisting of laminin receptor, antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7), protein DJ-1 and transglultaminase A chain Composition for detecting obesity biomarker of adipose tissue comprising an antibody that specifically binds to at least one obesity biomarker.
The method of claim 1,
The antibody is a composition for detecting obesity biomarker of adipose tissue, characterized in that at least one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, fragments of antibodies, recombinant antibodies and chemically modified antibodies. .
Select from the group consisting of laminin receptor, antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7), protein DJ-1 and transglultaminase A chain Obesity biomarker kit for detecting adipose tissue comprising an antibody that specifically binds to at least one obesity biomarker.
The method of claim 3,
The antibody is a kit for detecting obesity biomarker of adipose tissue, characterized in that at least one selected from the group consisting of monoclonal antibodies, polyclonal antibodies, multispecific antibodies, fragments of antibodies, recombinant antibodies and chemically modified antibodies .
The method of claim 3,
The diagnostic kit is a secondary antibody, a carrier, a cleaning buffer, a sample diluent, an enzyme substrate, a reaction stopper, and whether it is vulnerable to obesity, the presence or absence of obesity, the progression of obesity, the screening or treatment of obesity or the treatment of obesity Kit for detecting obesity biomarker of adipose tissue further comprising one or more selected from the group consisting of instructions teaching progress.
Select from the group consisting of laminin receptor, antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7), protein DJ-1 and transglultaminase A chain Obesity diagnostic composition comprising an antibody that specifically binds to the obesity biomarker.
Select from the group consisting of laminin receptor, antiquitin (aldehyde dehydrogenase family 7, aldehyde dehydrogenase family 7), protein DJ-1 and transglultaminase A chain Obesity diagnostic kit comprising an antibody that specifically binds to the obesity biomarker.
(a) feeding a high calorie diet to the test animal to screen for obesity vulnerable test animals, (b) feeding any substance to the selected obese vulnerable test animal with a high calorie diet, and (c) the material Laminin receptors, antiquitin (aldehyde dehydrogenase family 7), protein DJ-1 and transglutaminase A Measuring the expression level of the obesity biomarker of the present invention selected from the group consisting of transglultaminase A chain, and (d) comparing the measured expression level of the obesity biomarker with the expression level of the normal group to prevent or Determining whether the treatment candidate is a substance
Method of screening for candidates for the prevention or treatment of obesity.
The method of claim 8,
The experimental animal screening method of the candidate substance for preventing or treating obesity, characterized in that the rat.

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