KR102142702B1 - Composition or kit for diagnosing behavioral addiction and method of detecting Prokineticin-1 for diagnosis of behavioral addiction using the same - Google Patents

Abstract

Provided according to an aspect are: a composition for diagnosing behavioral addiction; a kit therefor; and a method of detecting prokineticin-1 (PROK1) or a fragment thereof for diagnosis of behavioral addiction using the same. According thereto, the present invention can be used to diagnose behavioral addiction, for example, Internet addiction or game addiction in a simple way with high accuracy and specificity.

Description

Composition or kit for diagnosing behavioral addiction and method of detecting Prokineticin-1 for diagnosis of behavioral addiction using the same}

The present invention relates to a composition, kit for diagnosing behavioral addiction, and a method for detecting biomarkers for diagnosis of behavioral addiction using the same.

Behavioral addiction is an addiction to any activity, process, or activity, such as gambling addiction, sexual addiction, or pathological Internet use, involving brain circuits, resistance and withdrawal can occur, and impulsiveness develops It plays an important role in the early stages and compulsiveness is key to maintaining behavior. Internet addiction, game addiction, and compulsive addiction, such as alcoholism and drug addiction, are prominent in modern society.

Biomarkers are known for psychiatric symptoms such as autism, Parkinson's disease, dementia, attention deficit hyperactivity disorder, alcoholism, drug addiction, and obsessive-compulsive disorder (US Publication 2005/0084880 A1 (2005.04.21) , Republic of Korea Registration Notice 10-1157526 (2016.06.27), etc.). However, in order to diagnose behavioral addiction, especially Internet addiction or game addiction, a brain imaging confirmation through magnetic resonance imaging (MRI) or a self-diagnosis method through a survey is mainly used, and an effective biomarker for diagnosis There is not yet.

Therefore, it is not an expensive and cumbersome brain imaging diagnosis or self-based survey method based on subjective judgment, but it is necessary to develop a biomarker that can diagnose behavioral addiction easily and inexpensively and has high accuracy and specificity of diagnosis. There is.

Provided is a composition for diagnosing behavioral addiction.

Provide a kit for diagnosing behavioral addiction.

It provides a method for detecting biomarkers for the diagnosis of behavioral addiction.

Provided is a composition for diagnosing behavioral addiction comprising an agent that measures the expression level of Prokineticin-1 (PROK1) or a fragment thereof.

The term "behavioral addiction" refers to an addiction to an action, process or activity. Behavioral addiction can lead to tolerance and withdrawal. The behavioral addiction may 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 a state of addiction impaired in physical, mental, or social life due to pathological or obsessive use of the Internet. The game addiction refers to a state of addiction impaired in physical, mental, or social life due to pathological or obsessive immersion in the game. The Internet or game includes use by all media such as computers, mobile phones, smart phones, and TVs.

Prokineticin-1 (Prokineticin-1: PROK1) is one of the prokineticin proteins and is a secretory protein that contracts gastrointestinal smooth muscle. PROK1 can induce the proliferation, migration, and fenestration of endocrine gland-derived capillary endothelial cells, or induce proliferation and differentiation of intestinal neurogonal cells. PROK1 can positively regulate PTGS2 (Prostaglandin-endoperoxide synthase 2) expression and prostaglandin synthesis. The PROK1 may also be referred to as Endocrine-gland-derived vascular endothelial growth factor (EG-VEGF) or Mambakine. The PROK1 may include the amino acid sequence of Uniprot No. P58294 in humans, and GenBank Accession No. It may include an amino acid sequence encoded by the nucleic acid sequence of NM_032414.

The fragment may be an immunogenic polypeptide as part of PROK1.

The agent may be an antibody or antigen-binding fragment thereof that specifically binds PROK1 or a fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. The term "antibody" can be used interchangeably with the term "immunoglobulin". The antibody can 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', scFv, diabody, nanobody, minibody, tetrabody, triabodies, V _H domain, or V _L domain. 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-PROK1 antibody or antigen-binding fragment.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to a polynucleotide encoding PROK1 or a fragment thereof. The nucleic acid can be a primer or a probe. The primer or probe may be labeled with a fluorescent material, chemiluminescent or radioactive isotope on its terminal or inside.

Another aspect provides a kit for diagnosing behavioral addiction comprising an agent that measures the expression level of PROK1 or a fragment thereof.

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

Another aspect includes measuring the expression level of PROK1 or a fragment thereof in a biological sample isolated from a subject suspected of behavioral addiction; And it provides a method for detecting PROK1 for the diagnosis of behavioral addiction comprising comparing the measured expression level with the protein expression level of the normal control.

The method includes measuring the expression level of PROK1 or a fragment thereof in a biological sample isolated from a subject suspected of behavioral addiction.

The subject can be a mammal, for example, a human, cow, horse, pig, dog, sheep, goat, or cat. The individual may be an individual suffering from or suspected of having behavioral 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, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof.

The measuring step may include the step of incubating the biological sample and an antibody specifically binding to PROK1 or a fragment thereof.

The measuring steps include electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, Northern blotting, polymerase amplification reaction (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 with the protein expression level of a normal control.

The normal control group is a group not affected by behavioral addiction and means a negative control group.

The method may further include the step of determining that the subject has or is more likely to suffer behavioral addiction when the measured expression level of PROK1 or a fragment thereof is reduced than the protein expression level of the normal control. Psychosocial treatment or drug treatment may be performed on an individual diagnosed with behavioral addiction, for example, Internet addiction or game addiction. The drug is, for example, naltrexone, disulfiram, methadone, chlordiazepoxide, acamprosate, bupropion, varenicline, Buprenorphine, or a combination thereof.

According to one aspect, PROK1 or a fragment thereof may be detected to provide information necessary for diagnosis of behavioral addiction.

According to a composition, kit for diagnosing behavioral addiction according to one aspect, and a method for detecting prokineticin-1 (PROK1) or a fragment thereof for diagnosis of behavioral addiction using the same, behavioral addiction, for example It can be used to diagnose Internet addiction or game addiction with simple, high accuracy and specificity.

1 is a graph showing the results of performing high performance liquid chromatography on a depleted serum sample (x-axis: time (minutes), y-axis: mass absorbance unit (mAU)).
Figure 2a is a SDS-PAGE gel image before and after depletion in the serum sample of the normal control, Figure 2b is an image before and after depletion SDS-PAGE gel in the serum sample of the Internet or game addiction group (left: before depletion, right: after depletion, M : Size marker (kDa), 1 to 4: Lanes 1 to 4).
Figure 3a is an image obtained by performing an antibody array on a protein of 507 species, Figure 3b is an image comparing the antibody array spot of prokineticin-1 (PROK1) in the control group and the Internet or game addiction group, 3c is a graph showing the relative intensity of the antibody array spot of PROK1.
4 is a graph showing the level of expression of PROK1 in blood in the normal control group and the Internet or game addiction group as the relative intensity of immunoblotting.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Internet or game Addicted Identification of differentially expressed proteins among serum proteins

1. Internet or game Addicted Sample preparation

Serum samples of patients with Internet or game addiction (n=4) were collected from Catholic University Seoul St. Mary's Hospital. Serum samples of normal subjects (n=4) who were not diagnosed with Internet or game addiction as negative controls were obtained.

To prevent proteolysis or modification of the serum sample, cOmplete, Mini EDTA-free protease inhibitor cocktail (Roche) as a protease inhibitor, and PhosSTOP (Roche) as a phosphatase inhibitor were added to the serum obtained immediately after the serum was obtained. Did. The inhibitor was dissolved in 200 μl of phosphate buffered saline (PBS) according to the manufacturer's protocol to make 50 X, and then added to each sample so that the final concentration of the inhibitor was 1.5 X. Serum samples with inhibitors were quantified and stored at -80°C until use in experiments.

2. Depletion of serum samples ( depletion )

The serum samples obtained were depleted to remove proteins present in serum, such as albumin or immunoglobulin G.

Specifically, the serum samples obtained in 1. were each prepared at a concentration of 54.8 µg/µl, the minimum amount of quantification, and experiments were performed under the same conditions. Prepared serum samples were diluted 6-fold with a buffer provided by the manufacturer, and the diluted samples were filtered through a 0.22 μm spin filter. The filtered 100 μl sample was injected into a Multiple Affinity Removal System (MARS) 14 column (Agilent), and the sample was depleted using an Agilent 1100 HPLC system. 14 types of proteins (albumin, immunoglobulin G, alpha-1-antitrypsin, immunoglobulin A, transferrin, haptoglobin, fibrinogen, alpha-2-macroglobulin, alpha-1-acidic glycoprotein, immunoglobulin) The filtrate from which M, apolipoprotein AI, apolipoprotein A-II, complement protein C3, and transtyretin) were removed, respectively, was collected. The results of real-time measurement of the absorbance of the filtrate depleted by the MARS 14 column are shown in FIG. 1 (--··--··--: indicates the point at which collection of the filtrate starts. ――――: filtration Indicates the point at which the collection of water ends).

3. Concentration and quantification of depleted serum samples

3K centrifugal filter (Amicon Ultra, Millipore) was prepared by adding twice with 400 μl of water (HPLC grade, J.T Baker) and twice with 400 μl of 10 mM HEPES buffer (pH8.0).

The serum samples depleted in 2. were diluted 5 times with 10 mM HEPES buffer. After adding the diluted sample to the prepared filter, the sample was concentrated by centrifugation at 10°C for 10 minutes at a rate of 14000x g. Then, the filter containing the sample was washed 5 times with 10 mM HEPES buffer, the filter containing the depleted serum sample was flipped over in a new tube, and concentrated by centrifugation at 10°C for 5 minutes at a rate of 3000xg. The recovered serum sample was recovered.

The concentrated sample was quantified by bicinchoninic acid (BCA) analysis. After separating the same amount of protein by SDS-PAGE, the electrophoresis gel was stained by silver staining. The images of the stained gels are shown in FIGS. 2A and 2B (FIG. 2A: SDS-PAGE gel images before and after depletion in serum samples of normal control, FIG. 2B: SDS-PAGE gels before and after depletion in serum samples of the internet or game addiction group) image).

4. Protein screening using antibody arrays

Serum samples from each of the normal control group and the Internet or game addiction group were pooled so that the protein concentration was 500 µg/total volume of 1.5 ml.

The reaction was terminated by adding 3.6 μl of biotinylating agent (RayBiotech, Inc.) to each pooled serum sample, incubating at room temperature for 30 minutes, and then adding 5 μl of the reaction termination solution (RayBiotech, Inc.). The reaction was filtered through a size exclusion column (RayBiotech, Inc.) to remove biotin remaining in the sample.

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

Thereafter, the array was washed with the wash buffer included in the antibody array kit, and streptavidin-HRP conjugation solution (RayBiotech, Inc.) was added to the array and incubated at room temperature for 2 hours. The array was again washed and the array signal was detected according to the manufacturer's instructions.

The results of the antibody array for 507 proteins are shown in FIG. 3A. As shown in FIG. 3A, proteins differentially expressed in samples of the normal control group and the Internet or game addiction group were identified. Among the proteins differentially expressed in the Internet or game addiction group, Prokineticin-1 (PROK1) was selected as a biomarker, and an antibody array image of the PROK1 protein is shown in FIG. 3B. Using TotalLab 1D analysis software (Nonlinear Dynamics), the relative intensity of spots in the antibody array image of PROK1 protein was quantified. The relative strength of the protein in the normal control group and the Internet or game addiction group is shown in FIG. 3C.

3B and 3C, PROK1 protein significantly decreased expression in the Internet or game addiction group compared to the normal control group.

5. Verification of differential expression using immunoblotting

4. The expression pattern of PROK1 protein selected as a biomarker candidate was verified. For verification, experiments were conducted using samples from 29 control groups from the Catholic University of Korea, St. Mary's Hospital and Boramae Hospital, and 19 from the Internet or game addiction groups.

Electrophoresis of 35 μg of samples depleted of 14 types of proteins present in the control group or the internet or game addiction group, and anti-prokineticin-1 antibody (R&D systems) and secondary antibody (Thermo Fisher Scientific) were used. Immunoblotting was performed. The images obtained by immunoblotting were analyzed, and the results of analysis by Mann-Whitney U-test are shown in FIG. 4 ( P =0.0002).

As shown in FIG. 4, the expression of PROK1 was significantly decreased in the Internet or game addiction group compared to the normal control group. Therefore, it was confirmed that PROK1 has excellent accuracy as a biomarker for Internet or game addiction.

Claims (11)

Prokineticin-1 (Prokineticin-1: PROK1) or a composition for diagnosing behavioral addiction comprising an agent that measures the expression level of a fragment thereof,
The behavioral addiction is a composition selected from the group consisting of Internet addiction and game addiction. delete The method according to claim 1, wherein the formulation
An antibody or antigen-binding fragment thereof that specifically binds PROK1 or a fragment thereof; or
A composition comprising a polynucleotide identical to or complementary to a polynucleotide encoding PROK1 or a fragment thereof. The composition according to claim 3, wherein the antibody is a polyclonal antibody or a monoclonal antibody. The composition according to claim 3, wherein the nucleic acid is a primer or a probe. A kit for diagnosing behavioral addiction comprising an agent that measures the expression level of PROK1 or a fragment thereof,
The behavioral addiction is a kit selected from the group consisting of Internet addiction and game addiction. Measuring the expression level of PROK1 or a fragment thereof, or a polynucleotide encoding them, in a biological sample isolated from a subject suspected of behavioral addiction; And
A method for detecting PROK1 for the diagnosis of behavioral addiction, comprising comparing the measured expression level with the expression level of a protein or fragment thereof, or a polynucleotide encoding the normal control protein,
The biological sample is blood, plasma, or serum,
The behavioral addiction is a method selected from the group consisting of Internet addiction and game addiction. delete The method of claim 7, wherein the measuring step comprises incubating the biological sample with an antibody that specifically binds PROK1 or a fragment thereof. The method according to claim 7, wherein the step of measuring is performed by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay: ELISA), protein chip, immunoprecipitation, microarray, or a combination thereof. Way. The method according to claim 7, If the measured expression level of PROK1 or a fragment thereof, or a polynucleotide encoding them, is lower than the expression level of a protein or a fragment thereof, or a polynucleotide encoding them, the subject is subject to behavioral addiction. And determining that there is a high likelihood of being caught.

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