KR20190027759A - A method for multiplexed detection of exosome miRNA and cell-surface protein by Magnetic beads and molecular beacon - Google Patents

Abstract

The present invention relates to a multi-detection method of exosomes using a molecular beacon, an antibody for detecting a target surface protein bound with fluorescent substances, and a magnetic bead, and to a kit for multiple detection thereof. More particularly, the method for multi-detection of the present invention can efficiently detect a target protein and a target gene from exosomes using the molecular beacon, the antibody for detecting the target surface protein to which the fluorescent substances are bound, and the magnetic bead having an antibody attached thereto, and thus can be used for diagnosis of a specific disease.

Description

[0001] The present invention relates to a method for multiplexing exosomes using magnetic beads and molecular beacons,

The present invention relates to a method for multiple detection of exosomes using magnetic beads and molecular beacons and a multiple detection kit for the same. More particularly, the present invention provides a multiple detection method using an antibody-attached magnetic bead and a molecular beacon Since the target protein and the target gene can be efficiently detected from the exosome, it can be used for diagnosis of a specific disease.

The ultimate goal in diagnostics is to diagnose the disease with high accuracy and simple processes. Various diagnostic methods, kits, and the like have been developed to achieve this purpose. However, there has been a growing interest in the multiple detection technology as a substitute for the single detection method.

The exosome is a small form of membrane vesicle that is mostly secreted from the cell. The exosome contains various kinds of proteins, genetic material (DNA, mRNA, miRNA) and lipids derived from the cell. These exosomes have different properties from the proteins and genetic materials present in the biological fluid. Specifically, exosomes are composed of spherical particles surrounded by a lipid bilayer. There are various cell-derived proteins on the surface of exosome, and cell-derived genetic materials are present inside. In particular, the presence of excess RNAase (RNase) in the serum makes it difficult for the genomic material secreted from the cell to be easily degraded and measured, while in the case of the genomic material present in the exosome, . Thus, it has been reported that tissue-derived exosomes can be used for diagnosis of diseases because they reflect the state of the tissue that secretes exosome.

As described above, there is a limit to a general diagnostic method for detecting a disease marker from a biological sample such as blood, saliva, or urine. Specifically, since a biological sample contains not only specific tissues but also various concentrations of proteins and genetic substances derived from various tissues, even if a marker for a specific disease (biomarker) is detected, I can not know. Furthermore, since the degree of expression of proteins and genetic material in each tissue is different, there is a limitation that an accurate diagnosis can not be made with a quantitative amount simply measured in a biological sample.

Accordingly, there is a desire to develop a multiple detection method capable of accurately diagnosing diseases from various biological samples by solving the above-mentioned problems.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

Patent Document 1: Korean Patent Laid-Open Publication No. 10-1997-0705753

The present inventors have tried to find a new detection method capable of easily, quickly, and accurately detecting a biomarker related to a specific disease from the tissue of a subject or a biological sample. As a result, a multiple detection method capable of efficiently detecting a plurality of biomarkers related to a specific disease from exosomes was devised. By using this method, CD63, EpCAM, EFGR, Survivin, IGF-1R surface protein and miR- 21 and miR-574-3p were detected, and their accuracy was confirmed, thereby completing the present invention.

It is therefore an object of the present invention to provide a multiple detection method capable of efficiently detecting a specific surface protein of a exosome and a specific gene present therein from a biological sample.

It is another object of the present invention to provide a method for providing information that can diagnose a specific disease from a biological sample.

It is still another object of the present invention to provide a multiple detection kit capable of efficiently detecting a specific protein and a specific gene of exosome from the biological sample.

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

According to one aspect of the present invention, the present invention provides a multiple detection method including the following steps.

1) simultaneously adding a molecular beacon for detecting a target gene and an antibody for detecting a target surface protein bound with a magnetic bead and a fluorescent substance to which the antibody is attached, to the tissue or biological sample of the subject;

2) measuring the fluorescence intensity from the mixed solution.

According to another aspect of the present invention, the present invention provides a multiple detection method including the following steps.

1 ') treating the tissue or biological sample of the subject with a magnetic bead with the antibody attached thereto;

2 ') adding to the mixture of step 1') an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene; And

3 ') measuring fluorescence intensity from the mixed solution.

And checking whether the surface protein and the target gene are present from the fluorescence intensity measured from the subject in the step 3 ').

The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof.

The magnetic beads to which the antibody is attached may be one having one or more antibodies specifically binding to the surface protein of the exosome.

The antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81.

Antibodies for detection of target surface proteins that detect exosomes bound to antibodies to magnetic beads include EpCAM (Epidermal Cell adhesion molecule), Epidermal growth factor receptor (EGFR), Survivin, Insulin-like growth factor 1 receptor Or an antibody against any one of the antigens selected from the group consisting of

The target gene may be miRNA, and the miRNA may be miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547.

The molecular beacon may be one in which a fluorescent substance and a quencher are labeled on both ends of a nucleic acid specifically binding to the target gene.

The nucleic acid may be a base sequence represented by any one of SEQ ID NO: 17 to SEQ ID NO: 32.

According to another aspect of the present invention, the present invention provides a method for providing information for diagnosing a specific disease from a subject, comprising the steps of:

(I) separating exosome with magnetic beads attached to the tissue or biological sample of the subject, adding an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene;

II) measuring fluorescence intensity from the mixed solution; And

III) comparing the fluorescence intensity in the subject measured in the step II) with the fluorescence intensity in the control subject.

According to another aspect of the present invention, the present invention provides a method for providing information for diagnosing a specific disease from a subject, comprising the steps of:

I ') treating the tissues or biological samples of the subject with magnetic beads with antibodies attached thereto;

II ') adding an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene to the mixture of step I');

III ') measuring the fluorescence intensity from the mixed solution; And

IV ') comparing the fluorescence intensity of the subject measured in the step (III') with the fluorescence intensity in the control.

The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof.

The magnetic beads to which the antibody is attached may be one having one or more antibodies specifically binding to the surface protein of the exosome.

The antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81.

The target surface protein detection antibody may be any one selected from the group consisting of EpCAM (Epidermal Cell adhesion molecule), Epidermal growth factor receptor (EGFR), Survivin, and Insulin-like growth factor 1 receptor Lt; / RTI >

The target gene may be a miRNA. Preferably, the miRNA may be miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547.

The molecular beacon may be one in which a fluorescent substance and a quencher are labeled on both ends of a nucleic acid specifically binding to the target gene.

The miRNA may be a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 16.

The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary CNS lymphoma, spinal cord tumor, brain tumor, brainstem glioma, and pituitary adenoma.

According to another aspect of the present invention, there is provided a magnetic bead comprising an antibody-bound magnetic bead; An antibody conjugated with a fluorescent substance; And a molecular beacon, wherein the molecular beacon comprises a nucleic acid complementary to a target gene existing in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher coupled to the other end of the nucleic acid The present invention also provides a kit for multiple detection.

The magnetic beads to which the antibody is attached may be one having one or more antibodies specifically binding to the surface protein of the exosome.

The antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81.

The target surface protein detection antibody may be any one selected from the group consisting of EpCAM (Epidermal Cell adhesion molecule), Epidermal growth factor receptor (EGFR), Survivin, and Insulin-like growth factor 1 receptor Lt; / RTI > Specifically, EpCAM (epidermal cell adhesion molecule) is a signaling molecule with tumor-promoting functions that is known to overexpress mRNA and protein in prostate cancer tissues and cell lines. EGFR (epithelial growth factor receptor) is known to be involved in colon cancer, lung cancer, , And is known to be overexpressed in the exosomes of patients with prostate cancer (in vitro) in vivo. Survivin is an inhibitor-of-apoptosis (LAP) protein that has been reported to cause poor prognosis and tumor recurrence when survivin is overexpressed in prostate cancer tumors. It has been reported that insulin-like growth factor 1 receptor (IGF-1R) stimulates prostate epithelial cell proliferation and plays an important role in the development of prostate cancer. Thus, the expression level of the target surface proteins is predictable It is a biomarker.

The target gene may be a miRNA. Preferably, the miRNA may be miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547.

The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary CNS lymphoma, spinal cord tumor, brain tumor, brainstem glioma, and pituitary adenoma.

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

(I) The present invention provides a method for multiply detecting a biomarker related to a specific disease from a tissue or a biological sample of a subject using antibodies and molecular beacons conjugated with antibody-attached magnetic beads and fluorescent beacons.

(Ii) The anti-invention also provides a kit for multiple detection using an antibody and a molecular beacon having a sphere-attached magnetic bead and a fluorescent substance bonded thereto.

(Iii) The multiple detection method of the present invention can easily and quickly detect the surface protein and the target gene of exosome from the tissue or biological sample of the subject, and can easily and quickly diagnose diseases such as cancer non-invasively through this method can do.

1 schematically shows a multiple detection method according to the present invention.
2 shows the result of analysis of the PC3 cell culture solution according to the multiple detection method (condition 1; reaction at 4 ° C for 2 to 12 hours) in Experimental Example 1. FIG.
Fig. 3 shows the result of analysis of the PC3 cell culture solution according to the multiple detection method (condition 2; reaction at 37 DEG C for 4 hours) of Experimental Example 1. Fig.
4 shows the results of the multiple detection method of Experimental Example 2 (Condition 1: analysis at 4 ° C for 12 hours) from various concentrations of exosome solution.
FIG. 5 shows the results of analysis of the multiple detection method of Example 2 (condition 2; reaction at 37 DEG C for 4 hours) from various concentrations of exosome solution.
FIG. 6 is a graph of fluorescence intensity measured by the multiple detection method of Experimental Example 3 according to presence or absence of exosome or antibody-attached magnetic particles. FIG.
FIG. 7 shows the result of performing the multiple detection method (CD63, miR-21) of Experimental Example 4 from the culture medium of normal prostate cells (RWPE-1), prostate cancer cells (DU145 and PC3).
FIG. 8 shows the result of performing the multiple detection method (EpCAM, miR-21 or EGFR, miR-21) of Experimental Example 4 from the culture medium of normal prostate cells (RWPE-1), prostate cancer cells (DU145 and PC3).
9 is a result of performing multiple detection (CD63, miR-21 or CD63, Mir-574-3p) of Experimental Example 5 from the culture medium of normal prostate gland cells (RWPE-1), prostate cancer cells (DU145 and PC3).
10 is a graph showing fluorescence intensities measured by performing multiple detection of Experimental Example 6 from urine of a normal person.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

One aspect of the invention relates to a multiple detection method comprising the steps of:

1 ') treating the tissue or biological sample of the subject with a magnetic bead with the antibody attached thereto;

2 ') adding to the mixture of step 1') an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene; And

3 ') measuring fluorescence intensity from the mixed solution.

The multiple detection method is schematically shown in FIG. Hereinafter, the present invention will be described in detail with reference to FIG.

The present inventors have made efforts to improve the accuracy of the detection method of biomarkers such as proteins or genes for diagnosis of diseases. As a result, the present inventors have found that antibody-attached magnetic beads and fluorescent molecules- It was confirmed that the bee polynucleotide can efficiently and precisely detect multiple proteins and genes used for diagnosis of a specific disease from exosomes.

First of all, the tissue or biological sample of the subject is treated with magnetic beads with antibodies attached thereto. This allows the detection of exosome- specific, disease-specific, or tissue-specific surface proteins from tissue or biological samples of the subject. Specifically, when the antibody-specific magnetic beads specifically bind to the exosome having the above-mentioned surface protein to form a complex, the complex can be separated through a magnet. Further, when the magnetic beads are separated from the complex, the exosomes having specific surface proteins can be detected not only in the primary but also in the separation and concentration.

The biological sample includes urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, aspiration nipple, lymphatic fluid, airway fluid, intestinal fluid, urinary reproductive fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, Body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof.

The magnetic beads to which the antibody is attached include one or more antibodies specifically binding to the surface protein of the exosome. The antibody is not particularly limited as long as it is an antibody capable of detecting or capturing exosome, An immunologically active fragment, an antibody heavy chain, an antibody light chain, a genetically engineered single chain molecule, a chimeric antibody or a humanized antibody, and the like. Preferably, the antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81, and more preferably an antibody against the CD63 antigen present in the membrane protein of the exosome.

The antibody and the magnetic bead may be directly bound or bound through a linker, wherein the linker is selected from the group consisting of protein G, protein A, protein A / G, Fc receptor, biotin, avidin, histidine, Ni- But are not limited to, one or more selected from the group consisting of avidin.

The antibody-immobilized magnetic beads can be used to target specific proteins (antigens) present on the surface of the exosomes by immobilizing the antibody on the surface of the magnetic beads, and can be easily collected and separated using magnetic separation.

When the magnetic beads with the antibody attached thereto are treated with tissue or a biological sample of the subject, the antibody attached to the magnetic beads binds to the surface protein (antigen) of the exosome existing in the tissue or sample. An exosome-antibody-magnetic bead complex having an antigen for the antibody is formed. Since the complex can be collected easily by magnetic force separation, nonspecific reaction can be reduced and detection efficiency can be maximized.

Specifically, the magnetic beads include any one or more selected from the group consisting of Fe (III), Mg, Mn, Ni, Zn, and Co. The magnetic beads are not particularly limited as long as they are magnetic beads commonly used. .

Next, an antibody for detecting a target surface protein to which a fluorescent substance is bound and a molecular beacon for detecting a target gene are added to the mixture of step 2 ') and step 1'). In this process, an antibody for detecting a target surface protein to which a fluorescent substance is bound and a molecular beacon for detecting a target gene are added to a mixture of exosomes obtained from step 1 ') to detect an exosome having both a target protein and a target gene can do.

In the present invention, a 'target gene' is a substance capable of diagnosing a specific disease from a subject, more preferably a nucleic acid biomarker, and most preferably, a miRNA. Since exosomes contain more RNA than DNA, miRNAs can be detected more efficiently than existing DNA technology.

MiRNAs such as miR-21 (hsa-miR-21), miR-574 (hsa-miR-574), miR- (hsa-miR-147), miR-141 (hsa-miR-141) and miR-547 (hsa-miR-547).

MiR-205 (hsa-miR-205) is an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2; miR-57 (hsa-miR- MiR-147 (hsa-miR-147) is a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 or 12, miR-141 (hsa- miR-141) is SEQ ID NO: 13 or 14, and miR-547 (hsa-miR-547) is SEQ ID NO:

In the present invention, the term "molecular beacon" is a nucleic acid forming a hairpin-like secondary structure, which changes its molecular beacon structure by specific hybridization of a complementary region and emits fluorescence. Hereinafter, the specific structure of the molecular beacon according to the present invention will be described.

The molecular beacon comprises a nucleic acid complementary to a target gene, which is a biomarker of a specific disease, present in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher quencher.

The molecular beacon consists of a loop part that can bind to the target gene and a stem part that forms a hairpin loop in an environment where the target gene is not present. The loop part can vary depending on the specific miRNA species and the target of the miRNA whole sequence, whether to target the part or not, and the stem part can be changed (depending on the efficiency) for each molecular beacon.

The nucleic acid may be linear DNA or RNA having a sequence capable of binding complementarily to a specific miRNA while being present in the exosome, and the RNA may be selected from the group consisting of mRNA, miRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, , Preferably having 5 'and 3' sequences complementary to both ends of the nucleic acid so as to form a hairpin loop in an environment in which no specific miRNA is present. Specifically, the nucleic acid may have a complementary 5 'and 3' sequence to form a hairpin structure at both ends with the main chain polynucleotide as a center. The backbone polynucleotide may be of a sequence sufficiently complementary to hybridize selectively to the target gene. It may be any one selected from the sequences represented by SEQ ID NOS: 17 to 32 of the present invention.

The term " complementary " in the present invention means having complementarity enough to selectively hybridize to the target gene of the present invention under any specific hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from perfectly complementary, and the nucleic acid of the present invention is miR-21 (hsa-miR-21), miR- (Hsa-miR-141) or miR-547 (hsa-miR-147), miR- -547), as long as it is capable of selectively hybridizing to the nucleotide sequence of the nucleotide sequence of SEQ ID NO.

In addition, a fluorophore and a quencher are respectively bonded to both ends of the nucleic acid. Specifically, a fluorescent substance is bonded to the end of the nucleic acid, and a quencher is bonded to the other end of the nucleic acid. More specifically, the fluorescent substance may be bound to the 5 'end of the nucleic acid, the quencher may be bonded to the 3' end, the fluorescent substance may be bonded to the 3 'end of the nucleic acid, and the quencher may be bonded to the 5' Preferably any one selected from those shown in SEQ ID NOS: 17 to 32 below.

The nucleic acid refers specifically to a linear oligomer of natural or modified monomers or linkages, which can hybridize specifically to a target gene, naturally occurring or artificially synthesized.

Wherein the fluorescent material is at least one selected from the group consisting of Cyanine family fluorescent molecules, Rodamine family fluorescent molecules, Alexa family fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) Preferably Fluorecein, fluorescein isothiocyanate, Biodipy-FL, Alexa Fluor Green, R-picoerythrin (R), and the like. R-phycoerythrin, TexasRed, Phycoerythrin-Texas Red, rhodamine, alexa, cyanine (Cy3, Cy3.5, Cy5), picoerythrin Phycoerythrin-cyanine 5, Phycoerythrin-cyanine 7, Peridinin-chlorophyll protein, Allophycocyanin, Allophycocyanin-cyanine 7, Allophycocyanin-cyanine7), o-phthalate May be any one or more selected from the group consisting of phthaldehyde, fluorescamine, BODIPY, coumarin and derivatives thereof, more preferably FAM (5-carboxy fluorescein) have.

The quencher may be any one or more selected from the group consisting of QSY dye, dabsyl, dabcyl, blackhole quencher (BHQ) and blackberry quencher (BBQ), preferably blackhole quencher BHQ), and more preferably BHQ2.

In the molecular beacon, the nucleic acid is bound to the hairpin structure (specifically, the primer portion) by an interaction between the two ends of the nucleic acid (specifically, the primer portion) . The molecular beacon does not emit fluorescence because the fluorescent material and the quencher, which are bound to both ends of the nucleic acid, are close to generate FRET.

When a target gene is present in a biological sample or a sample (mixture after first detection with magnetic beads with antibody attached thereto), the molecular beacon and the target gene hybridize to collapse the hairpin secondary structure of the molecular beacon, As the structure is opened, the distance between the fluorescent substance bound to the end of the nucleic acid and the quencher distance becomes large. As a result, the molecular beacon fluoresces because the FRET effect of the quencher that affects the fluorescent material is lost.

The nucleic acid of the molecular beacon can be appropriately selected in consideration of the level of the signal to noise ratio that can be obtained using the molecular beacon of the present invention and is preferably selected to be specific to the target gene selected from the following SEQ ID NOS: Or a nucleic acid that is capable of binding to a target nucleic acid.

The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, vulvar cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma, vulvar carcinoma Cancer of the kidney or kidney, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, cancer of the central nervous system (CNS), cancer of the central nervous system , Primary CNS lymphoma, spinal cord tumor, brain tumor, brain stem glioma, or pituitary adenoma. Preferably, the disease is lung cancer, ovarian cancer, skin cancer, colon cancer It may be any one selected from the group consisting of pituitary adenomas.

The above steps 1 ') and 2') may be performed sequentially, or the above steps may be carried out simultaneously to rapidly diagnose disease-specific or tissue-specific exosome and diagnosis of a specific disease in one step.

When the above steps 1 ') and 2') are simultaneously carried out, it is preferable that 1) the antibody for target surface protein detection, in which antibody-attached magnetic beads and fluorescent substance are bound, to the tissue or biological sample of the subject, Adding a molecular beacon; And 2) measuring fluorescence intensity from the mixed solution.

Finally, in step 3 '), the presence or absence of the surface protein and the target gene is checked from the fluorescence intensity measured from the subject. When the target gene is present in the sample or the sample, the molecular beacon hybridizes with the target gene to collapse the hairpin secondary structure of the molecular beacon, and the structure is linearly opened, and the fluorescent substance And the quadrature distance. Since the FRET effect of the quencher that affects the fluorescent material is lost, the molecular beacon emits fluorescence. Therefore, the fluorescence intensity of the molecular beacon is measured to determine whether the target gene exists in the exosome having the surface protein .

Another aspect of the present invention provides a method of providing information for diagnosing a specific disease from a subject comprising the steps of: detecting an antibody for a target surface protein bound with a magnetic bead and a fluorescent substance, Molecular beacons may be added sequentially or simultaneously to the tissues or biological samples of the subject. When added at the same time, the disease-specific or tissue-specific exosome capturing and diagnosis of a specific disease can be rapidly performed in one step.

(I) adding an antibody for detecting a target surface protein and a molecular beacon for detecting a target gene, to which a magnetic bead and a fluorescent substance with an antibody are bound, to a tissue or a biological sample of the subject; II) measuring fluorescence intensity from the mixed solution; And (III) comparing the fluorescence intensity in the subject measured in the step (II) with the fluorescence intensity in the control.

In the case of sequential addition, treating the tissue or biological sample of the subject with magnetic beads with the antibody attached thereto; II ') adding an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene to the mixture of step I'); III ') measuring the fluorescence intensity from the mixed solution; And (IV ') comparing the fluorescence intensity of the subject measured in the step (III') with the fluorescence intensity in the control.

First of all, the tissue or biological sample of the subject is treated with magnetic beads with antibodies attached thereto. This allows the detection of exosome- specific, disease-specific, or tissue-specific surface proteins from tissue or biological samples of the subject. Specifically, when the antibody-specific magnetic beads specifically bind to the exosome having the above-mentioned surface protein to form a complex, the complex can be separated through a magnet. Further, when the magnetic beads are separated from the complex, the exosomes having specific surface proteins can be detected not only in the primary but also in the separation and concentration.

The biological sample includes urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, aspiration nipple, lymphatic fluid, airway fluid, intestinal fluid, urinary reproductive fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, Body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof.

The magnetic beads to which the antibody is attached include one or more antibodies specifically binding to the surface protein of the exosome. The antibody is not particularly limited as long as it is an antibody capable of detecting or capturing exosome, An immunologically active fragment, an antibody heavy chain, an antibody light chain, a genetically engineered single chain molecule, a chimeric antibody or a humanized antibody, and the like. Preferably, the antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81, and more preferably an antibody against the CD63 antigen present in the membrane protein of the exosome.

The antibody and the magnetic bead may be directly bound or bound through a linker, wherein the linker is selected from the group consisting of protein G, protein A, protein A / G, Fc receptor, biotin, avidin, histidine, Ni- But are not limited to, one or more selected from the group consisting of avidin.

The antibody-immobilized magnetic beads can be used to target specific proteins (antigens) present on the surface of the exosomes by immobilizing the antibody on the surface of the magnetic beads, and can be easily collected and separated using magnetic separation.

When the magnetic beads with the antibody attached thereto are treated with tissue or a biological sample of the subject, the antibody attached to the magnetic beads binds to the surface protein (antigen) of the exosome existing in the tissue or sample. An exosome-antibody-magnetic bead complex having an antigen for the antibody is formed. Since the complex can be collected easily by magnetic force separation, nonspecific reaction can be reduced and detection efficiency can be maximized.

Specifically, the magnetic beads include any one or more selected from the group consisting of Fe (III), Mg, Mn, Ni, Zn, and Co. The magnetic beads are not particularly limited as long as they are magnetic beads commonly used. .

Next, an antibody for detecting a target surface protein to which a fluorescent substance is bound and a molecular beacon for detecting a target gene are added to the mixture of step II ') and step I'). In this process, an antibody for detecting a target surface protein to which a fluorescent substance is bound and a molecular beacon for detecting a target gene are added to a mixture of exosomes having surface proteins obtained from the step (I) The exosome can be detected.

In the present invention, the term "target surface protein" refers to a substance capable of diagnosing a specific disease from an examinee, more preferably a protein that is widely distributed on the surface of exosomes.

The target surface protein may be EpCAM (epidermal cell adhesion molecule), EGFR (epidermal growth factor receptor), Survivin, or Insulin-like growth factor 1 receptor (IGF-1R).

In the present invention, a 'target gene' is a substance capable of diagnosing a specific disease from a subject, more preferably a nucleic acid biomarker, and most preferably, a miRNA. Since exosomes contain more RNA than DNA, miRNAs can be detected more efficiently than existing DNA technology.

The miRNAs are miR-21 (hsa-miR-21), miR-574 (hsa-miR-574) miR-205 (hsa-miR-205), miR- hsa-miR-147), miR-141 (hsa-miR-141) and miR-547 (hsa-miR-547).

MiR-205 (hsa-miR-205) is an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2; miR-57 (hsa-miR- MiR-147 (hsa-miR-147) is a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 or 12, miR-141 (hsa- miR-141) is SEQ ID NO: 13 or 14, and miR-547 (hsa-miR-547) is SEQ ID NO:

The term 'molecular beacon' in the present invention refers to a nucleic acid forming a hairpin-like secondary structure, which changes its molecular beacon structure by specific hybridization of a complementary region and emits fluorescence. Hereinafter, the specific structure of the molecular beacon according to the present invention will be described.

The molecular beacon comprises a nucleic acid complementary to a target gene, which is a biomarker of a specific disease, present in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher quencher.

The nucleic acid may be linear DNA or RNA having a sequence capable of binding complementarily to a specific miRNA while being present in the exosome, and the RNA may be selected from the group consisting of mRNA, miRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, , Preferably having 5 'and 3' sequences complementary to both ends of the nucleic acid so as to form a hairpin loop in an environment in which no specific miRNA is present. Specifically, the nucleic acid may have a complementary 5 'and 3' sequence to form a hairpin structure at both ends with the main chain polynucleotide as a center. The backbone polynucleotide may be of a sequence sufficiently complementary to hybridize selectively to the target gene.

The term " complementary " in the present invention means having complementarity enough to selectively hybridize to the target gene of the present invention under any specific hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from perfectly complementary, and the nucleic acid of the present invention is miR-21 (hsa-miR-21), miR- (Hsa-miR-141) or miR-547 (hsa-miR-147), miR- -547), as long as it is capable of selectively hybridizing to the nucleotide sequence of the nucleotide sequence of SEQ ID NO.

In addition, a fluorescent substance and a fluorescent substance are respectively bonded to both ends of the nucleic acid. Specifically, a fluorescent substance is bonded to the end of the nucleic acid, and a quencher is bonded to the other end of the nucleic acid. More specifically, a fluorescent substance may be bound to the 5 'end of the nucleic acid, a quencher may be bonded to the 3' end, a fluorescent substance may be bonded to the 3 'end of the nucleic acid, and a quencher may be bonded to the 5' end.

The nucleic acid refers specifically to a linear oligomer of natural or modified monomers or linkages, which can hybridize specifically to a target gene, naturally occurring or artificially synthesized.

Wherein the fluorescent material is at least one selected from the group consisting of Cyanine family fluorescent molecules, Rodamine family fluorescent molecules, Alexa family fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) Preferably Fluorecein, fluorescein isothiocyanate, Biodipy-FL, Alexa Fluor Green, R-picoerythrin (R), and the like. R-phycoerythrin, TexasRed, Phycoerythrin-Texas Red, rhodamine, alexa, cyanine (Cy3, Cy3.5, Cy5), picoerythrin Phycoerythrin-cyanine 5, Phycoerythrin-cyanine 7, Peridinin-chlorophyll protein, Allophycocyanin, Allophycocyanin-cyanine 7, Allophycocyanin-cyanine7), o-phthalate May be any one or more selected from the group consisting of phthaldehyde, fluorescamine, BODIPY, coumarin and derivatives thereof, more preferably FAM (5-carboxy fluorescein) have.

The quencher may be any one or more selected from the group consisting of QSY dye, dabsyl, dabcyl, blackhole quencher (BHQ) and blackberry quencher (BBQ), preferably blackhole quencher BHQ), and more preferably BHQ2.

The nucleic acid is formed into a hairpin structure as the molecular beacons interact with each other closely to each other to form a bond in an environment in which a target gene (preferably a specific miRNA) is not present . The molecular beacon does not emit fluorescence because the fluorescent material and the quencher, which are bound to both ends of the nucleic acid, are close to generate FRET.

When a target gene is present in a biological sample or a sample (mixture after first detection with magnetic beads with antibody attached thereto), the molecular beacon and the target gene hybridize to collapse the hairpin secondary structure of the molecular beacon, As the structure is opened, the distance between the fluorescent substance bound to the end of the nucleic acid and the quencher distance becomes large. As a result, the molecular beacon fluoresces because the FRET effect of the quencher that affects the fluorescent material is lost.

The nucleic acid of the molecular beacon can be appropriately selected in consideration of the level of the signal to noise ratio that can be obtained using the molecular beacon of the present invention, and preferably, any one of bases selected from SEQ ID NOS: 17 to 32 Sequence.

The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary cNS lymphoma, spinal cord tumor, brain tumor, brainstem glioma, or pituitary adenoma, and preferably the disease is lung cancer, ovarian cancer, skin cancer, colon cancer, Pituitary adenoma, and the like.

The above steps (I ') and (II') may be carried out sequentially, or the above steps may be carried out simultaneously to rapidly diagnose a disease-specific or tissue-specific exosome and diagnosis of a specific disease in one step.

Finally, the fluorescence intensity of the subject measured in the step IV ') and the step III') is compared with the fluorescence intensity in the comparator to provide information for diagnosing a specific disease.

When the target gene is present in the sample or the sample, the molecular beacon hybridizes with the target gene to collapse the hairpin secondary structure of the molecular beacon, and the structure is linearly opened, and the fluorescent substance And the quadrature distance. Since the FRET effect of the quencher that affects the fluorescent material is lost, the molecular beacon emits fluorescence. Therefore, the fluorescence intensity of the molecular beacon is measured to determine whether the target gene exists in the exosome having the surface protein . This allows disease-specific or tissue-specific exosome capture and diagnosis of certain diseases to be performed.

Yet another aspect of the invention is a magnetic bead conjugated with an antibody; An antibody for detecting a target surface protein bound with a fluorescent substance; And a molecular beacon, wherein the molecular beacon comprises a nucleic acid complementary to a target gene existing in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher coupled to the other end of the nucleic acid The present invention relates to a multiple detection kit.

The magnetic beads to which the antibody is attached include one or more antibodies specifically binding to the surface protein of the exosome. The antibody is not particularly limited as long as it is an antibody capable of detecting or capturing exosome, An immunologically active fragment, an antibody heavy chain, an antibody light chain, a genetically engineered single chain molecule, a chimeric antibody or a humanized antibody, and the like. Preferably, the antibody may be an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81, and more preferably an antibody against the CD63 antigen present in the membrane protein of the exosome.

The antibody and the magnetic bead may be directly bound or bound through a linker, wherein the linker is selected from the group consisting of protein G, protein A, protein A / G, Fc receptor, biotin, avidin, histidine, Ni- But are not limited to, one or more selected from the group consisting of avidin.

The antibody-immobilized magnetic beads can be easily collected and separated using magnetic separation by targeting a specific protein (antigen) present on the surface of the exosome by immobilizing the antibody on the surface of the magnetic bead.

When the magnetic beads with the antibody attached thereto are treated with tissue or a biological sample of the subject, the antibody attached to the magnetic beads binds to the surface protein (antigen) of the exosome existing in the tissue or sample. An exosome-antibody-magnetic bead complex having an antigen for the antibody is formed. Since the complex can be collected easily by magnetic force separation, nonspecific reaction can be reduced and detection efficiency can be maximized.

Specifically, the magnetic beads include any one or more selected from the group consisting of Fe (III), Mg, Mn, Ni, Zn, and Co. The magnetic beads are not particularly limited as long as they are magnetic beads commonly used. .

In the present invention, the term "target surface protein" refers to a substance capable of diagnosing a specific disease from an examinee, more preferably a protein that is widely distributed on the surface of exosomes.

The target surface protein may be EpCAM (epidermal cell adhesion molecule), EGFR (epidermal growth factor receptor), Survivin, or Insulin-like growth factor 1 receptor (IGF-1R).

In the present invention, a 'target gene' is a substance capable of diagnosing a specific disease from a subject, more preferably a nucleic acid biomarker, and most preferably, a miRNA. Since exosomes contain more RNA than DNA, miRNAs can be detected more efficiently than existing DNA technology.

The miRNAs are miR-21 (hsa-miR-21), miR-574 (hsa-miR-574) miR-205 (hsa-miR-205), miR- hsa-miR-147), miR-141 (hsa-miR-141) and miR-547 (hsa-miR-547).

MiR-205 (hsa-miR-205) is an amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2; miR-57 (hsa-miR- MiR-147 (hsa-miR-147) is a nucleotide sequence selected from the group consisting of SEQ ID NO: 11 or 12, miR-141 (hsa- miR-141) is SEQ ID NO: 13 or 14, and miR-547 (hsa-miR-547) is SEQ ID NO:

The term 'molecular beacon' in the present invention refers to a nucleic acid forming a hairpin-like secondary structure, which changes its molecular beacon structure by specific hybridization of a complementary region and emits fluorescence. Hereinafter, the specific structure of the molecular beacon according to the present invention will be described.

The molecular beacon comprises a nucleic acid complementary to a target gene, which is a biomarker of a specific disease, present in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher quencher.

The nucleic acid may be linear DNA or RNA having a sequence capable of binding complementarily to a specific miRNA while being present in the exosome, and the RNA may be selected from the group consisting of mRNA, miRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, , Preferably having 5 'and 3' sequences complementary to both ends of the nucleic acid so as to form a hairpin loop in an environment in which no specific miRNA is present. Specifically, the nucleic acid may have a complementary 5 'and 3' sequence to form a hairpin structure at both ends with the main chain polynucleotide as a center. The backbone polynucleotide may be of a sequence sufficiently complementary to hybridize selectively to the target gene.

The term " complementary " in the present invention means having complementarity enough to selectively hybridize to the target gene of the present invention under any specific hybridization or annealing conditions. Thus, the term " complementary " has a different meaning from perfectly complementary, and the nucleic acid of the present invention is miR-21 (hsa-miR-21), miR- (Hsa-miR-141) or miR-547 (hsa-miR-147), miR- -547), as long as it is capable of selectively hybridizing to the nucleotide sequence of the nucleotide sequence of SEQ ID NO.

In addition, a fluorophore and a quencher are respectively bonded to both ends of the nucleic acid. Specifically, a fluorescent substance is bonded to the end of the nucleic acid, and a quencher is bonded to the other end of the nucleic acid. More specifically, a fluorescent substance may be bound to the 5 'end of the nucleic acid, a quencher may be bonded to the 3' end, a fluorescent substance may be bonded to the 3 'end of the nucleic acid, and a quencher may be bonded to the 5' end.

The nucleic acid refers specifically to a linear oligomer of natural or modified monomers or linkages, which can hybridize specifically to a target gene, naturally occurring or artificially synthesized.

Wherein the fluorescent material is at least one selected from the group consisting of Cyanine family fluorescent molecules, Rodamine family fluorescent molecules, Alexa family fluorescent molecules, FITC (fluorescein isothiocyanate) fluorescent molecules, FAM (5-carboxy fluorescein) Preferably Fluorecein, fluorescein isothiocyanate, Biodipy-FL, Alexa Fluor Green, R-picoerythrin (R), and the like. R-phycoerythrin, TexasRed, Phycoerythrin-Texas Red, rhodamine, alexa, cyanine (Cy3, Cy3.5, Cy5), picoerythrin Phycoerythrin-cyanine 5, Phycoerythrin-cyanine 7, Peridinin-chlorophyll protein, Allophycocyanin, Allophycocyanin-cyanine 7, Allophycocyanin-cyanine7), o-phthalate May be any one or more selected from the group consisting of phthaldehyde, fluorescamine, BODIPY, coumarin and derivatives thereof, more preferably FAM (5-carboxy fluorescein) have.

The quencher may be any one or more selected from the group consisting of QSY dye, dabsyl, dabcyl, blackhole quencher (BHQ) and blackberry quencher (BBQ), preferably blackhole quencher BHQ), and more preferably BHQ2.

In the molecular beacon, the nucleic acid is bound to the hairpin structure (specifically, the primer portion) by an interaction between the two ends of the nucleic acid (specifically, the primer portion) . The molecular beacon does not emit fluorescence because the fluorescent material and the quencher, which are bound to both ends of the nucleic acid, are close to generate FRET.

When a target gene is present in a biological sample or a sample (mixture after first detection with magnetic beads with antibody attached thereto), the molecular beacon and the target gene hybridize to collapse the hairpin secondary structure of the molecular beacon, As the structure is opened, the distance between the fluorescent substance bound to the end of the nucleic acid and the quencher distance becomes large. As a result, the molecular beacon fluoresces because the FRET effect of the quencher that affects the fluorescent material is lost.

The nucleic acid of the molecular beacon can be appropriately selected in consideration of the level of the signal to noise ratio that can be obtained using the molecular beacon of the present invention, and preferably, any one of bases selected from SEQ ID NOS: 17 to 32 Sequence.

According to one embodiment of the present invention, after treatment with antibody-attached magnetic beads, changes in molecular beacon structure and increase in fluorescence intensity due to hybridization with the miR-21 base sequence in the isolated exosome were confirmed (Experimental Example 1 , 2).

The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, stomach cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's gyn's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, vulvar cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma, vulvar carcinoma, Cancer of the kidney or kidney, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, cancer of the central nervous system (CNS), cancer of the central nervous system , Primary CNS lymphoma, spinal cord tumor, brain tumor, brainstem glioma, and pituitary adenoma.

Another aspect of the present invention relates to a composition for detecting a surface protein and a target gene from an exosome comprising a magnetic bead to which the antibody is attached and a molecular beacon for detecting a target gene.

The composition according to the present invention may comprise a carrier and a vehicle commonly used in the medical field. Specific examples thereof include ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffer substances (eg various phosphates, glycine, sorbic acid, potassium sorbate, (E.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts, etc.), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose But are not limited to, matrix, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol, and the like. The composition of the present invention may further include, in addition to the above components, a lubricant, a wetting agent, an emulsifying agent, a suspending agent, or a preservative.

The composition of the present invention may be prepared as an aqueous solution for parenteral administration, and preferably a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used . Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or desran.

In addition, preferred embodiments of the compositions of the present invention may be in the form of sterile injectable preparations of sterile injectable aqueous or oily suspensions. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (ex. Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used in the present invention include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. Any non-volatile oil that is irritating, including synthetic mono- or diglycerides, may be used for this purpose.

As used herein, the term " diagnosis " means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, it is possible to diagnose a related disease through multiple detection for detecting proteins and genes existing in exosomes. Preferably, the diseases and diseases are diagnosed in lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, Cancer of the lungs, cancer of the lungs, bone cancer, pancreatic cancer, head or neck cancer, skin or intra-ocular melanoma, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, fallopian tube carcinoma, endometrial carcinoma, Cancer of the prostate, chronic or acute leukemia, cancer of the prostate, cancer of the prostate, cancer of the prostate, cancer of the prostate, cancer of the prostate, cancer of the breast, cancer of the breast, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, (CNS), primary CNS lymphoma, spinal cord tumors, brain tumors, brain stem glioma, and pituitary adenoma. The term " peripheral nervous system " But is not limited thereto. Preferably, the disease is any one selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer and pituitary adenoma.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.

Example 1. Cell preparation

The PC3 (prostate cancer cells) cells used in the experiments were RPMI 1640 Medium (w / HEPES, Gibco ^TM ) containing 10% FBS (fetal bovine serum, Biowest, France) and penicillin / streptomycin And cultured in a 100 mm dish at 37 ° C and 5% CO ₂ .

Examples 2 and 3. Molecular beacon (MB) design and manufacture

For the cancer cells used in the experiment, miRNA-21 or miRNA-574, which can be used as biomarkers, were selected. In order to synthesize specific molecular beacons capable of detecting selected microRNAs, the following sequences were designed and synthesized by Integrated DNA Technologies, Inc.

[miR-21-MB-Cy5 molecular beacon SEQ ID NO: 17]

Cy5-GCGCGTCAACATCAGTCTGATAAGCTACGCGC-BHQ2

[miR-574-3p-MB-Cy5 molecular beacon SEQ ID NO: 20]

Cy5-GCGCGTTGTGGGTGTGTGCATGAGCGTGACGCGC-BHQ2

The molecular beacon is bound to both ends of a nucleic acid that forms a specific binding with miRNA-21 or miRNA 574, and a fluorescent substance and a kense are bonded to each other. Cy5 is used as a fluorescent substance, and BHQ2 is used as a KENCHO.

Experimental Example 1. Analysis of CD63 and miR-21 through multiple detection according to reaction conditions from prostate cancer cells (PC3)

1) Antibody-coated magnetic bead treatment

To isolate and detect exosomes from cells, magnetic beads with antibodies attached were treated. At this time, Exosome-human CD63 separation / detection kit (ExoCap ^™ Exosome Isolation and Enrichment Kits) (MBL) was used for the magnetic nanoparticles to which the antibody was attached.

Before the reaction, the magnetic nanoparticles to which the antibody was attached were suspended by vortexing for 30 seconds, and then 30 μl of the antibody-coated magnetic nanoparticles were immersed in 1.5 ml eppendorf tube (Axygen) (exosome isolation buffer: PBS solution with 0.1% BSA concentration, and then filtered through a 0.2 μm syringe filter). The washing method is performed by placing the eppendorf tube in a DynaMag (TM) -2 Magnet (Thermo Fisher Scientific Inc., 12321D) and then contacting the magnet for 1 minute to remove only the supernatant while leaving the pellet.

After the washing process was completed, the magnet was removed, and the exosomes obtained from the prostate cancer cells (PC3) were mixed with the exosomal isolation buffer at a concentration of 1 x 10 ⁸ particles and a total volume of 500 μl, The magnetic nanoparticles were reacted at 4 ° C (Condition 1) or 37 ° C (Condition 2) for various times. During the reaction time, the eppendorf tube containing the reaction solution was rotated (mode: 90, RPM: 60) with MyLab Intelli Mixer without rack (Surlyn Bio, SLRM-3). Next, 200 쨉 l of exosome-separating buffer was added and the mixture was vigorously pipetted. Exosomes trapped in the magnetic beads were separated through a magnet in the same manner as described above, and suspended in 45 쨉 l of buffer for exosome separation 1 'exosome capture').

2) Antibody for detection of target surface protein bound with fluorescent substance and molecular beacon detection for target gene detection

21-MB-Cy5 (Example 2) was added to the mixture obtained from the above step 1) (prostate cancer cell (PC3) exosome and antibody-attached magnetic bead) Target miRNA detection ',' target protein detection ', and' miRNA-1 ', which are present on the surface of exoemia, and miR-21, which is a disease marker (biologic marker for tumor diagnosis) Reference).

Concretely, 5 μl of a 10 μM concentration of miR-21-MB-Cy5 (Example 1) was added to 45 μl of the mixture obtained from the above step 1 (exocomes isolated / concentrated from PC3 cells) Lt; / RTI > for 1 hour. ^{Next, CD63 Antibody, Alexa Fluor ® 488} (MEM-259, Thermo Co.) for 1 to ㎕ added and reacted for 1 hour under a light shield 37 ℃ condition.

The antibody conjugated with the fluorescent substance and the molecular beacon for detecting the target gene of Example 2 may be added at the same time but they are added sequentially in this experiment for precise reaction.

3) Fluorescence analysis

After the reaction, to remove CD63 Antibody, Alexa Fluor 488 or molecular beacon that did not bind to exosomes, 200 μl of exosome separation buffer was added, and after weak pipetting, the supernatant was removed using a magnet Were repeated two times. In order to detect exosomes having cancer marker (CD63, miR21) isolated by the multiple detection method of the present invention, Varioskan Flash Spectral Scanning Multimode Reader (Thermo Fisher Scientific Inc., Billerica, MA, USA) Fluorescence intensity was measured. Specifically, CD63 was measured at excitation: 650 nm, emmision: 670 nm, and miR21 was measured at excitation: 495 nm and emmision: 519 nm.

4) Multiple detection analysis of PC3 (prostate cancer cells)

FIG. 2 shows the result of analysis of the PC3 cell culture solution according to the multiple detection method (Condition 1: reaction at 4 ° C for 2 to 12 hours) of Experimental Example 1, FIG. 3 shows the results of the multiple detection method 2; 37 ° C, 4 hours reaction). At this time, a PBS buffer solution containing no exosomes was used as the control group '0'.

2 and 3, the process of isolating and concentrating exosomes in a cell culture with magnetic beads having an antibody attached thereto was performed at 4 ° C for 12 hours (condition 1) The value of fluorescence intensity was about 2 ~ 6 times higher than that of the control group. Thus, it can be seen that the presence of the cancer biomarker CD63 (surface protein) and miR21 (target gene) can be simultaneously confirmed from the prostate cancer cells through the multiple detection method according to the present invention. In particular, it was confirmed that it is preferable to carry out the magnetic bead with antibody for at least 12 hours at 4 ° C and more than 4 hours at 37 ° C.

In addition, when the content of the surface protein and the target gene for a specific disease in a sample measured by the multiple detection method of the present invention is analyzed by fluorescence intensity and compared with a normal control group, It can be understood that highly reliable information can be provided.

Experimental Example 2. Analysis of CD63 and miR-21 through multiple detection from various concentrations of exosome solution

It was confirmed in Experimental Example 1 that exosomes were firstly separated by magnetic beads with antibodies attached thereto, and that surface proteins and genes, which are biomarkers for specific diseases, could be detected. The sensitivity of the multiple detection method of the present invention according to the concentration of exosomes present in the sample was examined.

As in Experimental Example 1, exosomes (Condition 1 or Condition 2) were firstly separated with magnetic beads having an antibody attached thereto, and a buffer solution (PBS) was added to the separated exosomes to prepare 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , And diluted to a concentration of 10 ⁹ exosomes / μl to prepare a stepwise exosome solution.

2) the antibody for detection of a target surface protein bound with a fluorescent substance, the molecular beacon treatment for detecting a target gene, and 3) the fluorescence analysis were performed in the same manner as in Experimental Example 1 except that the various concentrations of exosome solutions were used , And the results of the analysis are shown in Figs.

4 shows the result of the multiple detection method of Experimental Example 2 (condition 1: 4 ° C, 12 hours reaction) from various concentrations of exosome solution, FIG. 5 shows the result of multiple detection (Condition 2, reaction at 37 ° C. for 4 hours). In this case, PBS buffer without exosome was used as a control group '0'.

4 and 5, it was confirmed that the multiple detection method according to the present invention had a high S / B value in proportion to the concentration of exosomes. It was confirmed that the multiple detection method of the present invention is capable of detection from exosomes at a concentration of 10 ^{6 to 7} cells / μl. That is, through the multiple detection method according to the present invention, the exosome was isolated from the cell culture medium, and the surface protein and miRNA for the specific disease were detected. The S / B value of the fluorescence intensity, As shown in Fig.

Experimental Example 3: Whether the multiple detection method of the present invention has specificity for exosomes

And to confirm whether the multiple detection method according to the present invention is specific to exosomes of a specific disease to be detected.

As in Experimental Example 1, the exosome (Condition 1) was firstly separated from the magnetic beads with antibody attached thereto, and a buffer solution (PBS) was added to the separated exosomes to prepare exosome solution prepared at a concentration of 10 ⁸ exosomes / 3) fluorescence analysis was carried out in the same manner as in Experimental Example 1, except that 2) an antibody for detecting a target surface protein bound with a fluorescent substance, a molecular beacon treatment for detecting a target gene, and 3) Respectively. This is illustrated by the capture Ab; O, Exosome; O.

In order to specifically and clearly confirm that the multiple detection method according to the present invention is specifically responsive to exosomes existing in the cells, the following comparative example was prepared and analyzed. First, in Comparative Example 1 (capture Ab; Fig. 1), which was carried out in the same manner as in Experimental Example 3, except that the PBS buffer was used in place of the exosomal solution in the step 1) of Example 1 and the magnetic particles not coated with the antibody were treated. X, Exosome; X), the capture Ab: O, Exosome; X using PBS buffer instead of the exosomal solution in the step 1) of Experimental Example 1, Comparative Example 3 (capture Ab; X, Exosome; O) treated with magnetic particles was prepared and fluorescence intensities were measured in the same manner as in Experimental Example 3, and S / B values were analyzed therefrom.

FIG. 6 is a graph of fluorescence intensity measured by the multiple detection method of Experimental Example 3 according to presence or absence of exosome or antibody-attached magnetic particles. FIG. At this time, PBS buffer (w / o exosome) containing no exosomes was used as a control PBS (w / o exosome).

As shown in FIG. 6, when no exosome was added or when magnetic particles having no antibody were used, it was confirmed that no signals were measured even when the multiple detection method of the present invention was used. On the other hand, when exosomes exist in the magnetic particles with antibody attached thereto, it was confirmed that the fluorescence intensity was detected by performing the multiple detection method according to the present invention. That is, the S / B value of the fluorescence intensity measured by the multiple detection method of the present invention is a signal of the surface protein and miRNA present in the exosome isolated from the cell culture solution, and is a nonspecific signal The signal is not due to the immune response. Since the multiple detection method according to the present invention detects only surface proteins and miRNAs that are specifically present in exosome of cells, it is more accurate and reliable than simply separating exosomes using antibody-attached magnetic particles (2) the time required for the detection of the target surface protein bound to the fluorescent substance and the molecular beacon treatment step for detecting the target gene is 1 to 2 hours), and since the experimental site is not affected by the apparatus, The possibility is very high.

Experimental Example 4. Analysis of CD63, EpCAM or EGFR surface proteins and miR-21 analysis by multiplex detection from prostate normal cells (RWPE-1) and prostate cancer cells (DU145, PC3)

The inventors of the present invention have made it clear that the multiple detection method according to the present invention can accurately compare normal cells with cells having a specific disease (cancer). The present inventors also analyzed whether the multiple detection method according to the present invention can detect various biomarkers of cancer (CD63, EpCAM or EGFR surface protein and miR-21 or miR574 target gene). At this time, prostate normal cells (RWPE-1) and prostate cancer cells (DU145, PC3) were prepared and cultured in the same manner as in Example 1.

A to a bit one exo obtained from the respective cells was added to buffer solution (PBS) 10 was made of some ⁸ exo / ㎕ concentration, fluorophore ^{(Alexa Fluor ® 488 (MEM-} 259, Thermo Co.)) binding antibody ( CD63 Antibody, EpCAM Antibody or EGFR Antibody) and a molecular beacon (Example 2), respectively.

FIG. 7 shows the result of performing the multiple detection method (CD63, miR-21) of Experimental Example 4 from the culture solution of normal prostate gland cells (RWPE-1), prostate cancer cells (DU145 and PC3) (EpCAM, miR-21 or EGFR, miR-21) of Experimental Example 4 from the culture medium of the prostate cancer cells (DU145 and PC3), and the control PBS (w / o exosome) Were used in PBS buffer without exosome.

 As shown in FIGS. 7 to 8, it was confirmed that the multiple detection method according to the present invention can accurately detect various cancer biomarkers existing in the exosome of a cell to provide information for discriminating normal cells and cancer cells . Specifically, when multiple detection is performed, the molecular beacons for miR-21 detection are fixed, and when the target surface protein detection antibody (CD63, EpCAM, EGFR) conjugated with a fluorescent substance is differently combined, fluorescence intensities Respectively.

Specifically, since CD63 is a surface protein existing in exosome rather than a biomarker of a specific disease, expression level of CD63 is expressed at a similar level without distinguishing normal cells and cancer cells. Through this, quantitative information of exosomes can be determined have. In addition, EpCAM (Epithelial cell adhesion molecule) and EGFR (epithelial growth factor receptor) are not observed in normal cells, but they are clearly expressed in cancer cells. Thus, cancer diseases or cancer cells can be diagnosed and identified.

That is, it is possible to provide disease information (EpCAM, EGFR) as well as exo sum quantitative information (CD63) of cells existing in the sample through the multiple detection method according to the present invention. That is, through the multiple detection method according to the present invention, it is possible to accurately and rapidly detect exosomes having a biomarker (exosome surface protein and miRNA) associated with a specific disease from a cell culture medium, , It can be understood that the specific disease can be used as a basis for diagnosis and discrimination in the corresponding sample.

Experimental Example 5. Analysis of CD63 and miR-21 or miR574-3p through multiple detection from normal prostate (RWPE-1) and prostate cancer cells (DU145, PC3)

The aim of the present study was to determine whether miRNAs can be accurately measured when targeting various miRNAs to normal cells and cells having a specific disease (cancer). In this experiment, prostate normal cells (RWPE-1) and prostate cancer cells (DU145, PC3) were prepared in the same manner as in Example 1, and each culture was used as a sample.

After preparing a little one exo obtained from each of the cells with a buffer solution (PBS) was added to 10-bit ^{to 8-exo} / ㎕ concentration, the fluorescent substance-binding antibody ^{(CD63 Antibody, Alexa Fluor ® 488} (MEM-259, Thermo Co. ) And a molecular beacon (Example 2 or Example 3) were used, respectively, and that the reaction time conditions were improved at 4 ° C for 12 hours at 37 ° C, And the multiple detection was performed in the same manner.

9 is a result of performing multiple detection (CD63, miR-21 or CD63, Mir-574-3p) of Experimental Example 5 from the culture medium of normal prostate gland cells (RWPE-1), prostate cancer cells (DU145 and PC3). At this time, PBS buffer (w / o exosome) containing no exosome was used as a control PBS (w / o exosome).

 As shown in FIG. 9, although the molecular beacon (Example 2 or Example 3) was different in the multiple detection method of the present invention, the measured fluorescence intensity S / B value (as compared with the control group) High). Specifically, when the target surface protein to be detected in the multiple detection method was fixed with CD63 and the target gene was changed to miR-21 (Example 2) or miR-574-3p (Example 3) High fluorescence intensity S / B values were measured in prostate cancer cells.

Thus, it was confirmed that exosomes spread from normal prostate cancer cells and prostate cancer cells can be quantified, and the expression level of disease specific biomarker (exosome surface protein, miRNA) can be clearly detected. When multiple detection methods of Experimental Examples 1 to 5 are applied to a biological sample or a cell culture solution, it is possible to simultaneously confirm whether or not cancer-related biomarkers (surface proteins and miRNAs) are present in these exosomes, (Fluorescence intensity or S / B value obtained from the fluorescence intensity) can be used as a basis for diagnosing whether or not cancer is caused by comparing with normal cells. Specifically, if cancer biomarker expression is higher than that of normal cells (fluorescence intensity is high), it can be diagnosed as cancer or diagnosed as cancer cells.

Experimental Example 6. Analysis of CD63 and miR-21 through multiple detection from biological sample (urine)

And to confirm whether the multiple detection method according to the present invention is applicable to a human-derived biological sample (urine).

15 ml of urine was obtained from normal human beings instead of the cell culture medium, and the mixture was concentrated to 500 μl using Amicon Ultra-15 Centrifugal Filter Units (Merck). The 1) step was carried out at 37 ° C for 0 to 4 hours Multiple detection was performed in the same manner as in Example 1 except that reaction time conditions were applied.

10 is a graph showing fluorescence intensities measured by performing multiple detection of Experimental Example 6 from urine of a normal person. At this time, PBS buffer (w / o exosome) containing no exosome was used as a control PBS (w / o exosome).

 As shown in FIG. 10, the presence of the surface protein (CD63) and the target gene (miR-21) in the exosome was detected from a normal human sample through the multiple detection method of the present invention. As a result, Respectively. That is, it can be seen that the multiple detection method of the present invention can be practically applied to a clinical sample (biological sample), and the surface protein and miRNA present in exosomes in a biological sample can be simultaneously detected and confirmed.

In order to diagnose cancer, miRNA 21 expression levels in normal subjects and patients can be compared with each other. Therefore, accurate measurement of miRNA expression level in normal subjects and patients should be preceded. Therefore, it has been confirmed in the present invention that miRNA21 is clearly detected in a normal human. Therefore, it can be seen from the present invention that the analysis of the disease-specific biomarker expression level present in the exosome segregation-exosome can be practically applied without any additional process have.

<110> INCHEON UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> A method for multiplexed detection of exosome miRNA and          cell-surface protein by Magnetic beads and molecular beacons <130> HPC7426 <160> 32 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-21-5p <400> 1 uagcuuauca gacugauguu ga 22 <210> 2 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-21-3p <400> 2 caacaccagu cgaugggcug u 21 <210> 3 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-574-5p <400> 3 ugagugugug ugugugagug ugu 23 <210> 4 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-574-3p <400> 4 cacgcucaug cacacaccca ca 22 <210> 5 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-205-5p <400> 5 uccuucauuc caccggaguc ug 22 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-205-3p <400> 6 gauuucagug gagugaaguu c 21 <210> 7 <211> 23 <212> RNA <213> Artificial Sequence <220> &Lt; 223 > Mature sequence hsa-miR-92a-1-5p <400> 7 agguugggau cgguugcaau gcu 23 <210> 8 <211> 22 <212> RNA <213> Artificial Sequence <220> &Lt; 223 > Mature sequence hsa-miR-92a-2-5p <400> 8 ggguggggau uuguugcauu ac 22 <210> 9 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-92b-5p <400> 9 agggacggga cgcggugcag ug 22 <210> 10 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-92b-3p <400> 10 uauugcacuc gucccggccu cc 22 <210> 11 <211> 20 <212> RNA <213> Artificial Sequence <220> Mature sequence hsa-miR-147a <400> 11 guguguggaa augcuucugc 20 <210> 12 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-147b <400> 12 gugugcggaa augcuucugc ua 22 <210> 13 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-141-5p <400> 13 caucuuccag uacaguguug ga 22 <210> 14 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence hsa-miR-141-3p <400> 14 uaacacuguc ugguaaagau gg 22 <210> 15 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence rno-miR-547-5p <400> 15 ucacuucagg auguaccacc ca 22 <210> 16 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> Mature sequence rno-miR-547-3p <400> 16 auugguacuu cuuuaaguga ga 22 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> miR-21 targeting MB: miR-21-5p sequence <400> 17 gcgcgtcaac atcagtctga taagctacgc gc 32 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> miR-21 targeting MB: miR-21-3p sequence <400> 18 gcgcgtacag cccatcgact ggtgttgacg cgc 33 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> miR-574 targeting MB <400> 19 gcgcgtacac actcacacac acacactcaa cgcgc 35 <210> 20 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-574 targeting MB <400> 20 gcgcgttgtg ggtgtgtgca tgagcgtgac gcgc 34 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-205 targeting MB: miR-205-5p sequence <400> 21 gcgcgtcaga ctccggtgga atgaaggaac gcgc 34 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> miR-205 targeting MB: miR-205-3p sequence <400> 22 gcgcgtgaac ttcactccac tgaaatcacg cgc 33 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> miR-92 targeting MB: miR-92a-1-5p sequence <400> 23 gcgcgtagca ttgcaaccga tcccaaccta cgcgc 35 <210> 24 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-92 targeting MB: miR-92a-2-5p sequence <400> 24 gcgcgtgtaa tgcaacaaat ccccacccac gcgc 34 <210> 25 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-92 targeting MB: miR-92b-5p sequence <400> 25 gcgcgtcact gcaccgcgtc ccgtccctac gcgc 34 <210> 26 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-92 targeting MB: miR-92b-3p sequence <400> 26 gcgcgtggag gccgggacga gtgcaataac gcgc 34 <210> 27 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> miR-147 targeting MB: miR-147a sequence <400> 27 gcgcgtgcag aagcatttcc acacacacgc gc 32 <210> 28 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-147 targeting MB: miR-147b sequence <400> 28 gcgcgttagc agaagcattt ccgcacacac gcgc 34 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-141 targeting MB: miR-141-5p sequence <400> 29 gcgcgttcca acactgtact ggaagatgac gcgc 34 <210> 30 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-141 targeting MB: miR-141-3p sequence <400> 30 gcgcgtccat ctttaccaga cagtgttaac gcgc 34 <210> 31 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-547 targeting MB: miR-547-5p sequence <400> 31 gcgcgttggg tggtacatcc tgaagtgaac gcgc 34 <210> 32 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> miR-547 targeting MB: miR-547-3p sequence <400> 32 gcgcgttctc acttaaagaa gtaccaatac gcgc 34

Claims (47)

1) simultaneously adding an antibody for detecting a target surface protein and a molecular beacon for detecting a target gene to which a magnetic bead and a fluorescent substance with antibodies attached thereto are simultaneously added to a tissue or a biological sample of the subject; And
2) measuring the fluorescence intensity from the mixed solution; and detecting the fluorescence intensity from the mixed solution by using the magnetic beads and the molecular beacon. The method according to claim 1,
Detecting the presence or absence of a surface protein and a target gene from the fluorescence intensity measured from the subject in the step 2). The method according to claim 1,
The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Wherein the method is any one selected from the group consisting of body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof. The method according to claim 1,
Wherein the magnetic bead to which the antibody is attached is attached with at least one antibody that specifically binds to the surface protein of the exosome. The method according to claim 1,
Wherein said antibody is an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81. The method according to claim 1,
Wherein the antibody for detecting a target surface protein is an antibody against any one antigen selected from CD63, EpCAM, EFGR, Survivin, and IGF-1R. The method according to claim 1,
Wherein the target gene is miRNA. 8. The method of claim 7,
Wherein the miRNA is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. The method according to claim 1,
Wherein the molecular beacon is labeled with a fluorophore and a quencher on both ends of a nucleic acid that specifically binds to the target gene. 9. The method of claim 8,
Wherein the miRNA is a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16. 1 ') treating the tissue or biological sample of the subject with a magnetic bead with the antibody attached thereto;
2 ') adding to the mixture of step 1') an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene; And
3 &apos;) measuring the fluorescence intensity from the mixed solution. 12. The method of claim 11,
Detecting the presence or absence of the surface protein and the target gene from the fluorescence intensity measured from the subject in the step 3 '). 12. The method of claim 11,
The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Wherein the method is any one selected from the group consisting of body fluid, ascites fluid, cystic tumor fluid, positive fluid, and combinations thereof. 11. The method of claim 10,
Wherein the magnetic bead to which the antibody is attached is attached with at least one antibody that specifically binds to the surface protein of the exosome. 11. The method of claim 10,
Wherein said antibody is an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81. 12. The method of claim 11,
Wherein the target surface protein detection antibody is an antibody against any one antigen selected from the group consisting of CD63, EpCAM, EFGR, Survivin and IGF-1R. 12. The method of claim 11,
Wherein the target gene is miRNA. 18. The method of claim 17,
Wherein the miRNA is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. 12. The method of claim 11,
Wherein the molecular beacon is labeled with a fluorophore and a quencher on both ends of a nucleic acid that specifically binds to the target gene. 19. The method of claim 18,
Wherein the miRNA is a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 16. (I) adding an antibody for detecting a target surface protein and a molecular beacon for detecting a target gene to which a magnetic bead and a fluorescent substance with an antibody are bound, to a tissue or a biological sample of the subject;
II) measuring fluorescence intensity from the mixed solution; And
III) comparing the fluorescence intensity in the subject measured in the step II) with the fluorescence intensity in the control subject, and providing information for diagnosing a specific disease from the subject. 20. The method of claim 19,
The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Wherein the method is any one selected from the group consisting of body fluids, ascites, cystic tumor body fluids, saline fluids, and combinations thereof. 20. The method of claim 19,
Wherein the magnetic bead to which the antibody is attached is attached with at least one antibody that specifically binds to the surface protein of the exosome. 22. The method of claim 21,
Wherein said antibody is an antibody against any one antigen selected from CD9, CD63 and CD81. 22. The method of claim 21,
Wherein the antibody for detecting a target surface protein is an antibody against any one antigen selected from CD63, EpCAM, EFGR, Survivin, and IGF-1R. 22. The method of claim 21,
Wherein the target gene is miRNA. 27. The method of claim 26,
Wherein the miRNA is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. 22. The method of claim 21,
Wherein the molecular beacon is labeled with a fluorophore and a quencher on both ends of a nucleic acid that specifically binds to the target gene. 28. The method of claim 27,
Wherein the miRNA is a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 16. 22. The method of claim 21,
The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary CNS lymphoma, spinal cord tumor, brain tumor, brain stem glioma, and pituitary adenoma. I &apos;) treating the tissues or biological samples of the subject with magnetic beads with antibodies attached thereto;
II ') adding an antibody for detecting a target surface protein bound with a fluorescent substance and a molecular beacon for detecting a target gene to the mixture of step I');
III ') measuring the fluorescence intensity from the mixed solution; And
Comparing the fluorescence intensity of the subject measured in step (IV ') with the fluorescence intensity of the subject in step (III'); and providing information for diagnosing a specific disease from the subject. 31. The method of claim 31,
The biological sample may be any one or more of urine, mucus, saliva, tears, blood, plasma, serum, sputum, spinal fluid, pleural effusion, nipple aspirate, lymphatic fluid, Wherein the method is any one selected from the group consisting of body fluids, ascites, cystic tumor body fluids, saline fluids, and combinations thereof. 29. The method of claim 28,
Wherein the magnetic bead to which the antibody is attached is attached with at least one antibody that specifically binds to the surface protein of the exosome. 32. The method of claim 31,
Wherein said antibody is an antibody against any one antigen selected from CD9, CD63 and CD81. 32. The method of claim 31,
Wherein the antibody for detecting a target surface protein is an antibody against any one antigen selected from CD63, EpCAM, EFGR, Survivin, and IGF-1R. 32. The method of claim 31,
Wherein the target gene is miRNA. 37. The method of claim 36,
Wherein the miRNA is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. 32. The method of claim 31,
Wherein the molecular beacon is labeled with a fluorophore and a quencher on both ends of a nucleic acid that specifically binds to the target gene. 39. The method of claim 37,
Wherein the miRNA is a base sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 16. 32. The method of claim 31,
The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary CNS lymphoma, spinal cord tumor, brain tumor, brain stem glioma, and pituitary adenoma. Magnetic beads coupled with antibodies; And
An antibody for detecting a target surface protein bound with a fluorescent substance; And
A molecular beacon;
The antibody is an antibody for detecting a target surface protein to which a fluorescent substance binding to a target surface protein present on the surface of the exosome is bound,
Wherein the molecular beacon comprises a nucleic acid complementary to a target gene present in the exosome, a fluorescent substance bound to the end of the nucleic acid, and a quencher coupled to the other end of the nucleic acid. For the kit. 42. The method of claim 41,
Wherein the magnetic beads to which the antibody is attached are attached with at least one antibody that specifically binds to the surface protein of the exosome. 42. The method of claim 41,
Wherein the antibody is an antibody against any one antigen selected from the group consisting of CD9, CD63 and CD81. 42. The method of claim 41,
Wherein the antibody for detecting a target surface protein is an antibody against any one antigen selected from CD63, EpCAM, EFGR, Survivin, and IGF-1R. 42. The method of claim 41,
Wherein the target gene is miRNA. 39. The method of claim 37,
Wherein the miRNA is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. 42. The method of claim 41,
The disease is selected from the group consisting of lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, Hodgkin's disease, esophageal cancer, small bowel cancer, lymphadenocarcinoma, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, pituitary cancer, uterine cancer, uterine cancer, uterine cancer, uterine cancer, vulvar carcinoma , Central nervous system (CNS) tumors, cancer of the central nervous system (CNS), cancer of the central nervous system (CNS), cancer of the central nervous system (CNS) Primary CNS lymphoma, spinal cord tumor, brain tumor, brain stem glioma, and pituitary adenoma.

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