KR101943517B1 - composition or kit for diagnosing behavioral addiction and method of detecting proheparin-binding epithelial growth factor-like growth factor for diagnosis of behavioral addiction using the same - Google Patents

Abstract

Provided are a composition or kit for diagnosing behavioral addiction and method of detecting proheparin-binding EGF (pro HB-EFG)-like growth factor for diagnosis of behavioral addiction using the same. According to the present invention, the composition or kit and method may be used for diagnosing behavioral addiction, such as Internet addiction or game addiction, with ease, high accuracy, and specificity.

Description

[0001] The present invention relates to a diagnostic composition, a kit, and a method for detecting pro-heparin-binding epidermal growth factor-like growth factor for the diagnosis of behavioral intoxication using the composition, kit for diagnosing behavioral addiction and method for detecting proheparin- like growth factor for diagnosis of behavioral addiction using the same}

The present invention relates to a diagnostic composition, a kit, and a method for detecting a pro-heparin-binding EGF-like growth factor (proB HB-EGF) for diagnosis of behavioral intoxication using the same.

Behavioral addiction is about addiction to certain behaviors, processes, and activities such as gambling addiction, sexual addiction, and pathological Internet use. It can involve brain circuits, cause tolerance and withdrawal, It plays an important role in the early stages, and compulsive is the key to maintaining behavior. In modern society, Internet addiction, game addiction, and obsessive addiction, such as alcoholism and drug addiction, are becoming more prominent.

Psychiatric symptoms Biomarkers are known for, for example, autism, Parkinson's disease, dementia, attention deficit hyperactivity disorder, alcoholism, drug addiction, and obsessive compulsive disorder (US Pub. 2005/0084880 A1 , Korean Registration Bulletin 10-1157526 (June 26, 2016)). However, in order to diagnose behavior addiction, especially internet addiction or game addiction, self-diagnosis method through brain image confirmation through magnetic resonance imaging (MRI) or questionnaire is mainly used, and an effective biomarker There is not yet.

Therefore, it is not a self-survey method based on costly and troublesome brain imaging or subjective judgment, but it is necessary to develop biomarkers that can diagnose behavior intoxication easily and cost-effectively, and have high diagnostic accuracy and specificity. .

A composition for diagnostic of behavioral toxicity is provided.

Provides a kit for behavioral intoxication.

A method for detecting a pro-heparin-binding EGF-like growth factor (proB HB-EGF) for diagnosis of behavioral intoxication.

One aspect provides a composition for diagnosing behavioral intoxication comprising an agent that measures the level of expression of a pro-heparin-binding EGF-like growth factor protein or fragment thereof.

The term "behavioral addiction" refers to any addiction to an action, process or activity. Behavioral addiction can lead to tolerance and withdrawal. The behavioral addiction can be selected from the group consisting of internet addiction, smartphone addiction, game addiction, gambling addiction, religious addiction, sex addiction, shopping addiction, exercise addiction, and work addiction. The Internet addiction refers to the addiction resulting from the use of the pathological or compulsive Internet, which is hindered by physical, mental, or social life. The game poisoning refers to a poisoning state that is hindered by physical, mental, or social life due to immersion in the game, either pathologically or compulsively. The Internet or game includes use by any medium such as a computer, a mobile phone, a smart phone, and a TV.

Proheparin-binding epithelial growth factor-like growth factor (proHB-EGF) is a growth factor that is effective through EGFR, ERBB2 and ERBB4, It is known as a protein required for cardiac function. ProB HB-EGF is known to promote mitosis in fibroblasts but not to promote epidermal cell division. In addition, proB HB-EGF can act as a diphtheria toxin receptor. ProB HB-EGF may be a protein expressed from the HBEGF gene. ProB HB-EGF can be cleaved with a heparin-binding EGF-like growth factor (HB-EGF) and a Diphtheria toxin receptor (DT-R). Pro-HB-EGF may contain the amino acid sequence of Uniprot number Q99075 in humans.

The fragment may be an immunogenic polypeptide as part of proB HB-EGF.

The agent may be an antibody or an antigen-binding fragment thereof that specifically binds to a pro-HB-EGF protein or fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. The term " antibody " can be used interchangeably with the term " immunoglobulin ". The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen binding fragment refers to a polypeptide comprising an antigen binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a surface of a metal chip, plate, or well. The antibody or antigen-binding fragment may be an anti-proB HB-EGF antibody or an antigen binding fragment.

The agent may be a nucleic acid comprising a polynucleotide that is the same as or complementary to a polynucleotide encoding the pro-HB-EGF protein or fragment thereof. The nucleic acid may be a primer or a probe. The primer or the probe may be labeled at the end or inside thereof with a fluorescent substance, a chemiluminescent substance or a radioactive isotope.

Another aspect provides a kit for behavioral intoxication comprising an agent that measures the level of expression of a pro-HB-EGF protein or fragment thereof.

The kit may further include a sample required for the behavioral intoxication diagnosis. The kit may comprise a substrate, a suitable buffer solution, a chromogenic enzyme, a secondary antibody labeled with a fluorescent substance, or a chromogenic substrate for immunological detection of a solid support, antibody or antigen-binding fragment. The kit may comprise a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent material, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Another aspect includes measuring the level of expression of a pro-HB-EGF protein or fragment thereof in a biological sample separated from a suspect subject; And comparing the measured expression level to a protein expression level of a normal control. The present invention provides a method for detecting pro-HB-EGF for the diagnosis of addiction.

The method comprises measuring the level of expression of a pro-HB-EGF protein or fragment thereof in a biological sample separated from an individual suspected of having an activity addiction.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual suffering from or susceptible to an addiction.

The biological sample refers to a sample obtained from the individual. The biological sample may be, for example, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, fluid, mucosal fluid, amniotic fluid, or a combination thereof.

The measuring step may comprise incubating the biological sample with an antibody that specifically binds to the pro-HB-EGF protein or fragment thereof.

The measuring step may be performed by any one of electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, Northern blotting, polymerase chain reaction (PCR), or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real time PCR or reverse transcription PCR.

And comparing the measured expression level to a protein expression level of a normal control.

In this method, the amount of the measured expression level of pro-HB-EGF can be reduced compared to the expression level of pro-HB-EGF derived from a sample of normal control. The normal control group refers to a negative control group as a group that does not suffer from addiction to the action. For example, if the amount of detected pro-HB-EGF protein is less than the amount of pro-HB-EGF protein of a normal control, the subject is more likely to have behavioral intoxication, such as Internet addiction or game addiction, Can be diagnosed. Psychosocial treatment or medication may be performed on individuals diagnosed with behavioral addiction, for example, Internet addiction or game addiction. The medicament may be, for example, naltrexone, disulfiram, methadone, chlordiazepoxide, acamprosate, bupropion, varenicline, Buprenorphine, or a combination thereof.

According to one aspect of the present invention, there is provided a composition, a kit, and a method for detecting pro-HB-EGF for diagnosing behavioral intoxication using the same, wherein the action addiction, for example, the Internet addiction or game addiction is simple, It can be used for road diagnosis.

FIG. 1 is a graph showing the results of performing a high performance liquid chromatography on a depressed serum sample (x axis: time (minute), y axis: mass absorption unit (milli Absorbance Unit: mAU)).
FIG. 2 (a) is an SDS-PAGE gel image before and after the depression in the normal control group, and FIG. 2 (b) is an SDS-PAGE gel image before and after the depression in the serum samples of the Internet or game addiction group : Size marker (KDa), 1 to 4: lanes 1 to 4).
FIG. 3A is an image obtained by performing an antibody array on 507 kinds of proteins, FIG. 3B is an image comparing an antibody array spot of proB HB-EGF in a control and internet or game addiction group, FIG. Lt; RTI ID = 0.0 > EGF < / RTI >
FIG. 4A is a graph showing an image and a membrane staining image obtained by immunoblotting the amount of pro-HB-EGF in the control group and the Internet or game addiction group, and FIG. 4B is a graph showing the relative strength of immunoblotting results.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1. Internet or games Addictive  Identification of Differentially Expressed Proteins in Serum Proteins

1. Internet or games Addictive  Preparation of sample

Serum samples from the internet or game addicts (n = 4) were collected from the Catholic University of Seoul, Korea. As a negative control group, normal (n = 4) serum samples not diagnosed by the Internet or game addiction were obtained.

To prevent protein degradation or deformation of the serum sample, the serum obtained immediately after obtaining the serum was added with protease inhibitor cOmplete, Mini EDTA-free protease inhibitor cocktail (Roche). The inhibitor was prepared by dissolving one tablet in 200 μl of phosphate buffered saline (PBS) according to the manufacturer's protocol to 50 X, and then adding the resulting solution to each sample to a final concentration of 1.5 X of the inhibitor. Serum samples containing inhibitors were quantified and stored at -80 ° C until used in the experiment.

2. Degradation of serum samples ( depletion )

In order to remove proteins such as albumin or immunoglobulin G, which are abundant in serum, the obtained serum samples were impaired.

Concretely, the serum samples obtained in 1. above were prepared at a concentration of 54.8 μg / μl, which is the minimum quantitative amount, and the experiment was carried out under the same conditions. The prepared serum sample was diluted 6-fold with the buffer provided by the manufacturer, and the diluted sample was filtered through a 0.22 탆 spin filter. 100 [mu] l of the filtered sample was injected into a Multiple Affinity Removal System (MARS) 14 column (Agilent) and the sample was attenuated using an Agilent 1100 HPLC system. It has been shown that the presence of a large number of 14 proteins (albumin, immunoglobulin G, alpha-1-antitrypsin, immunoglobulin A, transferrin, haptoglobin, fibrinogen, alpha-2-macroglobulin, M, apolipoprotein AI, apolipoprotein A-II, complement protein C3, and trans-retinol) were collected, respectively. Figure 1 shows the results of real-time measurement of the absorbance of the filtrate from the membrane, which is attenuated by the MARS 14 column (- - - - -: indicates when the collection of the filtrate starts ----: filtration Indicating when the collection of water ends).

3. Concentration and quantification of impaired serum samples

A 3 k centrifugal filter (Amicon Ultra, Millipore) was prepared by adding twice with 400 μl of water (HPLC grade, J.T Baker) and 400 μl of 10 mM HEPES buffer (pH 8.0) twice.

In step 2, the depleted serum samples were diluted 5-fold with 10 mM HEPES buffer. The diluted sample was added to the prepared filter, and the sample was concentrated by centrifugation at 14000 x g at 10 ° C. Thereafter, the filter containing the sample was washed 5 times with 10 mM HEPES buffer, and the filter containing the deteriorated serum sample was inserted into a new tube. The filter was centrifuged at 3000 x g for 2 minutes at 10 ° C, And recovered.

Concentrated samples were quantitated by bicinchoninic acid (BCA) assay. The same amount of protein was separated by SDS-PAGE, and the electrophoresed gel was stained by silver staining. Images of the stained gel are shown in FIGS. 2A and 2B (FIG. 2A: SDS-PAGE gel images before and after attenuation in serum samples of normal control group, FIG. 2b: SDS-PAGE gels before and after attenuation in serum samples of internet or game addiction group image).

4. Protein screening using antibody array

Serum samples from each of the control and internet or game addiction groups were pooled to a protein concentration of 500 μg / total volume of 1.5 ml.

To each pooled serum sample, 3.6 μl of a biotinylating agent (RayBiotech, Inc.) was added and incubated for 30 minutes at room temperature. Then, 5 μl of reaction termination solution (RayBiotech, Inc.) was added to terminate the reaction. The reaction was filtered through a size exclusion column (RayBiotech, Inc.) to remove the remaining biotin from the sample.

An antibody array was prepared by adding a blocking buffer (RayBiotech, Inc.) to an antibody 507 array (RayBiotech, Inc.) and incubating at room temperature for 1 hour. 1.5 ml of the biotin-labeled sample was diluted with a blocking buffer to a total volume of 14 ml. Samples were added to the prepared array and incubated overnight at about 4 ° C.

The array was then washed with the wash buffer contained in the antibody array kit, the array was loaded with streptavidin-HRP conjugate solution (RayBiotech, Inc.) and incubated at room temperature for 2 hours. Again the array was cleaned and signals of the array were detected according to the manufacturer's instructions. The antibody array results for 507 proteins are shown in Figure 3a.

As shown in FIG. 3A, proteins differentially expressed in the normal control group and the samples of the Internet or game addiction group were identified. Proheparin-binding epithelial growth factor-like growth factor (proHB-EGF) was selected as a biomarker among the differentially expressed proteins in the Internet or game addiction group, and proB HB-EGF Lt; RTI ID = 0.0 > 3B. ≪ / RTI > The relative array intensity of the antibody HB-EGF antibody array was quantified using TotalLab 1D analysis software (Nonlinear Dynamics) and the relative intensity of the protein quantified in the control and internet or game addiction groups is shown in FIG. 3c.

As shown in FIG. 3C, the pro-HB-EGF expression was significantly decreased in the Internet or game addiction group as compared with the control group.

5. Immune blotting  Verification of differential expression using

4. The expression profile of pro-HB-EGF selected as a biomarker candidate was verified. For the verification, we used 28 samples from the normal control group and 28 samples from the internet or game addiction group.

10 μg of the sample, in which 14 proteins were abundant in the normal control group and the Internet or the game addiction group, were electrophoresed, and the anti-proB HB-EGF antibody (R & D systems) and the secondary antibody (Thermo Fisher Scientific) Immunoblotting was performed (Fig. 4A) (QC: Quality control sample). Immunoblotting was performed and the images obtained were analyzed. For normalization, the band strength of QC samples was used. The results of the Mann-Whitney U-test are shown in FIG. 4b (poisoning group: internet or game addiction group, P = 0.0072).

As shown in FIG. 4B, it was confirmed that the expression of proB HB-EGF was significantly decreased in the Internet or game addiction group compared to the normal control group, and thus, the proB HB-EGF was excellent as a biomarker for internet or game addiction Respectively.

Claims (10)

1. A composition for the diagnosis of behavioral toxicity comprising an agent for measuring the expression level of a proheparin-binding epithelial growth factor-like growth factor (proHB-EGF) protein or fragment thereof,
Wherein the behavioral addiction is selected from the group consisting of internet addiction and game addiction. delete The composition according to claim 1,
An antibody or an antigen-binding fragment thereof that specifically binds to a pro-HB-EGF protein or fragment thereof; or
Wherein the nucleic acid is a polynucleotide comprising a polynucleotide identical or complementary to a polynucleotide encoding a pro-HB-EGF protein or fragment thereof. 4. The composition of claim 3, wherein the antibody is a polyclonal antibody or a monoclonal antibody. 4. The composition of claim 3, wherein the nucleic acid is a primer or a probe. An activity-inducing diagnostic kit comprising an agent that measures the level of expression of a pro-HB-EGF protein or fragment thereof,
Wherein the behavioral addiction is selected from the group consisting of internet addiction and game addiction. Measuring the level of expression of the pro-HB-EGF protein or fragment thereof in a biological sample separated from the suspect subject;
Comparing the measured expression level to a protein expression level of a normal control; And
Detecting a decrease in the measured expression level relative to the level of protein expression of the normal control, comprising the steps of:
Wherein said behavioral addiction is selected from the group consisting of internet addiction and game addiction. 8. The method of claim 7, wherein the biological sample is blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, fluid, mucosal fluid, amniotic fluid, or a combination thereof. 8. The method of claim 7, wherein said measuring step comprises incubating said biological sample with an antibody that specifically binds to a pro-HB-EGF protein or fragment thereof. [7] The method of claim 7, wherein the measuring step comprises the steps of: electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, Northern blotting, A polymerase chain reaction (PCR), or a combination thereof.

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