KR101160153B1 - Method for diagnosing type 2 diabetes mellitus by measuring the level of beta-amyloid autoantibody in body fluids and diagnostic kit for type 2 diabetes mellitus using same - Google Patents

Abstract

The present invention relates to a method for diagnosing type 2 diabetes by measuring the concentration of beta-amyloid (β) autoantibodies in body fluids, and to a diagnostic kit for type 2 diabetes using the method. Methods and diagnostic kits make it easier to diagnose type 2 diabetes early.

Description

METHOD FOR DIAGNOSING TYPE 2 DIABETES MELLITUS BY MEASURING THE LEVEL OF BETA-AMYLOID AUTOANTIBODY IN BODY FLUIDS AND DIAGNOSTIC KIT FOR TYPE 2 DIABETES MELLITUS USING SAME}

The present invention relates to a method for diagnosing type 2 diabetes by measuring the concentration of beta-amyloid (Aβ) autoantibodies in body fluids and a diagnostic kit for type 2 diabetes using the method.

Diabetes mellitus (DM) is one of the most common diseases and is characterized by an increase in metabolic disorders as blood glucose levels rise (Alberti KG, Zimmet PZ (1998), Diabet Med 15 , 539-553). Diabetes appears to be a hyperglycemic state but can also lead to microscopic or macrovascular complications associated with individual morbidity and mortality. Moreover, diabetes is also associated with central obesity, hypertension and dyslipidemia, which collectively constitute metabolic syndrome. The components of metabolic syndrome are related to each other, each of which is a life-threatening risk factor such as myocardial infarction and cerebral vascular disease. Diabetes is classified into type 1 diabetes (T1DM) and type 2 diabetes (T2DM). In type 1 diabetes, pancreatic beta cells are reduced by non-ideal autoimmune reactions, and in type 2 diabetes, insulin resistance increases due to insulin resistance, thereby preventing the proper signaling response. However, as type 2 diabetes develops, pancreatic beta cells may also decrease.

Alzheimer's disease (AD) is a degenerative neurological disease that develops with age, with neurofibrillary tangles derived from senile plaques and hyperphosphorylated tau proteins formed from Aβ peptides. Observed (Yankner BA (1996) Neuron. 16, 921-932).

These diabetes and Alzheimer's disease initially appeared to be pathologically unrelated, but there is increasing evidence that they are strongly related to each other (Ott A, et al., (1999), Neurology 53 , 1937-1942; And Pelia R, et al., (2002), Diabetes 51 , 1256-1262). For example, the incidence of Alzheimer's disease is two to five times higher in type 2 diabetic patients than in normal people (Li L, Hlslscher C (2007), Brain Res Rev 56 , 384-402). Diseases exhibit similar pathologies, such as, for example, age-related, similar metabolic changes, degenerative diseases, and aggregation of amyloid beta. The pathological feature of Alzheimer's disease is Aβ spots, and islet amyloid is associated with the destruction of pancreatic islet cells mainly in type 2 diabetes. In addition, pancreatic amyloid is generated from islet amyloid β precursor protein (IAβPP) having high homology with amyloid beta precursor protein (AβPP). That is, in the same way that Aβ is derived from AβPP, pancreatic amyloid is derived from IAβPP (Cooper GJ, et. Al., (1987), Proc Natl Acad Sci USA 84 , 8628-8632). In addition, pancreatic islet amyloid is associated with induction of inflammatory responses and neurotoxicity (Gotz J, et. Al., (2009), Cell Mol Life Sci. 66 , 1321-1325). Therefore, recently, injection of streptozotocin, a diabetes-causing compound, into an intracerebroventricular has been used to prepare a new animal model of Alzheimer's disease. In contrast, it has been suggested that Alzheimer's disease represents a "brain-specific form of diabetes" (ie, type 3 diabetes) (Lester-Coll N, et. Al., (2006), J Alzheimers Dis 9 , 13- 33; Steen E, et. Al., (2005), J Alzheimers Dis 7 , 63-80; and de la Monte SM, et. Al., (2006), J Alzheimers Dis. 10 , 89-109).

Meanwhile, according to Sohn JH et al., (2007), Biochem Biophyus Res Commun 361, 800-804, the concentration of Aβ autoantibodies in serum was decreased in Tg 2576 mice, the Alzheimer's disease animal model. The concentration of Aβ autoantibodies in serum was also reduced in old mice with high Aβ concentrations. Another study similarly reported a decrease in Aβ autoantibody levels in Alzheimer's disease patients (Sohn JH et al., (2009), Front Biosci 14, 3879-3883; Weksler ME et al., ( Exp Gerontol 37, 943-948; and Moir RD et al. (2005), J Biol Chem 280, 17458-17463).

Immunization with Αβ peptide also prevents the accumulation of amyloid and can remove plaque that has already been generated from the brain (Bard F, et. Al., (2000), Nat Med 6 , 916-919; DeMattos RB, et. al., (2002), Science 295 , 2264-2267; Dickstein DL et al., (2006), FASEB J 20 , 426-433; and Schenk D, et. al., (1999), Nature 400 , 173-177), Decreased concentrations of Aβ autoantibodies will further promote Aβ accumulation by weakening the ability to remove Aβ from the brain of Alzheimer's disease patients (Pan W, et. Al., (2002), Exp Biol Med ). (Maywood) 227 , 609-615).

In the present invention, it was found that Αβ autoantibodies were continuously decreased in the serum of Alzheimer's disease patients but increased in patients with type 2 diabetes. This is in stark contrast to the findings that surprisingly prove that diabetics develop Alzheimer's disease.

Accordingly, the present inventors have attempted to investigate the relationship between Aβ autoantibodies and type 2 diabetes and to develop a test method for effective early diagnosis of type 2 diabetes. As a result, beta-amyloid present in the blood of type 2 diabetes patients The present invention was completed by confirming that type 2 diabetes can be effectively diagnosed by measuring the concentration of beta-amyloid antibody in the blood by antigen-antibody binding reaction using an antigen protein that specifically binds to an autoantibody.

Accordingly, it is an object of the present invention to provide a method for diagnosing type 2 diabetes by measuring the concentration of beta-amyloid autoantibodies in body fluids.

Another object of the present invention is to provide a diagnostic kit for diagnosing type 2 diabetes, comprising a component of an antigen protein specific for beta-amyloid autoantibodies.

In order to achieve the above object, the present invention provides a method for diagnosing type 2 diabetes by measuring the concentration of beta-amyloid autoantibodies in body fluids.

In order to achieve the above another object, the present invention comprises an antigen protein to which beta-amyloid autoantibodies specifically bind as a component, and by measuring the concentration of Aβ autoantibodies in the body fluid sample through antigen-antibody binding reaction It provides a diagnostic kit for diagnosing type 2 diabetes.

By using the diagnostic method and diagnostic kit of the present invention, it is easier to diagnose type 2 diabetes early.

Figure 1a graphically shows the concentration of Aβ autoantibodies in the normal and diabetic patients (*** p <0.001).
FIG. 1B is a graph showing the concentration of Aβ autoantibodies according to gender among normal and diabetic patients (*** p <0.001;### p <0.001;## p <0.01).
Figure 1c graphically shows the concentration of total IgG in normal and diabetic patients.
Figure 2a is a graph showing the cholesterol concentration according to the concentration of Aβ autoantibodies in the diabetic group (R = 0.264, p = 0.011).
Figure 2b is a graph showing the LDL concentration according to the concentration of Aβ autoantibodies in the diabetic group (R = 0.244, p = 0.020).
Figure 2c is a graph showing the TG concentration according to the concentration of Aβ autoantibodies in the diabetic group (R = 0.217, p = 0.039).

The present invention provides a method for diagnosing type 2 diabetes by measuring the concentration of beta-amyloid autoantibodies in a bodily fluid sample.

The present invention is based on the fact that the concentration of Aβ autoantibodies is increased in the body fluids of type 2 diabetic patients than the body fluids of normal persons, and when the concentration of Aβ autoantibodies is about 15% or more than that of normal body fluids, type 2 Diabetes can be diagnosed.

Diagnosis method of type 2 diabetes of the present invention comprises the steps of 1) putting a body fluid sample in a reactor coated with an antigen protein to which beta-amyloid autoantibodies specifically bind; 2) detecting the antigen-antibody reactant produced by the reaction using a colorant substrate solution of a secondary antibody-labeled conjugate and a label; And 3) confirming the concentration of the beta-amyloid autoantibody from the detection result of the sample.

The reactor may be a nitrocellulose membrane, a 96 well plate synthesized with a polyvinyl resin, a 96 well plate synthesized with a polystyrene resin, a slide glass made of glass, or the like.

The antigenic protein is specifically recognized by beta-amyloid autoantibodies. In order to prepare the antigenic protein, beta-amyloid protein must be obtained first. Beta-amyloid proteins may be synthesized using known amino acid sequences or produced in the form of recombinant proteins by genetic engineering methods, or may be commercially available. The antigenic protein used in the present invention is preferably characterized in that the beta-amyloid 1-42 protein.

In addition, the antigenic protein is preferably coated in the reactor in an appropriate amount such that the concentration of the beta-amyloid antibody in type 2 diabetic patients can be measured significantly, and coated in the reactor with 1 ng to 100 μg / 100 μl. Is preferred.

In the method for measuring the concentration of beta-amyloid antibody according to the present invention, the body fluid used as a sample is not particularly limited in its extraction site, and blood, lymph, cerebrospinal fluid, etc. can be used, but in consideration of ease of extraction and the like. Preferably it is blood.

The label of the secondary antibody is preferably a conventional color developing agent that performs a color reaction. ), Labels such as fluorescents and dyes such as rhodamine-B-isothiocyanate (RITC) and dyes may be used.

The substrate for inducing color development is preferably used according to the labeling reaction, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid), and OPD (o-phenylenediamine). At this time, the color development substrate is more preferably provided in a dissolved state in a buffer solution (0.1 M NaAc, pH 5.5). A chromogenic substrate such as TMB is decomposed by HRP used as a label of the secondary antibody conjugate to generate a chromosome deposit, and the concentration of the beta-amyloid antibody in the sample is determined by visually confirming the deposition degree of the chromosome deposit.

The antigen-antibody reactant produced by binding the antigen-specific antibody to the beta-amyloid autoantibody produced as described above with the antibody in the bodily fluid sample was prepared using the secondary antibody-label conjugate and the color substrate solution of the label. As the detection method, any immunological method known in the art may be used without limitation, but preferably, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a sandwich measuring method ( sandwich assay, Western blot on polyacrylamide gel, immunoblot analysis, immunofluorescence assay (IFA), immunochemiluminescence assay (Immunochemiluminescence Assay) or immunochromatography (Immunochromatography, Rapid) desirable.

Thereafter, by confirming the detection result, the concentration of beta-amyloid antibody in the body fluid sample can be determined to diagnose type 2 diabetes.

In addition, in the present invention, a component comprising an antigen protein to which beta-amyloid autoantibodies specifically bind as a component, and diagnoses type 2 diabetes by measuring the concentration of Aβ autoantibodies in the body fluid sample through the antigen-antibody binding reaction Provides diagnostic kit for

The diagnostic kit of the present invention may be prepared by a production method known in the art, and typically includes a lyophilized antibody, a buffer, a stabilizer, an inactive protein, and the like. The antibody can be labeled by radionuclides, fluorescors, enzymes, and the like.

More preferably, the diagnostic kit of the present invention comprises: 1) a reactor coated with an antigen protein to which Aβ autoantibodies specifically bind; 2) secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; 3) a chromogenic substrate solution to be colored and reacted with the label; 4) wash solution to be used for each reaction step; And 5) enzyme stopping solution.

The diagnostic kit is useful for diagnosing type 2 diabetes through an antigen-antibody binding reaction, wherein the antigen-antibody binding reaction can be carried out through the same method as mentioned above by immunological methods known in the art. Can be.

The reactor of the diagnostic kit, the type of antigen protein coated on the reactor, the amount of the antigen protein coated, the label of the secondary antibody and the chromogenic substrate are the same as mentioned above.

The wash solution preferably comprises phosphate buffer, NaCl and Tween 20, more preferably a buffer solution consisting of 0.02 M phosphate buffer, 0.13M NaCl and 0.05% Tween 20. The washing solution is reacted with the secondary antibody to the antigen-antibody conjugate after the antigen-antibody binding reaction, and then washed in an appropriate amount in a reactor for 3 to 6 times. The blocking solution is preferably a phosphate buffer solution containing 0.1% BSA, and the reaction stopping solution is preferably 2N sulfuric acid solution.

[Example]

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 Analysis of Samples

Serum samples of diabetic patients (92) were collected from Seoul National University Hospital (SNUH), and serum samples of the same age (106; control group) were collected from Seoul National University Hospital Healthcare Gangnam Center. The subject's height, weight, body mass index (BMI), waist circumference, systolic blood pressure (SBP), diastolic blood pressure (DBP), and fasting blood glucose (fasting) with the approval of an institutional review board (IRB). blood sugar), HbAlc, total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), low density lipoprotein cholesterol (LDL) and creatinine were measured and analyzed by unpaired t-test. 1 is shown. In this case, ^*** indicates p <0.001, ^** indicates p <0.01, and ^* indicates p <0.05.

Normal person (n = 106) Diabetic (n = 91) Gender Male Female) (62:44) (53:38) age 55.1 ± 7.1 55.8 ± 8.0 Height (cm) 163.2 ± 7.0 163.2 ± 8.8 Weight (kg) 62.6 ± 9.0 64.3 ± 11.3 BMI (kg / m ² ) 23.4 ± 2.4 24.0 ± 3.0 Triglycerides (mg / dL) 103.8 ± 42.9 130.9 ± 71.7 ^** Total Cholesterol (mg / dL) 197.5 ± 28.8 192.3 ± 33.8 HDL (mg / dL) 55.0 ± 12.5 49.4 ± 12.3 ^** LDL (mg / dL) 121.7 ± 27.7 116.8 ± 30.1 Systolic Blood Pressure (mmHg) 118.7 ± 16.6 123.0 ± 16.2 Diastolic blood pressure (mmHg) 75.4 ± 11.2 76.3 ± 9.3 Fasting Blood Sugar (mg / dL) 90.0 ± 6.3 136.30 ± 37.5 ^*** HbA1c (ng / mL) 5.5 ± 0.2 7.0 ± 1.2 ^*** Creatinine (mg / mL) 1.0 ± 0.2 1.0 ± 0.2

As shown in Table 1, diabetics and normal people did not show a significant difference in age, BMI, waist circumference, blood pressure, blood cholesterol concentration, blood LDL concentration and creatinine concentration. However, diabetic patients showed higher values in fasting blood glucose, HbAlc (p <0.001) and triglycerides (p <0.005) than normal, and HDL (p <0.005) showed lower values than normal.

Example 2 Measurement of Aβ Autoantibodies

Concentrations of Aβ autoantibodies in serum samples of diabetic and normal subjects were determined using enzyme-linked immunosorbent assay (ELISA) in the manner described in de la Monte SM et al., (2006) J Alzheimers Dis 10 , 89-109. It was measured by. Diluted 1 μg of human Aβ1-42 (Bachem, Bubendorf, Switzerland) per well in 0.1M bicarbonate sodium buffer (pH 9.6) into microtiter wells (Maxisorp, Nunc, Roskilde, Denmark) Coated at 4 ° C. overnight. The wells of the plates are then washed with wash buffer (0.05% Tween20 containing phosphate buffer (PBS)) and the plates blocked with PBS (FBS / PBS buffer) containing 10% FBS for 1 hour at room temperature. It was. The plate was washed, and serum (sera) diluted 1: 100 in FBS / PBS buffer was added to each well and reacted at 37 ° C. for 1 hour. After washing, the well was added with a 1: 2,000 dilution of HRP-conjugated anti-human IgG antibody (Amershan Pharmacia Biotech, Buckinghamshire, UK) and reacted at 37 ° C. for 30 minutes. The wells were washed, and TMB (Pierce, Rockford, IL), a chromogenic substrate, was added to each well. The degree of color reaction was evaluated by measuring the absorbance at 450 nm wavelength.

Relative concentrations of Aβ autoantibodies measured by ELSIA were calculated using the average value of the normal (control) as 100%. In addition, the unpaired t-test was used to determine whether the difference in Aβ autoantibody concentrations between diabetic and control groups was statistically significant.

As a result, as shown in Figure 1a, the concentration of Aβ autoantibodies in the type 2 diabetic patient group showed a higher concentration by 45.4 ± 7.7% compared to the normal (p <0.001). In addition, as shown in FIG. 1B, women in type 2 diabetes showed 63.0 ± 13.7% higher levels of Aβ autoantibodies than men, and Aβ autoantibodies in women (12.7) were also normal (p = 0.048). +/- 6.4%). In conclusion, although the magnitude of the difference among women was large, the values of Aβ autoantibodies in type 2 diabetic patients were both in women (74.9 ± 12.0%; p <0.001) and in men (24.6 ± 8.7%; p = 0.006). Statistically significant increase compared to normal.

Example  3: total IgG  Concentration measurement

The total IgG concentration in serum of type 2 diabetic patients and normal subjects was measured by direct-ELISA method according to the manufacturer's protocol described in the human IgG kit (Standard Diagnostics INC., Korea). Is shown in Figure 1c. As a result, the total IgG concentration was 14.6 ± 0.7mg / mL in the type 2 diabetic group, and 13.8 ± 0.6mg / mL in the normal group did not show a significant difference (p = 0.237).

This difference in total IgG concentration between type 2 diabetic patients and normal groups indicates that the overall B cell immune response does not change in type 2 diabetic patients. Thus, a significant increase in the concentration of Aβ autoantibodies in patients with type 2 diabetes means that the B-cell response is dependent on the concentration of Aβ antigens.

Furthermore, given that the mean age of the test subjects of the present invention is about 55 years younger than 20 years of age typical of Alzheimer's disease, increased Aβ autoantibody concentration in type 2 diabetes reflects the concentration of Aβ antigen. In addition, it can be used as a diagnostic marker for type 2 diabetes and early diagnosis markers associated with the risk of developing Alzheimer's disease.

Example 4: Statistical Analysis

Correlations with Aβ autoantibodies and other variables in diabetic patients were analyzed by correlation analysis and linear regression. Statistical analysis was performed using SPSS 12.0 (SPSS Inc., Chicago, IL).

As a result, as shown in FIGS. 2A to 2C, in the type 2 diabetic patients, the concentration of Aβ autoantibodies was high in total cholesterol (R = 0.264, p = 0.011), low density lipoprotein (R = 0.244, p = 0.020), and triglycol. Positive correlations were found with variables such as serides (R = 0.217, p = 0.039), whereas no correlation was found in normal subjects (total cholesterol: R = -0.064, p = 0.513; LDL: R = -0.040, p = 0.684; and TG: R = -0.040, p = 0.686). In this case, R represents Pearson's correlation coefficient.

As such, the increase in total cholesterol, low density lipoprotein, and triglyceride in the diabetic group correlates with the increase in the concentration of Aβ autoantibodies, thereby causing chronic dyslipidemia to increase the concentration of Aβ, which in turn stimulates B cells. It can be seen that it produces more autoantibodies against Aβ.

Claims (17)

delete delete delete delete delete delete delete delete delete A component comprising an antigen protein specifically bound to beta-amyloid (β-amyloid) autoantibodies, and type 2 diabetes by measuring the concentration of Aβ autoantibodies in body fluid samples through antigen-antibody binding reactions. Diagnostic kit for diagnosis. 11. The method of claim 10,
Diagnostic kit, characterized in that the body fluid is blood. 11. The method of claim 10,
The diagnostic kit is 1) a reactor coated with an antigen protein specific for Aβ autoantibodies; 2) secondary antibody conjugates to which a label that is developed by reaction with a substrate is conjugated; 3) a chromogenic substrate solution to be colored and reacted with the label; 4) wash solution to be used for each reaction step; And 5) a diagnostic kit comprising an enzyme reaction stopping solution. The method of claim 12,
Diagnostic kit, characterized in that the antigenic protein is beta-amyloid 1-42 protein. The method of claim 12,
The diagnostic kit, characterized in that the beta-amyloid antigen protein is coated in the reactor 1 ng to 100 ㎍ / 100 ㎕. The method of claim 12,
And the reactor is selected from the group consisting of nitrocellulose membranes, well plates synthesized with polyvinyl resin, well plates synthesized with polystyrene resin, and slide glass made of glass. The method of claim 12,
The diagnostic kit of the secondary antibody conjugate is selected from the group consisting of HRP, basic dephosphatase, colloidal gold, fluorescent materials and pigments. The method of claim 12,
The chromogenic substrate was composed of TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (o-phenylenediamine). Diagnostic kit, characterized in that selected from the group.

Images