KR101681217B1 - Diagnostic method for type 1 diabetes using autoantibodies - Google Patents
Diagnostic method for type 1 diabetes using autoantibodies Download PDFInfo
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- KR101681217B1 KR101681217B1 KR1020150035280A KR20150035280A KR101681217B1 KR 101681217 B1 KR101681217 B1 KR 101681217B1 KR 1020150035280 A KR1020150035280 A KR 1020150035280A KR 20150035280 A KR20150035280 A KR 20150035280A KR 101681217 B1 KR101681217 B1 KR 101681217B1
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- eef1a1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Abstract
The present invention relates to a composition for the diagnosis of type 1 diabetes comprising an agent for measuring the level of autoantibodies against EEF1A1 or UBE2L3, a type 1 diabetes diagnostic kit comprising said composition, and information necessary for the diagnosis of type 1 diabetes Lt; RTI ID = 0.0 > EEF1A1 < / RTI > or UBE2L3. Use of an autoantibody against EEF1A1 or UBE2L3 provided by the present invention can be used for the treatment of diabetes more effectively because it can specifically diagnose the incidence of type 1 diabetes.
Description
The present invention relates to a method for diagnosing
Diabetes is one of the most common diseases and is characterized by increased metabolic disturbances as blood glucose levels rise. Diabetes is a hyperglycemic condition, but it can also lead to microvascular or macrovascular complications associated with individual morbidity and mortality.
These complications are dangerous and life-threatening factors that reduce quality of life. The most common type of diabetes is
As such, a common result of the onset of diabetes is an increase in blood glucose level, but the exact diagnosis is very important because the treatment depends on the cause of the disease. (GADA), autoantibodies (GADA), and autoantibodies to protein tyrosine phosphatase autoantibodies (GADA), which are clinically used for the diagnosis of
In addition, the limitations of autoantibodies associated with
Under these circumstances, the present inventors have made extensive efforts to develop a method for specifically diagnosing
It is an object of the present invention to provide a composition for the diagnosis of
Another object of the present invention is to provide a kit for the diagnosis of
Another object of the present invention is to provide a method for detecting autoantibodies against EEF1A1 or UBE2L3 to provide information necessary for the diagnosis of
The inventors have focused on autologous antibodies while carrying out various studies to develop a method for specifically diagnosing
In one embodiment for achieving the above object, the present invention provides a composition for the diagnosis of
The "eukaryote translation elongation factor 1α1" and the "ubiquitin-conjugating enzyme 2L3" of the present invention may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 1 (EEF1A1) and SEQ ID NO: 2 (UBE2L3) , But is not limited thereto. EEF1A1 is NCBI Accession No. 2. NP_001393 (GenBank Accession NM_001402), UBE2L3 is the sequence described in NCBI Accession No. It may be the sequence described in CAG30492 (GenBank Accession NM_003347). In addition, EEF1A1 and UBE2L3 each include their functional equivalents. The functional equivalent may be a polypeptide having at least 70%, preferably at least 80%, more preferably at least 90% sequence homology (i.e., identity) to the amino acid sequence shown in SEQ ID NOs: 1 and 2 .
The term "autoantibodies (AAb) " of the present invention refers to an antibody that is expressed in the body and specifically reacts with an in vivo component.
In the present invention, the autoantibody may be an autoantibody to EEF1A1 or an autoantibody to UBE2L3, which is produced at the onset of
The term "
In the present invention, the
The term "diagnosing" of the present invention means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming the incidence of
The term "agent for measuring the level of antibody" of the present invention means an agent used for measuring the level of an antibody against a target protein contained in a sample, preferably Western blotting, ELISA enzyme linked immunosorbent assay (RIA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay ), A complement fixation assay (FACS), a FACS and a protein chip assay, and the like.
The term "antigen" of the present invention means a proteinaceous immunogen capable of binding an antibody and an antigen antibody, wherein the antigen is obtained by cloning each gene into an expression vector according to a conventional method, , And can be prepared from the obtained protein by a conventional method.
The antigen used in the present invention may be EEF1A1 or UBE2L3 described above.
The term "antibody" of the present invention means a proteinaceous molecule capable of specifically binding to an antigenic site of a protein or peptide molecule. Such an antibody can be prepared by cloning each gene into an expression vector according to a conventional method, A protein encoded by the marker gene can be obtained and can be produced from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and any immunoglobulin antibody may be included, Specific antibodies of the invention. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and can be Fab, F (ab ') 2, F (ab') 2, Fv and the like.
In the present invention, the antibody may be an antibody capable of specifically binding to an autoantibody to EEF1A1 or UBE2L3, preferably a polyclonal antibody capable of specifically binding to an autoantibody to EEF1A1 or UBE2L3 An anti-body antibody, a monoclonal antibody, or a part thereof.
The term "aptamer " of the present invention refers to a substance capable of specifically binding to a target substance to be detected in a sample, and is a single stranded nucleic acid (DNA, RNA, or modified nucleic acid) , And the presence of the target substance in the sample can be specifically confirmed through the binding. The preparation of the aptamer can be carried out by synthesizing an oligonucleotide having a selective and high binding ability to a target protein to be identified according to a general method of preparing an aptamer and then synthesizing an oligonucleotide having a 5'end or 3'end of the oligonucleotide To -SH, -COOH, -OH or -NH 2 so as to be able to bind to the functional group of the linker.
In the present invention, the aptamer may be an aptamer capable of specifically binding to an autoantibody to EEF1A1 or UBE2L3, preferably a DNA aptamer capable of specifically binding to an autoantibody to EEF1A1 or UBE2L3 It can be.
As another embodiment for achieving the above object, the present invention provides a kit for the diagnosis of
The kit of the present invention can be used to diagnose the incidence of
The term "sample" of the present invention means a direct object of measuring the expression level of an autoantibody to EEF1A1 or UBE2L3 separated from an individual suffering from
The term "individual" of the present invention may include, without limitation, mammals including rats, livestock, humans, etc., who are at risk for developing
As a specific example, the kit of the present invention may be an ELISA kit for measuring the level of autoantibodies against EEF1A1 or UBE2L3, which is not particularly limited, but may be any suitable kit for the immunological detection of antibodies, , A secondary antibody labeled with a chromogenic enzyme or a fluorescent substance, a chromogenic substrate, and the like. The substrate is not particularly limited, but a nitrocellulose membrane, a 96-well plate synthesized with polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass can be used, and the coloring enzyme is not particularly limited The fluorescent substance may be FITC, RITC or the like, and the coloring substrate liquid is not particularly limited, but ABTS (2, 3, 4, 5, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or OPD (o-phenylenediamine), TMB (tetramethylbenzidine).
As another example, the kit of the present invention may include reagents necessary for performing negative and positive control reactions, or reagents such as buffers necessary for the hybridization reaction. The optimal amount of reagent used in a particular reaction can be readily determined by those skilled in the art having the benefit of this disclosure. For example, the kit of the present invention may comprise the components described above in a separate package or compartment.
As another embodiment for achieving the above object, the present invention provides a method for detecting an autoantibody to EEF1A1 or UBE2L3 in order to provide information necessary for diagnosis of
Specifically, the method for detecting an autoantibody to EEF1A1 or UBE2L3 provided by the present invention comprises the steps of: (a) measuring the level of autoantibodies against EEF1A1 or UBE2L3 from a biological sample of an individual to be tested; And (b) comparing the level of the measured protein to a level measured from a normal individual biological sample. At this time, the biological sample is not particularly limited, but may be blood, serum, plasma, tissue sample, and the like.
In the method, if the level of autoantibody to EEF1A1 or UBE2L3 measured in the biological sample is significantly increased compared to the level measured from a biological sample of a normal individual, And if it is not significantly increased, it can be determined that the
Use of an autoantibody against EEF1A1 or UBE2L3 provided by the present invention can be used for the treatment of diabetes more effectively because it can specifically diagnose the incidence of
FIG. 1 is a schematic diagram showing a procedure of a method of locating an autoantibody contained in a serum of a diabetic patient.
2A is a Venn diagram showing the distribution of autoantibodies commonly expressed in T1DM patients, T2DM patients, and normal individuals.
FIG. 2B is a heat map showing the results of analysis of changes in the expression levels of autoantibodies contained in T1DM patients, T2DM patients, and normal individuals according to the hierarchical cluster analysis method, in which yellow indicates that the expression level is increased, Indicating that the amount of expression is decreased.
FIG. 3A is a two-dimensional PLS-DA plot showing the results of performing PLS-DA using an autoantibody to distinguish T1DM patients from T2DM patients and normal individuals, and the dotted line indicates the discrimination between T2DM patients and normal individuals and T1DM patients Function.
FIG. 3B is a graph showing the variable importance in projection (VIP) value of each autoantibody determined by PLS-DA, and the dotted line indicates 1.59 as a VIP lower limit.
Figure 3c is a heat map showing the expression levels of the respective autoantibodies (EEF1A1-AAb, UBE2L3-AAb and c7orf53-AAb) expressed in T1DM patients, T2DM patients and normal subjects.
FIG. 4 is a photograph showing the results of immunofluorescence staining analysis using anti-EEF1A1 antibody, anti -UBE2L3 antibody or anti-c7orf53 antibody in pancreatic tissues, wherein A shows the result of immunofluorescence staining using anti-EEF1A1 antibody ; B, G, and L show immunofluorescence staining results with anti-insulin antibody; C, H, and M show immunofluorescence staining results with anti-glucagon antibody; D, I, and N represent DAPI stained nuclei; F shows immunofluorescence staining results with anti-UBE2L3 antibody; K represents the result of immunofluorescence staining using anti-c7orf53 antibody; E is a photograph synthesized from A to D; J is a photograph synthesized from F to I, and O is a photograph synthesized from K to N.
FIG. 5A is a graph showing the results of measurement of the amounts of EEF1A1-AAb and UBE2L3-AAb contained in sera of patients with T1DM, normal human, T2DM, and Graves' disease.
FIG. 5B is a graph showing the receiver operating characteristic curve for EEF1A1-AAb and UBE2L3-AAb.
FIG. 5C is a graph showing the results of measurement of the amounts of EEF1A1-AAb and UBE2L3-AAb contained in serum of T1DM child patients and normal children.
FIG. 6A is a Venn diagram showing the distribution of each autoantibody (GADA, EEF1A1-AAb and UBE2L3-AAb) possessed by a T1DM patient.
FIG. 6B is a graph showing the results of analysis of the content of EEF1A1-AAb or UBE2L3-AAb in serum according to the onset age of T1DM.
FIG. 6c is a graph showing the results of analysis of the difference in the content of EEF1A1-AAb in serum according to the onset age and the duration of onset of T1DM.
FIG. 6D is a graph showing the results of analysis of the content of UBE2L3-AAb in serum according to the onset age and the duration of onset of T1DM.
FIG. 6E is a graph showing the results of analysis of differences in content of EEF1A1-AAb or UBE2L3-AAb in serum according to the level of C-peptide upon fasting.
FIG. 6F is a graph showing the results of analysis of differences in the content of serum EEF1A1-AAb in patients with general T1DM, fulminant T1DM, or patients with latent autoimmune diabetes (LADA) .
FIG. 6G is a graph showing the results of analysis of the difference in the content of UBE2L3-AAb in serum in a general T1DM patient, a fulminant T1DM patient, or a LADA patient.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.
Example
1: Detection of autoantibodies contained in serum of diabetic patients
The autologous antibodies contained in the sera of the diabetic patients were uncovered according to the method disclosed in Fig. FIG. 1 is a schematic diagram showing a procedure of a method of locating an autoantibody contained in a serum of a diabetic patient.
First, respective sera were obtained from a primary test group consisting of 16 patients with
PBS was added to each of the obtained sera to obtain a sample diluted 1: 500. The sample was added to protein microarray platform version 5.0 (ProtoArray; Invitrogen), and reacted for 90 minutes. After the reaction was completed, Alexa Fluor 647-conjugated anti-human IgG was added and then scanned with a GenePix 4000B fluorescent scanner (Molecular Devices) to detect 3,060 autoantibodies (AAb) bound to the microarray.
Among these autoantibodies, autoantibodies strongly expressing in T1DM patients were selected. In T1DM patients, 103 and 27 autoantibodies, which were highly correlated with normal T2DM patients, were compared. The autoantibodies, which were significantly higher in T1DM patients than those in the autoantibodies [normal group + T2DM group] , The last 29 autoantibodies were selected (Figure 2a).
Histological cluster analysis was performed on the identified autoantibodies. First, the intensity of the signal detected in the microarray is applied to ProtoArray Prospector, which is a computer software, to calculate a normalized value, and the calculated value is stored in the Gene Expression Omnibus database (http: //www.ncbi.nlm.nih.gov/gds) M-statistic test was used to calculate the z value. When the z value was > 1.64, the presence of the autoantibody was considered. The result was indicated by a heat map (Fig. 2B).
FIG. 2B is a heat map showing the results of analysis of changes in the expression levels of autoantibodies contained in T1DM patients, T2DM patients, and normal individuals according to the hierarchical cluster analysis method, in which yellow indicates that the expression level is increased, Indicating that the amount of expression is decreased. Therefore, the intensity of the selected autoantibody was higher in T1DM patients than in T2DM patients or normal subjects.
The relative contribution of these autoantibodies was assessed in order to select autoantibodies that can characteristically distinguish T1DM patients. Specifically, by performing the empirical PLS-DA using the 69 autoantibody expression levels, a variable importance in projection (VIP) value indicating the relative group contribution of each autoantibody isolated from T1DM patients, T2DM patients, Respectively. As a result of estimating the empirical distribution of VIP for the null hypothesis, the VIP lower limit of 1.59, which is 90% of the empirical VIP distribution, was determined, and thus the autoantibody of VIP> 1.59 was separated from the T1DM, T2DM, Were considered to have a significant distribution (Figs. 3A and 3B).
FIG. 3A is a two-dimensional PLS-DA plot showing the results of performing PLS-DA using an autoantibody to distinguish T1DM patients from T2DM patients and normal individuals, and the dotted line indicates the discrimination between T2DM patients and normal individuals and T1DM patients FIG. 3B is a graph showing a variable importance in projection (VIP) value of each autoantibody determined by PLS-DA, and a dotted line indicates a VIP lower limit of 1.59. As shown in FIG. 3B, it was confirmed that the number of autoantibodies having a VIP value higher than the VIP lower limit was 50.
Among the 50 autoantibodies identified, the autoantibodies that were not expressed in the normal subjects were searched for three antibodies: EEF1A1 (eukaryote translation elongation factor 1α1), UBE2L3 (ubiquitin-conjugating enzyme 2L3) or c7orf53 (chromosome 7 open reading frame 53 (EEF1A1-AAb, UBE2L3-AAb, and c7orf53-AAb) (Fig. 3C).
Figure 3c is a heat map showing the expression levels of the respective autoantibodies (EEF1A1-AAb, UBE2L3-AAb and c7orf53-AAb) expressed in T1DM patients, T2DM patients and normal subjects. As shown in FIG. 3C, each selected autoantibody was found to be increased in T1DM patients rather than T2DM patients and normal subjects.
Example
2:
Immunofluorescent staining
analysis
To determine whether the three autoantibodies (EEF1A1-AAb, UBE2L3-AAb and c7orf53-AAb) selected in Example 1 were expressed in the pancreas, which is an insulin secretory organ directly affecting T1DM, Dyeing analysis was performed.
Specifically, anti-EEF1A1 antibody, anti -UBE2L3 antibody or anti-c7orf53 antibody was added to the pancreas tissue obtained from a patient who underwent partial pancreatectomy due to serous adenoma and Alexa Fluor 488-conjugated secondary antibody was added And further reacted. As a control, an anti-insulin antibody was added to the reaction mixture, followed by further reaction with a secondary antibody conjugated with Alexa Fluor 647, followed by reaction with an anti-glucagon antibody, followed by addition of Alexa Fluor 594-conjugated secondary antibody Lt; / RTI > The immunofluorescent staining was performed and the results were confirmed using a confocal microscope (Olympus FluoView FV1000 confocal laser scanning microscope) (Fig. 4).
FIG. 4 is a photograph showing the results of immunofluorescence staining analysis using anti-EEF1A1 antibody, anti -UBE2L3 antibody or anti-c7orf53 antibody in pancreatic tissues, wherein A shows the result of immunofluorescence staining using anti-EEF1A1 antibody ; B, G, and L show immunofluorescence staining results with anti-insulin antibody; C, H, and M show immunofluorescence staining results with anti-glucagon antibody; D, I, and N represent DAPI stained nuclei; F shows immunofluorescence staining results with anti-UBE2L3 antibody; K represents the result of immunofluorescence staining using anti-c7orf53 antibody; E is a photograph synthesized from A to D; J is a photograph synthesized from F to I, and O is a photograph synthesized from K to N. As shown in Fig. 4, EEF1A1 and UBE2L3 were present in large amounts in islet cells of pancreatic islet contained in pancreatic tissue, while c7orf53 was exceptionally found in alpha cells.
Therefore, EEF1A1-AAb and UBE2L3-AAb, which are autoantibodies that bind to a large amount of antigen present in the pancreatic β-cells of insulin-secreting pancreas, were finally selected.
Example
3: Validation of the effect of autoantibody
Example
3-1: Comparison of autoantibody content
In order to verify the autoantibodies finally selected in Example 2, 95 patients with T1DM, 49 patients with T2DM, 11 patients with latent autoimmune diabetes in adults (LADA), 20 patients with Graves disease and 66 patients with normal ≪ / RTI > NGT) were used. ELISA was performed on the serum samples, and the amounts of EEF1A1-AAb and UBE2L3-AAb contained in each serum sample were measured (FIG. 5A). At this time, the ELISA was performed by adding a serum sample (1: 1,000-1: 10,000) diluted with PBS to a plate coated with EEF1A1 or UBE2L3 and reacting with a chlorine-derived anti-human IgG-HRP antibody Respectively.
FIG. 5A is a graph showing the results of measurement of the amounts of EEF1A1-AAb and UBE2L3-AAb contained in sera of patients with T1DM, normal human, T2DM, and Graves' disease. As shown in FIG. 5A, it was confirmed that EEF1A1-AAb and UBE2L3-AAb were relatively higher in T1DM patients than normal and T2DM patients ( ** P <0.01). In particular, it was found that the levels of T1DM patients were relatively higher than those of patients with Graves' disease, which was used as a control for autoimmune diseases ( ** P <0.01).
Example
3-2: Antigen inducing autoantibody
Positive rate
compare
Positive rates of EEF1A1 and UBE2L3 were measured in the second test group (Table 1).
T2DM (n = 49)
LADA (n = 11)
Graves disease (n = 20)
NGT (n = 66)
1 (2.0)
0
2 (10.0)
1 (1.5)
1 (2.0)
0
4 (20.0)
1 (1.5)
T1DM vs. T2DM
T1DM vs. LADA
T1DM vs. Graves disease
<0.001
0.035
0.094
<0.001
0.015
0.201
As shown in Table 1, the positive rates of EEF1A1-AAb and UBE2L3-AAb in T1DM patients were 29.5% and 35.8%, respectively, but 2% in T2DM patients and 1.5% in normal patients.
Example
3-3: ROC curve analysis
To quantitatively evaluate the T1DM prediction accuracy, a receiver operating characteristic curve for an autoantibody was generated, from which areas under the curve (AUC) were calculated (FIG. 5B).
FIG. 5B is a graph showing the receiver operating characteristic curve for EEF1A1-AAb and UBE2L3-AAb. As shown in FIG. 5B, it was confirmed that the AUC value calculated from the ROC curve for EEF1A1-AAb was 0.73 and the AUC value calculated from the ROC curve for UBE2L3-AAb was 0.78. Therefore, each of the autoantibodies predicts T1DM And it shows a high level of accuracy.
Example
3-4: Verification of the effect of autoantibody in children
To determine whether the method of predicting T1DM using the two autoantibodies could be applied to non-adult children, a tertiary test group comprising 33 children with T1DM and 34 normal children was used. Serum samples were obtained from the tertiary test group and ELISA was performed on the serum samples to determine the amounts of EEF1A1-AAb and UBE2L3-AAb contained in each serum sample (FIG. 5C).
FIG. 5C is a graph showing the results of measurement of the amounts of EEF1A1-AAb and UBE2L3-AAb contained in serum of T1DM child patients and normal children. As shown in FIG. 5C, it was found that EEF1A1-AAb and UBE2L3-AAb were detected at a significantly higher level in serum of children with T1DM than in normal children ( * P <0.05, ** P <0.01).
In summary, the results of Examples 3-1 to 3-4 show that the autoantibodies EEF1A1-AAb and UBE2L3-AAb provided in the present invention are specifically detected in the sera of not only adults but also children T1DM patients, It can be used as a marker for diagnosis.
Example
4: Clinical characterization of autoantibodies
The clinical characteristics of EAD1A1-AAb and UBE2L3-AAb positive T1DM patients were compared with those of GADA (an autoantibody to GAD65), a representative autoantibody of T1DM patients (Table 2). At this time, whether or not the EEF1A1-AAb and UBE2L3-AAb were positive was measured by the diagnostic kit provided in the present invention.
(n = 71)
(n = 22)
(n = 28)
(n = 67)
(n = 34)
(n = 61)
(17-69)
(19-58)
(17-33)
(17-69)
(17-37)
(17-69)
(13-66)
(10-58)
(13-33)
(10-66)
(14-36)
(10-66)
3.0
3.2
2.7
3.7
2.7
3.7
(72.7)
(35.3)
(40.3)
(41.2)
(39.3)
Level (ng / mL)
(50.0%)
(10.7%)
(11.9%)
(5.9%)
(14.8%)
(14.3%)
(18.5%)
(21.2%)
(21.1%)
(20.0%)
(75.0%)
(76.9%)
(79.4%)
(74.6%)
(31.8%)
(92.9%)
(11.9%)
(31.8%)
(76.5%)
(3.3%)
First, the distribution of each autoantibody possessed by T1DM patients was analyzed (Fig. 6A).
FIG. 6A is a Venn diagram showing the distribution of each autoantibody (GADA, EEF1A1-AAb and UBE2L3-AAb) possessed by a T1DM patient. As shown in FIG. 6A, 38.7% (36/93) of the patients in T1DM patients were positive for EEF1A1-AAb or UBE2L3-AAb. In GADA-negative T1DM patients, the proportion of patients positive for either EEF1A1-AAb or UBE2L3-AAb autoantibodies was 40.9% (9/22), and GADA, EEF1A1-AAb or UBE2L3-AAb 86% (80/93) were positive for any of the three autoantibodies.
Using the previously known GADA, it was possible to diagnose about 76% of T1DM patients, but it was found that the diagnosis rate of T1DM patients could be increased to about 86% by using EEF1A1-AAb and UBE2L3-AAb in addition to GADA there was.
Second, the content of EEF1A1-AAb or UBE2L3-AAb in serum according to the age of onset of T1DM was analyzed (FIG. 6B).
FIG. 6B is a graph showing the results of analysis of the content of EEF1A1-AAb or UBE2L3-AAb in serum according to the onset age of T1DM. As shown in FIG. 6B, the content of EEF1A1-AAb or UBE2L3-AAb in serum increased with the age at onset of T1DM, but GADA did not show such a characteristic.
Third, the content of EEF1A1-AAb or UBE2L3-AAb in serum was analyzed according to the duration of T1DM (FIGS. 6C and 6D).
FIG. 6c is a graph showing the results of analysis of the difference in the content of EEF1A1-AAb in serum according to the onset age and the duration of T1DM. FIG. 6d shows the difference in the content of UBE2L3-AAb in serum according to the onset age and duration of T1DM The graph showing the result of the analysis. As shown in FIGS. 6C and 6D, no significant correlation was found between the duration of T1DM and the content of EEF1A1-AAb or UBE2L3-AAb in serum, but the serum EEF1A1-AAb or UBE2L3 The tendency of the change in the content of AAA was maintained.
Fourth, the content of EEF1A1-AAb or UBE2L3-AAb in serum was analyzed according to C-peptide level on fasting (Fig. 6E).
FIG. 6E is a graph showing the results of analysis of differences in content of EEF1A1-AAb or UBE2L3-AAb in serum according to the level of C-peptide upon fasting. As shown in FIG. 6E, it was confirmed that the content of EEF1A1-AAb or UBE2L3-AAb increased with decreasing C-peptide level on fasting.
Fifth, the contents of EEF1A1-AAb or UBE2L3-AAb in serum according to the subtypes of T1DM were analyzed (FIGS. 6F and 6G).
FIG. 6f is a graph showing the results of analysis of the difference in the content of serum EEF1A1-AAb in a general T1DM patient, a fulminant T1DM patient, or a LADA patient. FIG. 6g is a graph showing the results of a general T1DM patient, a fulminant T1DM patient, Of the total amount of UBE2L3-AAb in the serum. As shown in FIGS. 6F and 6G, no EEF1A1-AAb or UBE2L3-AAb was detected in the serum of the control LADA patient, and a small amount of EEF1A1-AAb or UBE2L3-AAb was detected in serum of fulminant T1DM patients.
It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. More variations are possible within a range that does not. Accordingly, the present invention may be embodied in other ways than those specifically described and illustrated herein, and it may be understood by those skilled in the art.
<110> SNU R & DB FOUNDATION KNU-Industry Cooperation Foundation Daegu Gyeongbuk Institute of Science and Technology <120> Diagnostic method for type 1 diabetes using autoantibodies <130> KPA141379-KR <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> recombinant EEF1A1 <400> 1 Met Gly Lys Glu Lys Thr His Ile Asn Ile Val Val Ile Gly His Val 1 5 10 15 Asp Ser Gly Lys Ser Thr Thr Thr Gly His Leu Ile Tyr Lys Cys Gly 20 25 30 Gly Ile Asp Lys Arg Thr Ile Glu Lys Phe Glu Lys Glu Ala Ala Glu 35 40 45 Met Gly Lys Gly Ser Phe Lys Tyr Ala Trp Val Leu Asp Lys Leu Lys 50 55 60 Ala Glu Arg Glu Arg Gly Ile Thr Ile Asp Ile Ser Leu Trp Lys Phe 65 70 75 80 Glu Thr Ser Lys Tyr Tyr Val Thr Ile Ile Asp Ala Pro Gly His Arg 85 90 95 Asp Phe Ile Lys Asn Met Ile Thr Gly Thr Ser Gln Ala Asp Cys Ala 100 105 110 Val Leu Ile Val Ala Ala Gly Val Gly Glu Phe Glu Ala Gly Ile Ser 115 120 125 Lys Asn Gly Gln Thr Arg Glu His Ala Leu Leu Ala Tyr Thr Leu Gly 130 135 140 Val Lys Gln Leu Ile Val Gly Val Asn Lys Met Asp Ser Thr Glu Pro 145 150 155 160 Pro Tyr Ser Gln Lys Arg Tyr Glu Glu Ile Val Lys Glu Val Ser Thr 165 170 175 Tyr Ile Lys Lys Ile Gly Tyr Asn Pro Asp Thr Val Ala Phe Val Pro 180 185 190 Ile Ser Gly Trp Asn Gly Asp Asn Met Leu Glu Pro Ser Ala Asn Met 195 200 205 Pro Trp Phe Lys Gly Trp Lys Val Thr Arg Lys Asp Gly Asn Ala Ser 210 215 220 Gly Thr Thr Leu Leu Glu Ala Leu Asp Cys Ile Leu Pro Pro Thr Arg 225 230 235 240 Pro Thr Asp Lys Pro Leu Arg Leu Pro Leu Gln Asp Val Tyr Lys Ile 245 250 255 Gly Gly Ile Gly Thr Val Val Gly Arg Val Glu Thr Gly Val Leu 260 265 270 Lys Pro Gly Met Val Thr Phe Ala Pro Val Asn Val Thr Thr Glu 275 280 285 Val Lys Ser Val Glu Met His His Glu Ala Leu Ser Glu Ala Leu Pro 290 295 300 Gly Asp Asn Val Gly Phe Asn Val Lys Asn Val Ser Val Lys Asp Val 305 310 315 320 Arg Arg Gly Asn Val Ala Gly Asp Ser Lys Asn Asp Pro Pro Met Glu 325 330 335 Ala Ala Gly Phe Thr Ala Gln Val Ile Ile Leu Asn His Pro Gly Gln 340 345 350 Ile Ser Ala Gly Tyr Ala Pro Val Leu Asp Cys His Thr Ala His Ile 355 360 365 Ala Cys Lys Phe Ala Glu Leu Lys Glu Lys Ile Asp Arg Arg Ser Gly 370 375 380 Lys Lys Leu Glu Asp Gly Pro Lys Phe Leu Lys Ser Gly Asp Ala Ala 385 390 395 400 Ile Val Asp Met Val Pro Gly Lys Pro Met Cys Val Glu Ser Phe Ser 405 410 415 Asp Tyr Pro Pro Leu Gly Arg Phe Ala Val Arg Asp Met Arg Gln Thr 420 425 430 Val Ala Val Gly Val Ile Lys Ala Val Asp Lys Lys Ala Ala Gly Ala 435 440 445 Gly Lys Val Thr Lys Ser Ala Gln Lys Ala Gln Lys Ala Lys 450 455 460 <210> 2 <211> 154 <212> PRT <213> Artificial Sequence <220> <223> recombinant UBE2L3 <400> 2 Met Ala Ala Ser Arg Arg Leu Met Lys Glu Leu Glu Glu Ile Arg Lys 1 5 10 15 Cys Gly Met Lys Asn Phe Arg Asn Ile Gln Val Asp Glu Ala Asn Leu 20 25 30 Leu Thr Trp Gln Gly Leu Ile Val Pro Asp Asn Pro Pro Tyr Asp Lys 35 40 45 Gly Ala Phe Arg Ile Glu Ile Asn Phe Pro Ala Glu Tyr Pro Phe Lys 50 55 60 Pro Pro Lys Ile Thr Phe Lys Thr Lys Ile Tyr His Pro Asn Ile Asp 65 70 75 80 Glu Lys Gly Gln Val Cys Leu Pro Val Ser Ser Ala Glu Asn Trp Lys 85 90 95 Pro Ala Thr Lys Thr Asp Gln Val Ile Gln Ser Leu Ile Ala Leu Val 100 105 110 Asn Asp Pro Gln Pro Glu His Pro Leu Arg Ala Asp Leu Ala Glu Glu 115 120 125 Tyr Ser Lys Asp Arg Lys Lys Phe Cys Lys Asn Ala Glu Glu Phe Thr 130 135 140 Lys Lys Tyr Gly Glu Lys Arg Pro Val Asp 145 150
Claims (8)
Wherein said EEF1Al comprises the amino acid sequence of SEQ ID < RTI ID = 0.0 > 1. ≪ / RTI >
Wherein said UBE2L3 consists of the amino acid sequence of SEQ ID NO: 2.
Wherein the kit is an ELISA kit.
(b) comparing the measured protein level to a level measured from a biological sample isolated from a normal subject.
A method for detecting an autoantibody to EEF1A1 or UBE2L3 to provide information necessary for type 1 diabetes-specific diagnosis.
Wherein the sample is a blood, serum, plasma, or tissue sample.
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| US7598031B2 (en) | 1999-01-06 | 2009-10-06 | Genenews Corporation | Method for the detection of gene transcripts in blood and uses thereof |
| KR101160153B1 (en) | 2009-12-10 | 2012-06-27 | 서울대학교산학협력단 | 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 |
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| US7598031B2 (en) | 1999-01-06 | 2009-10-06 | Genenews Corporation | Method for the detection of gene transcripts in blood and uses thereof |
| KR101160153B1 (en) | 2009-12-10 | 2012-06-27 | 서울대학교산학협력단 | 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 |
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| KR102337743B1 (en) * | 2020-06-04 | 2021-12-13 | 한국과학기술연구원 | Biomarker for predicting cardiovascular risk factor in a psoriasis patient and use thereof |
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