KR20160137859A

KR20160137859A - Novel Biomarker Indicative of Diabetes and Their Uses

Info

Publication number: KR20160137859A
Application number: KR1020150072096A
Authority: KR
Inventors: 안성수; 정혜린
Original assignee: 가천대학교 산학협력단
Priority date: 2015-05-22
Filing date: 2015-05-22
Publication date: 2016-12-01
Also published as: KR101893244B1

Abstract

The present invention relates to novel biomarkers for diabetes and uses thereof. The marker of the present invention can be used to conveniently, promptly and accurately diagnose diabetes as a blood marker.

Description

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The present invention relates to novel biomarkers for diabetes and uses thereof.

Diabetes mellitus is a type of metabolic disease that lacks insulin secretion or does not function normally. It is characterized by hyperglycemia in which the concentration of glucose in the blood increases. Hyperglycemia causes various symptoms and signs, and glucose is released from the urine . In modern times, changes in dietary life and lack of exercise caused a great change in the energy metabolism process of the human body, and the prevalence of diabetes is increasing. It is known that the prevalence of diabetes in Korea is 5-10%, and it is continuously increasing.

Diabetes is divided into type 1 and type 2, type 1 diabetes was formerly called 'child diabetes' and is caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively insufficiently insulin-dependent, is characterized by insulin resistance (insulin resistance that lowers blood sugar and cells do not burn glucose effectively). Although type 2 diabetes is associated with environmental factors such as high caloric value, high fat, high protein diet, lack of exercise and stress due to westernization of dietary habits, diabetes can also be caused by defects of specific genes, Infection, or drug. More than 95% of diabetes is type 2 diabetes.

To determine diabetes, measure urine glucose, measure blood sugar at fasting, and perform glucose test by oral or intravenous injection. In the case of urine glucose measurement, there is diarrhea (urinary diabetes) in which urinary sugar appears when the diabetes mellitus is mild, and if there is a decrease in the diabetic discharge threshold of the kidney even if it is not diabetic, This is a difficult problem. Diabetes mellitus is diagnosed when the fasting capillary blood glucose level is more than 140 mg / dl before breakfast. However, in the case of mild cases, it may be within the normal range, so it is necessary to perform additional glucose tolerance test.

In order to overcome the disadvantages of the conventional diabetes diagnosis method, there is a need for a technique capable of accurately diagnosing diabetes early and thus a new biomarker for diagnosis of diabetes is being developed.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 10-2010-0093562 (published on August 25, 2010) Korean Patent No. 10-1160153 (published on June 16, 2011)

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of diabetes. As a result, the present inventors have completed the present invention by confirming that diabetes can be diagnosed early by detecting all multimeric amylin in blood or amylin in the form of amyloid oligomer as a biomarker.

Accordingly, it is an object of the present invention to provide a diabetic diagnostic kit.

Another object of the present invention is to provide a method for detecting a diabetic marker so as to provide information necessary for diabetic diagnosis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the invention, the present invention provides a capture agent which binds to an epitope of amylin to capture a monomeric or multimeric form of amylin present in a biological sample of a human; And a detection agent that binds to an epitope that is overlapped with an epitope to which the capture agent binds, or that binds to an amyloid oligomer of a multimeric amylin trapped by the capture agent, wherein the capture and detection agent is an antibody or an app The kit for diagnosing diabetes is provided.

The present inventors have tried to find a novel biomarker capable of rapidly and accurately molecular diagnosis of diabetes. As a result, it was confirmed that amylin in the form of a whole multimeric amylin or amyloid oligomer in the blood as a biomarker can be used for the early diagnosis of diabetes.

As used herein, the expression "diabetes diagnostic kit" means a kit containing a diabetic diagnostic composition. Therefore, the expression "diabetes diagnostic kit" may be used in combination with or in combination with "diabetic diagnostic composition ".

As used herein, the term "diagnosing" is intended to include determining the susceptibility of an object to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, Determining the prognosis of an object (e.g., predicting the stage of diabetes or predicting its responsiveness to treatment), or determining the status of an object to provide information on therametrics Monitoring).

As used herein, the term "diabetic diagnostic marker or diabetic marker" refers to a biomolecule capable of diagnosing diabetes and showing an increased pattern in individuals suffering from diabetes compared to a normal person. For purposes of the present invention, the diabetes diagnostic marker is a multimeric amylin (full multimeric amylin, or amylin in the form of an amyloid oligomer of multimeric amylin).

As used herein, the term "monomer form of amylin" refers to amylin as a single monomolecular molecule and the term "multimeric form of amylin" Amylin < / RTI > complex. Amylin is a peptide hormone consisting of 37 amino acid residues, which is secreted with insulin in the pancreatic β-cells. The amino acid sequence of amylin is shown in SEQ ID NO: 2. Amylin is a monomeric amylin that can form a dimer, a tetramer or a fibril, and may form amyloid oligomers similar to prions. .

As used herein, the term "amyloid oligomer or amylin in the form of amyloid oligomer" refers to an oligomeric form of amylin complex formed by aggregation of amylin present in monomer form.

Amylin in the form of such amyloid oligomers can be detected using an antibody that recognizes commercially available amyloid oligomers (e.g., A11 antibody). The antibody recognizing the amyloid oligomer can recognize the oligomer of the protein or peptide regardless of the base sequence, and does not recognize the monomer or fibril of the protein or peptide.

According to one embodiment of the present invention, the kit of the present invention detects a diabetic marker (whole multimeric amylin, or multimeric amylin amyloid oligomer) by an immunoassay method, that is, an antigen-antibody reaction method Can be used.

In this case, the detection of the diabetic marker may be accomplished by (i) a capture agent (antibody or aptamer) which binds to an epitope on the amylin phase and captures a monomeric or multimeric amylin, and an epitope on which the capture agent binds (Antibody or aptamer) that binds to the < RTI ID = 0.0 > epitope < / RTI > Or (ii) a capture agent that binds to an epitope on the amylin phase to capture a monomeric or multimeric amylin, and a detection agent that binds to an amyloid oligomer in a multimeric amylin. In the former case, the entire multimeric amylin present in the biological sample can be detected. In the latter case, amylin in the form of amyloid oligomer present in the biological sample can be detected.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For purposes of the present invention, the antibody refers to an antibody that specifically binds to the marker of the present invention or the constituent protein of the marker, and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

Since the novel diabetes diagnostic markers have been identified as described above, the production of antibodies using monomeric or multimeric amylin (marker protein antigens) can be easily performed using techniques well known in the art .

The polyclonal antibody can be produced by a method well known in the art for obtaining serum containing the antibody by injecting the marker protein antigen into the animal and collecting it from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

The monoclonal antibody may be prepared by a hybridoma method (see Kohler and Milstein, European Jounal of Immunology 6: 511-519, 1976) or a phage antibody library (Clackson et al. Nature , 352: 624 -628, 1991; Marks et al., J. Mol. Biol. , 222: 58, 1-597, 1991).

The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

The kit of the present invention can be applied to a conventional immunoassay to diagnose diabetes. Such immunoassay methods are various quantitative or qualitative immunoassay methods conventionally developed. Immunoassay formats include enzyme linked immunosorbent assay (ELISA), sandwich ELISA, radioimmunoassay (RIA) Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Immunoassay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Immunodiffusion, Ouchterlony Immunodiffusion, Rocket Immunoelectrophoresis, Activated Cell Sorter (FACS), and protein chip.

Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, when the kit of the present invention is applied to a radioactive immunoassay, the detection agent in the kit is labeled with radioactive isotopes (e.g., C ¹⁴ , I ¹²⁵ , P ^32, and S ³⁵ ).

According to one embodiment of the present invention, the kit of the present invention may be applied to a Multimer Detection System (abbreviated as MDS) to be used for detecting a diabetic marker. When the kit of the present invention is applied to MDS, the kit of the present invention may further comprise, in addition to the capture agent capturing a monomeric or multimeric amylin present in a human biological sample by binding to an epitope on the amylin phase, An antibody or an aptamer that binds to an epitope overlapped with an amylin phase epitope to which the capture agent binds.

The term " overlapped with "means that the detection agent and the epitope sequence recognized by the capture agent are completely or partially overlapped. As described above, since the MDS employs capturing agents and detection agents long-lived epitopes, the detection agent can not bind to the monomer-type amylin bound to the capturing agent but can bind to the multimeric amylin. Therefore, MDS Can detect all multimeric forms of amylin other than the monomeric form of amylin present in the biological sample.

According to another embodiment of the present invention, the kit of the present invention may be applied to a sandwich ELISA and used to detect diabetic markers. In a specific example, such a sandwich ELISA comprises the steps of (a) coating an antibody capable of binding to an epitope on the amylin as a capture antibody to the surface of a solid substrate; (b) reacting the capture antibody with the biological sample; (c) reacting the result of step (b) with a detection antibody capable of binding to an amyloid oligomer in a multimeric amylin bound to a label generating signal and captured by the capture antibody; And (d) measuring a signal originating from the label.

The detection agent has a label that generates a detectable signal. The label may be labeled with a chemical (e. G., Biotin), an enzyme (alkaline phosphatase,? -Galactosidase, horseradish peroxidase and cytochrome P450), a radioactive material, But are not limited to, luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press,

Measurement of the activity of the final enzyme or measurement of the signal in the MDS and ELISA can be performed according to various methods known in the art. This detection of the signal enables a qualitative or quantitative analysis of the marker of the present invention. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

According to one embodiment of the invention, the epitope to which the capture agent binds comprises the amino acid sequence of the first sequence of the sequence listing.

According to another variant of the invention, an aptamer can be used instead of an antibody. The aptamer is an oligonucleic acid or peptide molecule, preferably a peptide molecule, and the general contents of aptamers are described in Bock LC et al. Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med . 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA . 95 (24): 142727 (1998).

By analyzing the intensity of the final signal by the above-described immunoassay procedure, diabetes can be diagnosed. Specifically, if the signal for the marker of the present invention in the sample is stronger than the normal sample, it can be diagnosed as diabetes (see Examples 1 and 2).

As described above, the kit of the present invention is a kit for immunoassay. For example, the kit of the present invention is an MDS or ELISA kit. In this case, the kit of the present invention may additionally include essential components necessary for performing MDS or ELISA.

The kits of the present invention may be made from a number of separate packaging or compartments including the components described above.

According to one embodiment of the present invention, the biological sample of the present invention is blood, plasma or serum.

According to one embodiment of the present invention, the diabetes of the present invention is type 2 diabetes.

According to another aspect of the present invention, the present invention provides a method for measuring the presence or amount of amyloid oligomers in a multimeric amylin or multimeric amylin in a human biological sample to provide information necessary for the diagnosis of diabetes A method for detecting a diabetic marker is provided.

Since the method for detecting the diabetic marker and the diabetic diagnostic kit use the same marker, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

The present invention judges diabetes by a molecular diagnostic method, not by a physician's opinion. The marker of the present invention is a biomolecule existing at a high concentration in a diabetic patient as compared with a normal person.

According to one embodiment of the present invention, the method of the present invention can be carried out by an antigen-antibody reaction method.

According to a specific example, the method comprises the steps of: (a) contacting a biological sample isolated from a human with a capture antibody immobilized on a solid phase matrix that is capable of binding to amylin; (b) contacting a detection antibody that binds to an epitope bound to an epitope to which said capture antibody binds, or a detection antibody that binds to amylin in the form of an amyloid oligomer, to the product of step (a); And (c) detecting the antigen-antibody complex formed in step (b).

Detection of the antigen-antibody complex formation can be carried out using a label that generates a detectable signal.

Since the marker of the present invention is a blood marker, the use of the marker of the present invention makes it possible to easily and quickly determine whether diabetes develops.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides novel molecular markers for diabetes.

(Ii) The marker of the present invention can be used to diagnose diabetes as a blood marker conveniently, promptly and accurately.

FIG. 1 shows the results of detection of the entire multimeric amylin present in the plasma using a Multimer Detection System (MDS). Minitab was used to obtain an individual value plot graph for the MDS results. The graph is shown as the mean value of two signals for each sample obtained by running the experiment twice.
Figure 2 shows the detection of amylin in the form of amyloid oligomers present in plasma using a sandwich ELISA. Minitab was used to obtain an individual value plot graph for the sandwich ELISA results. The graph is shown as the mean value of two signals for each sample obtained by running the experiment twice.

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

Example

Example 1. Detection of amylin of the entire multimeric form in plasma using MDS (Multimer Detection System)

1-1. Experimental material

Carbonate-bicarbonate: for dilution of capture antibody

PBS (0.01 M, pH 7.4): for dilution of plasma and streptavidin-poly HRP

PBS (0.01 M, pH 7.4) + 0.05% Tween: Wash buffer

25% BlockAce: Blocking Buffer

2N H ₂ SO ₄ : Stop solution

1-2. Plate preparation

After incubation at 4 ° C overnight, 100 μl (1 μg / ml) of anti-amylin amide pAb (Peninsula Laboratories International, Inc., T-4157.0400) was diluted with carbonate- bicarbonate, (300 [mu] L). 300 [mu] l of blocking solution was treated and incubated at 37 [deg.] C for 90 minutes and then washed three times with wash buffer (300 [mu] l).

1-3. Detection of total multimeric amylin in plasma using MDS

Blood samples were taken from 9 normal type 2 diabetic patients and 5 healthy subjects for use as controls. Specifically, 10 ml of blood was collected from 9 diabetic patients and 5 normal patients using a heparin tube. Immediately after the blood collection, the anticoagulant was slowly mixed so that the mixture was sufficiently mixed. The tubes were then stored at room temperature and centrifuged at 850 xg for 30 minutes within 1 hour after collection. Plasma was separated from the blood without mixing with blood cells, and the tubes were dispensed into 1.5 ml tubes and stored at -80 ° C.

Plasma samples were diluted 2-fold with PBS (0.01 M, pH 7.4) and then treated with 100 μl of the plate on which the capture antibody was immobilized. After treatment, the cells were incubated at 25 DEG C for 2 hours. It was then washed three times with wash buffer (300 l). The same detection antibody (biotinylated anti-amylin amide pAb; Peninsula Laboratories International, Inc., T-4157.0400) as that of the capture antibody was diluted with PBS (0.01 M, pH 7.4) and then treated with 100 μl of the plate (100 ng / ) And incubated at 25 [deg.] C for 1 hour and a half. It was then washed three times with wash buffer (300 l). Streptavidin-poly HRP (Pierce, 21140) was diluted X10,000-fold with PBS (0.01 M, pH 7.4) and then treated (100 μl) on plates and incubated at 25 ° C for 30 minutes. It was then washed three times with wash buffer (300 l). Then, 100 μl of TMB (3,3 ', 5,5'-tetramethyl benzidine) was treated on the plate, incubated for 20 minutes at 37 ° C, and then terminated with 50 μl of termination solution. Optical density values were measured at 450 nm using a microplate reader at 0.1 second intervals.

Statistical analysis was performed using Student's t-test in MiniTab version 14 (Minitab Inc., State College, PA, USA). The results were expressed as means and standard deviations. Two sample t-tests were used to compare the results.

As described above, the total amount of multimeric amylin present in the blood plasma of normal and diabetic patients was measured by the MDS method. The results of the experiment are shown in FIG. As a result, as shown in Fig. 1, a large amount of whole multimeric amylin was detected in the blood of diabetic patients (Fig. 1) as compared with normal persons. These results indicate that diabetes can be diagnosed quickly and accurately by measuring the total amount of multimeric amylin present in the blood.

Example 2. Amylin detection in the form of amyloid oligomers using a sandwich ELISA

2-1. Experimental material

Carbonate-bicarbonate: for dilution of capture antibody

PBS (0.01 M, pH 7.4): for dilution of plasma and streptavidin-poly HRP

PBS (0.01 M, pH 7.4) + 0.05% Tween: Wash buffer

25% BlockAce: Blocking Buffer

2N H ₂ SO ₄ : Stop solution

2-2. Plate preparation

2-3. Amylin detection in the form of amyloid oligomers using sandwich ELISA

Plasma samples collected as described in Example 1 were diluted 2-fold with PBS (0.01 M, pH 7.4), and then 100 μl was treated with a plate on which the capture antibody was immobilized. After treatment, the cells were incubated at 25 DEG C for 2 hours. It was then washed three times with wash buffer (300 l). (100 ng / ml) was treated with 100 [mu] l of the detection antibody (biotinylated anti-amyloid antibody A11; Biorbyt, orb152853) in PBS (0.01 M, pH 7.4) and incubated for 1 hour and 30 minutes at 25 [ . It was then washed three times with wash buffer (300 l). Streptavidin-poly HRP (Pierce, 21140) was diluted X10,000-fold with PBS (0.01 M, pH 7.4) and then treated (100 μl) on plates and incubated at 25 ° C for 30 minutes. Then, 100 μl of TMB (3,3 ', 5,5'-tetramethylbenzidine) was treated on the plate, incubated for 20 minutes at 37 ° C, and then terminated with 50 μl of the final solution. Optical density values were measured at 450 nm using a microplate reader at 0.1 second intervals.

All statistical analyzes were performed using Student's t-test in MiniTab version 14 (Minitab Inc., State College, PA, USA), and the results were expressed as mean and standard deviation. For comparison, two sample t-tests were used.

As described above, the amount of amylin in the form of amyloid oligomer present in the blood plasma was measured using a blood of a normal person and a diabetic patient by a sandwich ELISA using an amyloid oligomer-specific antibody (A11 antibody) Respectively. As a result, as shown in Fig. 2, amylin in the form of amyloid oligomer was detected in the blood of diabetic patients (Fig. 2). These results indicate that the amount of amylin in the form of amyloid oligomer present in the blood can be measured to quickly and accurately diagnose diabetes.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Gachon University of Medicine and Science Industry-Academic Cooperation Foundation <120> Novel Biomarker Indicative of Diabetes and Their Uses <130> PN150196 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> epitope of amylin <400> 1 Ala Ile Leu Ser Ser Thr Asn Val Gly Ser Asn Thr Tyr 1 5 10 <210> 2 <211> 37 <212> PRT <213> human amylin <400> 2 Lys Cys Asn Thr Ala Thr Cys Ala Thr Gln Arg Leu Ala Asn Phe Leu 1 5 10 15 Val His Ser Ser Asn Asn Phe Gly Ala Ile Leu Ser Ser Thr Asn Val 20 25 30 Gly Ser Asn Thr Tyr 35

Claims

A capture agent which binds to an epitope of amylin to capture a monomeric or multimeric amylin present in a biological sample of a human; And
Wherein the capture agent binds to an epitope bound to an epitope to which the capture agent binds, or binds to an amyloid oligomer in a multimeric amylin trapped by the capture agent,
Wherein the capture and detection agent is an antibody or an aptamer.

2. The kit of claim 1, wherein said epitope is the amino acid sequence of SEQ ID NO: 1.

The kit according to claim 1, wherein the biological sample is blood, plasma or serum.

The kit according to claim 1, wherein the diabetes is type 2 diabetes.

A method for detecting a diabetic marker through the step of measuring the presence or amount of amyloid oligomers in a multimeric type amylin or multimeric amylin in a human biological sample to provide information necessary for the diagnosis of diabetes.

6. The method of claim 5, wherein the method comprises the steps of: (a) contacting a biological sample isolated from a human with a capture antibody immobilized on a solid phase matrix capable of binding to amylin; (b) contacting a detection antibody that binds to an epitope bound to an epitope to which said capture antibody binds, or a detection antibody that binds to amylin in the form of an amyloid oligomer, to the product of step (a); And (c) detecting an antigen-antibody complex formed in step (b).