KR20180032830A - Composition of detecting biomarker for diagnosis degree of obesity, rapid diagnostic kit using the same and method for diagnosis using the same - Google Patents

Abstract

The present invention relates to a composition of detecting a biomarker for diagnosing the degree of obesity, and to a rapid diagnostic kit and a method for determining the degree of obesity, and specifically, to a diagnostic kit provided to diagnose the degree of obesity using urine as a sample, wherein the diagnostic kit comprises: a membrane on which a monoclonal antibody of an obesity-specific antigen selected from adiponectin and FGF21 is adsorbed; a gold pad in which the obesity-specific antibody is attached to gold particles, and dried by being impregnated with an antibody-gold conjugate; and a diagnostic strip in which a sample pad and an absorption pad supporting the flow of the sample are laminated. According to the present invention, it is possible to diagnose obesity and obesity complications through coloring by obesity-specific antigens and an antibody reaction and to diagnose polarity of obesity quickly and simply.

Description

TECHNICAL FIELD The present invention relates to a composition for detecting a biomarker for the diagnosis of obesity, a rapid diagnostic kit for the diagnosis of obesity using the same, and a method for determining the degree of obesity using the same.

The present invention relates to a composition for detecting a biomarker for the diagnosis of obesity, a rapid diagnostic kit for the diagnosis of obesity, and a method for determining the degree of obesity using the diagnostic kit.

Obesity means a state of excessive accumulation of fat in the body, and it occurs not only as a cause of one cause but as a combination of genetic and environmental factors. Bouchard et al. (1990) investigated whether there is a correlation between individual differences in body weight gain and genotype. In a study of identical twins, we found that individual differences in body weight and body fat change patterns for overeating were due to genetic factors. Levine et al. (2005) suggested that non-excercise activity thermogenesis is a major factor in individual differences in weight control programs, and approximately 40-70% of human obesity-related phenotypes are genetic (Comuzzie, 1998). Therefore, it is very important to find gene markers that can diagnose obesity quickly and accurately.

As a result of continuing research to develop a gene marker capable of diagnosing obesity, the present inventors have completed the present invention by identifying a gene whose expression is specifically increased in an obesity model with increased body weight.

1. Korean Patent No. 10-1262496

It is an object of the present invention to provide a composition for detecting obesity or obesity diagnostic biomarkers comprising adiponectin and an agent for measuring the level of mRNA of the FGF 21 gene or the protein encoded by the gene.

It is still another object of the present invention to provide a rapid diagnostic kit for the diagnosis of obesity by an immunochromatographic method from a urine sample.

It is still another object of the present invention to provide a method for determining the result of the rapid diagnosis diagnostic rabbit diagnostic kit.

According to one aspect of the present invention, there is provided a method for producing a protein having mRNA of at least one obesity-specific gene selected from the group consisting of adiponectin, FGF21, and a mixture thereof, And a biomarker composition for diagnosing obesity.

According to another aspect of the present invention, there is provided a diagnostic kit for diagnosing obesity with urine as a sample, comprising: a monoclonal antibody or a polyclonal antibody of an obesity-specific antigen selected from adiponectin and FGF21, Which is capable of diagnosing the degree of obesity through color development resulting from a reaction between the specific antigen and the antibody.

According to another aspect of the present invention, there is provided a method for evaluating the results of a rapid diagnostic kit for obesity diagnosis, comprising the steps of: applying 80 to 120 μl of urine to a sample pad of a diagnostic kit, 15 to 20 minutes after the control line C In the case of negative samples, only the control line is colored and displayed in reddish purple color. In the positive sample, both the control line and the resultant line are reddish purple, and the color of the test line is measured. Of the rapid diagnosis kit.

According to the present invention, an immunochromatography-based diagnostic kit using monoclonal and polyclonal antibodies against recombinants of adiponectin and FGF21 can be easily measured from a sample that can be easily collected, such as urine, and the biomarker can be quantified It is possible to diagnose obesity and complications, and it is possible to diagnose voice of obesity quickly and easily.

Figure 1 is a standard curve of the adiponectin ELISA.
2 is a structure of a rapid diagnostic kit for measuring obesity according to an embodiment of the present invention.
FIG. 3 is a color chart and a reaction result of the detection limit experiment of adiponectin according to an example of the present invention.
FIG. 4 is an image showing measurement results of adiponectin and FGF-21 of a rapid diagnostic kit for measuring obesity according to an example of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately It should be interpreted in accordance with the meaning and concept consistent with the technical idea of the present invention based on the principle that it can be defined.

Therefore, the constitutions shown in the drawings and the embodiments described in the present specification are merely the most illustrative examples of the present invention and are not intended to represent all of the technical ideas of the present invention, so that various equivalents And variations are possible.

Diagnosis of obesity Biomarker  Composition

The present invention provides a biomarker composition for diagnosing obesity, comprising mRNA of at least one gene selected from the group consisting of adiponectin, FGF21, and a mixture thereof, or an agent for measuring the level of protein encoded by the gene.

In the present invention, "diagnosis" means confirming a pathological condition. For the purposes of the present invention, the diagnosis is to ascertain whether an obesity or obesity complication is present.

In the present invention, the term "diagnostic biomarker" refers to a polypeptide or nucleic acid having a significant increase or decrease in the level of gene expression or protein expression in an individual having obesity or obesity complaints compared to a normal control (non-obese individual) mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.

For the purpose of the present invention, the biomarker for the diagnosis of obesity may be at least one selected from adiponectin and FGF21.

The adiponectin and FGF21 gene according to the present invention are characterized in that the expression of the FGF21 gene is significantly increased in the group inducing obesity as compared with the normal group (control group), and thus it can be usefully used for the diagnosis of obesity.

In the present invention, the agent for measuring the level of the protein encoded by the adiponectin and the FGF21 gene comprises an antibody specific to the protein encoded by the gene.

&Quot; Antibody " in the present invention means a specific protein molecule indicated for an antigenic site, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

The polyclonal antibody can be produced by a method well known in the art for obtaining an antibody-containing serum by injecting an antigen of the gene into an animal and collecting it from an animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, and the like. Monoclonal antibodies may also be prepared using hybridoma methods (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519) or phage antibody libraries (Clackson et al, Nature, The antibodies of the present invention can also be prepared using two full length antibodies of the full length (SEQ ID NOs: 1, 2, 3, 4, 5, 6, Functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and Fab, F (ab ') 2, ), F (ab ') 2, and Fv.

The biological sample includes tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine, but preferably urine (urine) may be used.

The level of mRNA expression may be determined by PCR, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay RPA (RNase protection assay), Northern blotting, and DNA chip, but the present invention is not limited thereto.

The protein expression level can be measured by an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (Radioimmunoassay) or immunoassay using an antigen-antibody binding reaction using an antibody that specifically binds to a protein encoded by an obesity- (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, But are not limited to, methods such as fluorescence activated cell sorter (FACS) and protein chip.

Through these methods, it was found that adiponectin, FGF21 (SEQ ID NO: 2), and FGF21 were significantly higher in the biological sample than the normal group (control) by comparing the level of adiponectin, the mRNA of the FGF21 gene or the protein level encoded by the gene to the mRNA or protein expression level in the normal A significant increase in the mRNA or protein expression level of a gene can be used to diagnose obesity as an obesity, and the degree of obesity can be diagnosed by quantitatively analyzing the increase.

In one example, the method for measuring obesity using the obesity-specific genes adiponectin and FGF21 biomarker of the present invention includes, but is not limited to, the following steps.

(a) measuring the level of protein expression of adiponectin, and FGF21, or the level of mRNA expression of adiponectin and FGF21 gene from a biological sample;

(b) comparing the level of mRNA expression of the adiponectin of said biological sample and the protein expression level of FGF21 or of the gene encoding adiponectin and FGF21 with the expression level of a normal sample; And

(c) diagnosing obesity if the expression level of the adiponectin and FGF21 protein or mRNA of the biological sample is higher than the expression level of the normal sample.

Diagnosis of obesity Rapid Diagnostic Kit

The present invention also provides an obesity diagnostic kit comprising the composition. Conventionally, since the diagnostic kit using blood requires special equipment such as ELISA reader and automation equipment and specialized manpower, it is required to go to the hospital to get the test result and obtain the test result, and to solve the complicated and long time- Provides a simple and rapid diagnostic kit that can verify your test results within 15-20 minutes.

The diagnostic kit of the present invention is designed based on an immunochromatographic assay in which urine is used as a specimen to diagnose obesity by coloring according to an antigen-antibody reaction of an obesity-specific antigen. Therefore, the principles of currently available diagnostic kits can be applied as they are. Immunochromatography is a test method that combines immunochemistry and chromatogrphic assay to detect the specific immunoreactivity of an antibody to an antigen , Colloidal gold, and molecular movement due to the capillary phenomenon of a porous membrane.

The diagnostic kit of the present invention comprises a monoclonal antibody or a polyclonal antibody of an obesity-specific antigen selected from among adiponectin and FGF21, and is capable of diagnosing obesity through color development depending on the reaction between the obesity-specific antigen and the antibody have. Specifically, the diagnostic kit of the present invention comprises a membrane on which a monoclonal antibody or polyclonal antibody of an obesity-specific antigen selected from adiponectin and FGF21 is adsorbed; A gold pad dried by attaching the obesity-specific antibody to gold particles and impregnating the antibody-gold conjugate; A sample pad for supporting the flow of the sample, and a hygroscopic pad.

Referring to FIG. 2, the rapid diagnosis kit for obesity diagnosis according to one preferred embodiment comprises: a membrane 110 adsorbing monoclonal antibody of selected obesity-specific antigen among adiponectin and FGF21; A gold pad 120 attached to the gold particles and impregnated with the antibody-gold conjugate; A sample pad 130 and a hygroscopic pad 140 for facilitating the flow of the sample.

The membrane that can be used in the production of the diagnostic strip in the present invention is a material used as a material of a conventional diagnostic strip. For example, various synthetic polymers such as nitrocellulose, celluloses, cellulose acetate, May be used, and the reagents used for the immunological analysis may be provided with a suitable carrier, a detection label capable of generating a detectable signal, a solubilizer, and a detergent. In addition, when the detection labeling agent is an enzyme, a substrate capable of measuring enzyme activity and a suitable carrier that can contain a reaction terminator include a soluble carrier such as a physiologically acceptable buffer (for example, PBS) or the like can be used.

The membrane can change its original color when it is in contact with urine and allows various test results to be known according to the converted color, which is well known in the art. It is obvious that the number of the inspection areas and the kinds of colors can be variously configured depending on the type of the urine strap.

The diagnostic antibody according to the present invention may be provided as a purified monoclonal antibody, a rabbit antibody, a goat anti-mouse IgG, a 45 nm gold colloidal gold raw material which is a raw material of a commercially available antibody that can be used for the production of strips You can use them. 45nm colloidal gold is a suitable coloring material and contains radioactive materials such as 125I, 131I, 111In, 99Tc14C, and 3H.

In one preferred example, the strip material is a nitrocellulose membrane, and the membrane is divided into two lines (1 line: Control, 2 lines: Test), and the first line (Control, 111) And in the second line (Test 112) monoclonal antibodies of selected obesity-specific antigens of adiponectin, FGF21 and mixtures thereof may be dispensed at a concentration of 0.5 to 1.5 mg / ml.

The obesity diagnostic kit and the diagnostic method of the present invention are characterized in that the obesity-specific antigen in the urine forms an immunological complex with the antibody against adiponectin or FGF-21 in the kit through an antigen-antibody reaction, Numerous immunoassays used to detect or quantitate antigens are known to those skilled in the art and are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988, 556-612 , Reiter RE., Gu Z., Watabe T., Thomas G., Prostate stem cell antigen: A cell surface marker overexpressed in prostate cancer. Proc Natl Acad Sci USA. 1998. Feb 17; 95 (4): 1735-40.

The immunochromatography can be performed in a one-step manner by integrating the sample dilution, washing, and the color development process through the reaction of the enzyme conjugate and the substrate required in a conventional multi-step immunoassay. And also has advantages in ease of being able to judge a test result without using specific equipment, economical efficiency, and quickness of reading test result.

When the amount of obesity-specific antigen in the urine is 2.75 ng / ml to 44 ng / ml, the sensitivity gradually increases in proportion to the concentration, thereby diagnosing obesity. For example, in the diagnostic kit of the present invention, the cutoff of adiponectin for obesity diagnosis is 5.6 g / ml and the cutoff of FGF-21 is 46 pg / ml. In addition, the diagnostic kit provided herein can utilize urine as a sample as well as blood to be used as a sample to measure an obesity-specific antigen by immunochromatography, and can be used not only for obesity but also for diabetes, insulin resistance syndrome, Osteoarthritis, osteoarthritis, non-alcoholic fatty liver disease, cardiovascular disease, polycystic ovary syndrome, and metabolic syndrome.

The present invention can be provided as a diagnostic kit capable of semi-quantitatively measuring the amount of adiponectin or FGF-21 in the urine and blood by the immunochromatography method. Further, in order to accurately diagnose the present invention And can be applied as a method of accurately diagnosing immune response to a body fluid component using a reader.

The rapid diagnostic kit according to the present invention may further include a plastic housing having a sample input port, a result display window, and a handle for commercialization and allowing the diagnostic strip to be assembled therein to observe test results.

The method for producing the rapid diagnostic kit according to the present invention is not particularly limited and may be carried out using methods known in the art, for example, the following steps.

(a) coating a goat anti-mouse antibody at a first line (Control) position;

(b) coating a second line of the membrane (Test) with an antibody against an obesity-specific antigen selected from adiponectin, FGF21 and mixtures thereof;

(c) preparing an antibody-gold conjugate by binding an antibody against purified adiponectin and an antibody against FGF-21 to a gold colloid; And

(d) attaching a dried moisture absorption pad to the top of the membrane.

Diagnosis of obesity Rapid Of the diagnostic kit  Judgment method

In addition, the present invention provides a method for determining the degree of obesity using the rapid diagnostic kit.

In one example, urine was added to the sample pad of the diagnostic kit in an amount of 80 to 120 mu l as a sample. After 15 to 20 minutes, the color of the control line C and the test line T was determined. And when the sample is a positive sample, both the control line and the resultant line are reddish purple, and the degree of obesity can be judged based on the color of the resultant line. For example, if the color is dark, it is judged to be high-level obesity. If the color is light, it can be judged to be obesity.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

[ Experimental Example  1] in human urine Adiponectin  Numerical verification

Adiponectin was quantitatively detected in a urine kit using an adiponectin ELISA kit (AdipoGen, Korea). Adiponectin concentration was 0.76 ng / ml in a normal urine specimen (1 case) The concentration of adiponectin in the urine was 44.3 ng / ml, indicating a significant difference between the obese and normal groups.

[ Manufacturing example  1] Recombinant antigen development

1-1. Adiponectin ( Adiponectin )

The amino acid 101 to 244 region containing the Complement component C1q domain was amplified by PCR (SEQ ID NO: 2) and cloned into an expression vector using the synthetic gene sequence (SEQ ID NO: 1) of Homo sapiens adiponectin (NM_001177800) as a template. E coli was used as a recombinant protein expression system and pQE vector was used. For E. coli , the cells were cultured in LB medium at 37 ° C and 220 rpm, and then induced with IPTG at a value of 0.5 at OD600. For purification, cell pellet except supernatant was disrupted, and insoluble protein was dissolved in 6M guanidine to perform Ni-affinity purification. The purified protein was dialyzed against 20 mM Carbonate buffer (pH 9.6).

1-3. FGF21

Amino acid sequences 29 to 209, which are mature forms except the signal sequence, were amplified by PCR using the synthetic gene sequence (SEQ ID NO: 3) of homo sapiens fibroblast growth factor 21 (FGF21, NM_019113) as a template (SEQ ID NO: Respectively. The subsequent process is the same as the development of Adiponectin antigen.

[ Manufacturing example  2] Antibody Development

2-1. Adiponectin

In step 1-1, the purified recombinant antigen was used as an immunogen to immunize BALB / c mice. Then, mouse spleen cells were isolated and conjugated with myeloma cells. Hybrid cell lines secreting monoclonal antibody against recombinant adiponectin antigen were selected by ELISA method and single cell group was established by step dilution method. The resulting cell line was inoculated into BALB / c mouse abdominal cavity in a large number of cell lines, and the IgG antibody was purified using protein A / G gel. The obtained antibody is a mouse monoclonal antibody prepared by using adiponcetin antigen as an immunogen.

2-2. FGF21

A mouse monoclonal antibody was obtained in the same manner as in Production Example 2-1, except that the recombinant antigen purified in the above step 1-3 was used as an immunogen and FGF21 was used instead of the adonectin in Production Example 2-1 .

[ Example  1] Manufacture of diagnostic strips

The test strip consists of a sample pad Gold Pad Membrane Moisture Absorbing Pad sequence as shown in FIG.

Membrane  Coating process

The test line of the nitrocellulose membrane was coated with an antibody specific for Adiponectin, Leptin or FGF21, and the goat anti-mouse antibody was coated on the control line. Herein, the antibody against Adiponectin, Leptin or FGF21 antigen was diluted with PBS (phosphate buffered saline) at a concentration of 1 mg / ml and coated at the inspection line position of the membrane.

(2) Antibody-gold conjugate manufacturing process

Antibodies against purified Adiponectin, antibodies against Leptin, and antibodies against FGF-21 (Gold colloid), respectively. Specifically, the gold chloride was reduced with a sodium citrate solution to prepare gold particles having a size of about 40 nm such that the absorbance at 532 nm was 10 +/- 1. The gold particles were stabilized with a PEG (poly (ethylene glycol)) solution, and then the purified antibody against Adiponectin, the antibody against Leptin and the antibody against FGF-21 were mixed at a predetermined ratio (for example, 10 μg / , Impregnated with polyester or glass fiber, and dried to prepare a gold-pad.

(3) Manufacture of absorbent pad and strip assembly

The dried hygroscopic pad was attached to the top of the nitrocellulose membrane to allow the reaction solution passed through the nitrocellulose membrane to be well absorbed.

[ Example  2] Quickly Immunochromatography  by Adiponectin  Diagnostic method

(1) All samples and reagents should be kept at room temperature for 15 ~ 30 minutes before starting the experiment.

(2) Open the ventilator and take out the test device and put it on a flat surface.

(3) Take 100 μl of urine with a suitable pipette and drop it on elliptical specimen.

(4) When the test starts 15-20 minutes after the start of the test, the test line (T) result is read.

All test results shall show purple lines on the contour line (C). If there are no bands in the tide, this test may be invalidated and retest with new reagent as there is a possibility that the test is wrong or there is a problem with the quality of the reagent.

[ Experimental Example  2] Quickly Immunochromatography  by Adiponectin  Diagnostic efficacy test

The samples were serial diluted to normal urine specimens (0.76 ng / ml) up to 1.38 ng / ml using an obesity urine specimen (20 pools, 44 ng / ml) (Fig. 3). Sensitivity was proportional to concentration from 44 ng / ml to 2.75 ng / ml.

[ Experimental Example  3] Diagnosis of obesity Rapid Diagnostic Kit  Obesity determination using

The diagnostic kit according to the present invention was used to determine the degree of obesity from the urine of a child / adolescent obese patient.

Urine samples were collected from 93 obese subjects with a BMI greater than 99 percent of age 13 to 15 years old and 92 control subjects with a BMI of 25 to 75 percentile and were kept at 80 ° C until use. Age and gender matched the obesity group and the control group. The biomarkers of the diagnostic kit used are adiponectin, FGF-21, gretinin (Cr) and albumin (Alb).

The urine sample stored at -80 ° C was thawed at room temperature 30 minutes before the start of the experiment and centrifuged at 3000 rpm for 10 minutes to remove the debris.

For the determination of adiponectin, urine samples were diluted 1/10 with dilution buffer and standard was prepared at 0, 0.5, 1, 2, 4, 8, 16, 32 ng / ml. Standard and samples were dispensed in duplicate on a 96-well plate coated with 100 μl of adiponectin antibody and incubated at 37 ° C for 1 hour. After washing with 300 μl of wash buffer three times, 100 μl of detection antibody was added and incubated at 37 ° C for 1 hour. After that, the plate was washed three times, 100 쨉 l of Detector was added, incubated at 37 째 C for 1 hour, and washed three times. 100 μl of the substrate solution was added and incubated for 20 minutes at room temperature. 100 μl of the stop solution was added thereto, and OD was measured at 450 nm in an ELISA reader within 30 minutes.

The result was calculated by the standard curve with the X axis as the concentration and the Y axis as the OD value, and the dilution factor of 10 was used to calculate the sample concentration.

For the measurement of FGF-21, standards were prepared at 0, 30, 60, 120, 240, 480, 960 and 1920 pg / ml. QC HIGH and QC LOW were prepared by dissolving with dilution buffer. Urine samples were not diluted. Standard, QC, and 100 μl of each sample were dispensed in duplicate on a 96-well plate coated with antibody, incubated in an orbital shaker at 300 rpm for 1 hour at room temperature, and washed three times with 350 μl of wash buffer. Then, 100 μl of Biotin labeled antibody was added, incubated at room temperature and 300 rpm for 1 hour, and washed three times. 100 ㎕ of Streptavidin-HRP Conjugate was then added, incubated at room temperature for 30 minutes at 300 rpm, and then washed three times. 100 μl of substrate solution was added and incubated for 15 minutes at room temperature. 100 μl of stop solution was added and the OD was measured at 450 nm / 630 nm in an ELISA reader within 5 minutes.

The results were calculated using the standard curve with the X axis as the concentration, the Y axis as the OD value, and the sample concentration with the four-parameter algorithm.

Cr and Alb were measured using an automated chemistry analyzer (Toshiba 200-FR).

The measured results were analyzed by the Wilcoxon rank test. The ROC AUC of each biomarker was analyzed by ROC analysis. The C-tree and Maxstat were used to determine the appropriate cutoff value for the diagnosis of obesity. And the results are shown in Table 2.

The median urinary adiponectin concentration was significantly higher in the obese group than in the control group (2.4 vs 3.8 ng / mL, p = 0.021). Urinary FGF-21 levels were significantly higher in the obese group (median = 43.7 in the control group and 59.0 pg / mL in the obese group (p = 0.006)). Adiponectin / Cr, FGF-21 / Cr, Adiponectin / Alb and FGF-21 / Alb were also significantly higher in the obese group than in the control group.

Adiponectin was 5.6 ng / mL and FGF-21 was 46 pg / mL in the cut-off for obesity diagnosis using Maxstat and C-tree.

FIG. 4 is a graph showing the FGF-21 (upper) and Adiponectin (lower) measurement results of the Rabbit's Diagnostic Kit for Obesity Measurement. From the left, 2 cases. BMI (body mass index, BMI) was used as the criterion for positive and negative specimens. BMI was more than 99% positive (high obesity) and 25 ~ 75% negative (normal). As can be seen from FIG. 4, the kit of the present invention is designed to be able to distinguish between high obesity and normal weight by accurately detecting FGF-21 and adiponectin.

<110> INJE UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION          SEOUL NATIONAL UNIVERSITY HOSPITAL          STANDARD DIAGNOSITCS, INC. <120> COMPOSITION OF DETECTING BIOMARKER FOR DIAGNOSIS DEGREE OF          OBESITY, RAPID DIAGNOSTIC KIT USING THE SAME AND METHOD FOR          DIAGNOSIS USING THE SAME <130> PN1411-349 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin <400> 1 atgctgttgc tgggagctgt tctactgcta ttagctctgc ccggtcatga ccaggaaacc 60 acgactcaag ggcccggagt cctgcttccc ctgcccaagg gggcctgcac aggttggatg 120 gcgggcatcc cagggcatcc gggccataat ggggccccag gccgtgatgg cagagatggc 180 acccctggtg agaagggtga gaaaggagat ccaggtctta ttggtcctaa gggagacatc 240 ggtgaaaccg gagtacccgg ggctgaaggt ccccgaggct ttccgggaat ccaaggcagg 300 aaaggagaac ctggagaagg tgcctatgta taccgctcag cattcagtgt gggattggag 360 acttacgtta ctatccccaa catgcccatt cgctttacca agatcttcta caatcagcaa 420 aaccactatg atggctccac tggtaaattc cactgcaaca ttcctgggct gtactacttt 480 gcctaccaca tcacagtcta tatgaaggat gtgaaggtca gcctcttcaa gaaggacaag 540 gctatgctct tcacctatga tcagtaccag gaaaataatg tggaccaggc ctccggctct 600 gtgctcctgc atctggaggt gggcgaccaa gtctggctcc aggtgtatgg ggaaggagag 660 cgtaatggac tctatgctga taatgacaat gactccacct tcacaggctt tcttctctac 720 catgacacca actga 735 <210> 2 <211> 432 <212> DNA <213> Artificial Sequence <220> <223> Adiponectin <400> 2 aaaggagaac ctggagaagg tgcctatgta taccgctcag cattcagtgt gggattggag 60 acttacgtta ctatccccaa catgcccatt cgctttacca agatcttcta caatcagcaa 120 aaccactatg atggctccac tggtaaattc cactgcaaca ttcctgggct gtactacttt 180 gcctaccaca tcacagtcta tatgaaggat gtgaaggtca gcctcttcaa gaaggacaag 240 gctatgctct tcacctatga tcagtaccag gaaaataatg tggaccaggc ctccggctct 300 gtgctcctgc atctggaggt gggcgaccaa gtctggctcc aggtgtatgg ggaaggagag 360 cgtaatggac tctatgctga taatgacaat gactccacct tcacaggctt tcttctctac 420 catgacacca ac 432 <210> 3 <211> 630 <212> DNA <213> Artificial Sequence <220> <223> FGF-21 <400> 3 gggttcgg cttctgctgg gagcctgcca ggcacacccc atccctgact ccagtcctct cctgcaattc 120 gggggccaag tccggcagcg gtacctctac acagatgatg cccagcagac agaagcccac 180 ctggagatca gggaggatgg gacggtgggg ggcgctgctg accagagccc cgaaagtctc 240 ctgcagctga aagccttgaa gccgggagtt attcaaatct tgggagtcaa gacatccagg 300 ttcctgtgcc agcggccaga tggggccctg tatggatcgc tccactttga ccctgaggcc 360 tgcagcttcc gggagctgct tcttgaggac ggatacaatg tttaccagtc cgaagcccac 420 ggcctcccgc tgcacctgcc agggaacaag tccccacacc gggaccctgc accccgagga 480 ccagctcgct tcctgccact accaggcctg ccccccgcac tcccggagcc acccggaatc 540 ctggcccccc agccccccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 600 cagggccgaa gccccagcta cgcttcctga 630 <210> 4 <211> 543 <212> DNA <213> Artificial Sequence <220> <223> FGF-21 <400> 4 caccccatcc ctgactccag tcctctcctg caattcgggg gccaagtccg gcagcggtac 60 ctctacacag atgatgccca gcagacagaa gcccacctgg agatcaggga ggatgggacg 120 gtggggggcg ctgctgacca gagccccgaa agtctcctgc agctgaaagc cttgaagccg 180 ggagttattc aaatcttggg agtcaagaca tccaggttcc tgtgccagcg gccagatggg 240 gccctgtatg gatcgctcca ctttgaccct gaggcctgca gcttccggga gctgcttctt 300 gaggacggat acaatgttta ccagtccgaa gcccacggcc tcccgctgca cctgccaggg 360 aacaagtccc cacaccggga ccctgcaccc cgaggaccag ctcgcttcct gccactacca 420 ggcctgcccc ccgcactccc ggagccaccc ggaatcctgg ccccccagcc ccccgatgtg 480 ggctcctcgg accctctgag catggtggga ccttcccagg gccgaagccc cagctacgct 540 tcc 543

Claims (9)

For the detection of an obesity diagnostic biomarker comprising an mRNA of at least one obesity-specific gene selected from the group consisting of adiponectin, FGF21, and a mixture thereof, or an agent for measuring the protein level encoded by the obesity-specific gene Composition. The method according to claim 1,
Wherein the agent is a monoclonal antibody or a polyclonal antibody produced by using an obesity-specific antigen selected from among adiponectin and FGF21 as immunogens. A diagnostic kit for diagnosing obesity using urine as a sample, comprising:
A monoclonal antibody or a polyclonal antibody of an obesity-specific antigen selected from the group consisting of adiponectin, FGF21, adiponectin, and FGF21, and is capable of diagnosing the degree of obesity by coloring upon reaction between the obesity- Diagnostic Rapid Diagnostic Kit. The method of claim 3,
Wherein the diagnostic kit comprises: a membrane on which a monoclonal antibody or a polyclonal antibody of an obesity-specific antigen selected from adiponectin and FGF21 is adsorbed; A gold pad dried by attaching the obesity-specific antibody to gold particles and impregnating the antibody-gold conjugate; A sample pad for supporting the flow of the sample, and a hygroscopic pad. The method of claim 3,
Wherein the diagnostic kit is characterized by being able to confirm the color development progressively in proportion to the concentration from 44 ng / ml to 2.7 ng / ml in obesity-specific antigen in the urine. The method of claim 3,
Wherein the cutoff of adiponectin for diagnosis of obesity is 5.6 g / ml and the cutoff of FGF-21 is 46 pg / mL. 5. The method of claim 4,
The strip material is a nitrocellulose membrane and two lines (1 line: Control, 2 lines: Test) are applied to the membrane. The goat anti-mouse antibody is dropped on the first line (control) ), A monoclonal antibody of an obesity-specific antigen selected from adiponectin, FGF21, and a mixture thereof is dropped at a concentration of 0.5 to 1.5 mg / ml. 5. The method of claim 4,
Further comprising a plastic housing having a sample inlet, a result display window, and a handle, and assembling the diagnostic strip therein to observe the inspection results. 9. A method for determining the results of a rapid diagnostic kit for the diagnosis of obesity according to any one of claims 3 to 8,
80-120 占 퐇 of urine was added to the sample pad of the diagnostic kit for 15-20 minutes and the color of the control line C and the test line T was determined. Red and purplish color. In the case of positive samples, both the control line and the resultant line are reddish purple.
And determining the degree of obesity based on the degree of color development of the resultant line.

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