KR102329816B1 - A Method for Separating and Concentrating Cells or Secretion thereof using Super Absorbent Beads - Google Patents

Abstract

The present invention relates to a method for effectively separating and concentrating cells or their secretions multiplied by an antibody and a nucleic acid molecule, and by using cells or their secretions by quickly and efficiently concentrating a desired cell or its secretion to amplify a fluorescence signal. It can be used for various studies, and in particular, it can be usefully used as a reliable diagnostic method for the disease by rapidly detecting cells or secretions thereof that are derived from various lesion tissues and cells and express a specific disease marker.

Description

A method for separating and concentrating cells or their secretions using a highly hygroscopic resin {A Method for Separating and Concentrating Cells or Secretion thereof using Super Absorbent Beads}

The present invention relates to a method for effectively separating and concentrating these cells or their secretions multi-detected with an antibody and a nucleic acid molecule in order to quickly and accurately detect a desired cell or a secretion thereof, and more particularly, using a superabsorbent resin to a method for separating and concentrating a detection target by removing unbound antibodies and nucleic acid molecules.

Devices for separating or concentrating nucleic acids, cells, or secretions thereof from blood, cells, or various biological samples are widely applied in various fields such as biology, biochemistry, molecular medicine, forensics, and diagnostic medicine. As the field of sample analysis and diagnosis has grown rapidly, the need for developing an analysis method for efficiently and rapidly separating, classifying, and quantifying cells containing a specific marker protein or gene or secretions isolated therefrom is increasing.

Extracellular vesicles are mostly small vesicles with a size between 50 and 1,000 nm secreted from cells, and are present in body fluids such as blood and urine. Contains genetic material (DNA, mRNA, miRNA), lipids, etc.

When isolating cells or their secretions from a sample, the most important thing is to increase the signal for the target gene or target surface protein and decrease the background signal to increase the signal to background (S/B) signal. have. However, cells or extracellular vesicles (especially exosomes) present in the sample are not only present in a small amount, but also have a very small size, so a sufficient amount cannot be obtained by a general separation method such as centrifugation. Even if a sufficient amount is obtained, the background signal becomes high due to the probe not bound to a biological material such as an extracellular vesicle during the detection process.

In order to remove the unreacted probe, methods such as ultrafiltration or ultracentrifugation have been used, but there are issues such as the use of consumable materials and expensive equipment, and in the concentration and washing process, a significant amount of extracellular Another problem existed, such as loss or contamination of the endoplasmic reticulum.

On the other hand, for accurate diagnosis of diseases from cells or secretions thereof, reliability can be increased by simultaneously detecting nucleic acids and surface proteins present therein. Conventionally, real-time PCR and ELISA (enzyme linked immunosorbent assay) have been used, respectively. Since simultaneous detection of nucleic acids and surface proteins is impossible, various processes and time are required, and the results vary depending on the skill level of the experimenter. There is a downside to falling.

Therefore, in the present invention, the present invention was completed by focusing on the ability to simultaneously detect a target gene and a surface protein from a cell secretion such as a cell or an extracellular vesicle by using a superabsorbent polymer.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Patent Literature 1. Korean Patent Publication No. 10-2012-0002943

The present inventors have made intensive research efforts to develop a method for isolating, concentrating, and detecting cells or their secretions from biological samples with high accuracy and reproducibility through antibodies and nucleic acid molecules. As a result, it is possible to recover cells including multiple biomarkers or their secretions with high purity and high quality, away from the conventionally used ultracentrifugation method or the isolation method by antibody binding, and through this, the amount of target cells or secretions thereof and By being able to more accurately detect the presence or absence, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a method for isolating and concentrating cells or secretions thereof from a biological sample.

Another object of the present invention is to provide a method for detecting a cell or a secretion thereof from a biological sample.

Another object of the present invention is to provide a kit for detecting cells or secretions thereof.

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: 1) an antibody that specifically binds to a target surface protein contained in a cell or a secretion thereof in a biological sample containing a biological material, and a target contained in the cell or secretion thereof adding a nucleic acid molecule that specifically binds to a gene or a combination thereof; and

2) separating and concentrating cells or secretions thereof by contacting the biological sample of 1) with a superabsorbent polymer;

The present inventors detect a specific antibody and nucleic acid molecule of a target cell or secretion thereof, amplify the fluorescence signal measured therefrom easily, quickly and efficiently, and reduce the background signal without a separate washing process, thereby providing high accuracy and reproducibility In order to develop a method for isolating, concentrating, and detecting a desired cell or its secretion from a biological sample, intensive research efforts were made. As a result, it is possible to recover cells containing multiple biomarkers or their secretions with high purity and high quality using a superabsorbent resin, away from the conventionally used ultra-high speed centrifugation method or the isolation method by antibody binding, and through this It has been found that the amount and presence of cells or secretions thereof can be more accurately detected.

In the present invention, 'isolation' refers to obtaining a target surface protein, a target gene, or cells or secretions thereof simultaneously containing the target surface protein, the target gene, from a biological sample by distinguishing them from impurities not containing the target surface protein and the target gene. At this time, as the antibody and/or nucleic acid molecule that is not bound to the target surface protein and/or the target gene is removed through the absorbent resin, the concentration and purity of the substance to be detected in the sample increase.

In the present invention, the term 'cell' is a circulating cell, circulating tumor cell, circulating endothelial cell, and circulating endothelial progenitor cell selected from the group consisting of It means any one or more, and may preferably be a circulating tumor cell.

In the present invention, circulating tumor cells (CTCs) are tumor cells circulating in the blood, and molecular characteristics analysis of circulating tumor cells is being used as a new diagnostic method for advanced cancer patients. Circulating tumor cells contain a very small amount in the blood, which made it difficult to detect them with the prior art. However, through the confined process of the present invention, rapid separation and concentration of circulating tumor cells containing specific surface proteins and genes is possible. do. Circulating tumor cells (CTCs) have a size of 10 to 25 μm.

In the present invention, the term 'cell secretion' refers to a series of substances that are separated from living cells and are responsible for certain biological and physiological actions in cells. For example, the cell secretion may be a protein, gene, nucleic acid, peptide, organelle, lipid, sugar or other substance to be isolated and concentrated in the present invention. The organelle may be a nucleus, mitochondria, chloroplast, endoplasmic reticulum, Golgi apparatus, secretory granule, extracellular endoplasmic reticulum, lysosome, phagosome, vacuole, or peroxisomal, preferably extracellular endoplasmic reticulum.

The extracellular ER is a small vesicle (membrane vesicle) with a size of 50 to 1,000 nm secreted from most cells, and is present in body fluids such as blood and urine, and various kinds of proteins derived from the cells inside the extracellular ER. , genetic material (DNA, mRNA, miRNA), and lipids.

First, 1) a biological sample containing a cell or a secretion thereof, an antibody that specifically binds to a target surface protein contained in the cell or secretion thereof, specifically binding to a target gene contained in the cell or secretion thereof A nucleic acid molecule or a combination thereof is added. It can also be referred to as a step in which a cell or a secretion thereof having a specific biomarker (surface protein and gene) binds to an antibody, a nucleic acid molecule, or a mixture thereof through step 1). In cells or secretions thereof that do not express the biomarker, the antibody and the nucleic acid molecule are not bound and remain in solution.

2) Contacting the biological sample of 1) with the superabsorbent resin to separate and concentrate the cells or their secretions. 1 and 2, when the superabsorbent polymer is brought into contact, unreacted substances (the antibody and the nucleic acid molecule, etc.) in the biological sample are absorbed together with water molecules, and the cells or secretions thereof captured by the antibody and the nucleic acid molecule are It is not absorbed into the superabsorbent polymer.

Accordingly, unreacted substances exhibiting a background signal are removed, and cells or secretions thereof associated with a specific disease are separated and concentrated in solution. Accordingly, it is possible not only to amplify the fluorescence intensity, but also to provide an accurate fluorescence signal by reducing the background signal, that is, noise.

According to a specific embodiment of the present invention, the cell or secretion thereof to be isolated and concentrated in the present invention may be a disease-related cell or secretion thereof derived from a lesion tissue and/or cell and expressing a specific disease marker, specifically blood It may be a circulating cell or an extracellular vesicle, and more preferably a circulating tumor cell (CTC) or an extracellular endoplasmic reticulum. When the cells or their secretions are circulating tumor cells (CTCs) or extracellular vesicles, highly reliable diagnosis of the disease through rapid separation, concentration and identification of cells or secretions containing disease-specific surface proteins and/or genes information can be obtained.

Conventionally, for accurate detection, expensive equipment and advanced methods have been devised to remove unreacted substances, but this incurs excessive costs, makes rapid processing impossible, and also causes a large amount of sample loss, which reduces signal accuracy. There was a problem with dropping.

However, the present invention can efficiently isolate and concentrate disease-related cells or secretions thereof only in a single process while overcoming the above-described problems and minimize sample loss, and unreacted substances (the antibody and the nucleic acid molecule, etc.) It is very useful because it can even be removed at once.

It is preferable that 100 μl of the biological sample of step 1) contains 15 to 100 mg of superabsorbent resin.

In the present invention, the biological sample is any one or more selected from the group consisting of whole blood, serum, plasma, saliva, urine, sputum, lymph and cells, or among the whole blood, serum, plasma, saliva, urine, sputum, lymph and cells. It may be a cell isolated and isolated from any one or a secretion thereof, but is not limited thereto.

In the present invention, the extracellular vesicles have various sizes, and in particular, the exosome with the smallest double size is a small cell membrane vesicle of 40 to 200 nm. Extracellular vesicles are secreted by cells of insects, plants, and microorganisms, as well as humans and animals. In particular, they contain specific molecules such as proteins, nucleic acids, lipids, and carbohydrates that cells have, and are surrounded by a lipid bilayer to stably protect these molecules and deliver them to other cells after secretion. Extracellular vesicles found in high concentrations in body fluids such as blood, urine, and saliva in vivo have potential as new biological materials due to these characteristics.

In the present invention, the extracellular vesicle is an endoplasmic reticulum produced in a cell and secreted out of the cell, and includes exosomes, microvesicles, microparticles, etc., and preferably has a size of 40 to 200 nm. It may be an exosome.

In the biological sample, normal cells or secretions thereof and disease-related live cells or secretions thereof are mixed. Since the method of the present invention provides the ability to isolate disease-related cells or secretions thereof having at least two disease-related biomarkers in a single process, directly separating and concentrating disease-related cells or secretions from cultured cells In order to do this, it is possible to omit procedures that require time, labor and cost as in the prior art.

In particular, the ability to separate the normal cells or secretions provided in the present invention and cells or secretions thereof containing two or more biomarkers for a specific disease can be obtained by confirming and detecting the presence of disease-related cells or secretions thereof, It is a very important ability in diagnosing a specific disease from a biological sample of In particular, in this regard, it was confirmed through an experiment that it was remarkably effective in isolating the extracellular vesicles simultaneously having tumor-related biomarkers (miR-21, CD63) from tumor cells. In addition, the method of the present invention is not limited to simply isolating disease-related cells or secretions thereof, and can be detected with a high signal versus background signal (S/B) value, so it can be said to be very useful in various fields.

The antibody that specifically binds to the target surface protein is preferably labeled with a fluorescent dye for ease of detection later.

The antibody is directed to any one antigen selected from the group consisting of CD9, CD63, CD81, EpCAM (Epithelial cell adhesion molecule), EGFR (Epidermal growth factor receptor), Survivin, and IGF-1R (Insulin-like growth factor 1 receptor). It may be an antibody to Specifically, EpCAM (Epithelial cell adhesion molecule) is a signaling molecule with tumor-promoting functions, and it is known that mRNA and protein are overexpressed in prostate cancer tissues and cell lines. It is known that overexpression causes tumors in the back, and it is also overexpressed in exosomes of prostate cancer cells (in vitro) and prostate cancer patients (in vivo). Survivin is an inhibitor-of-apoptosis (LAP) protein, and it has been reported that overexpression of survivin in prostate cancer tumors causes poor prognosis and tumor recurrence. IGF-1R (insulin-like growth factor 1 receptor) stimulates prostate epithelial cell proliferation and has been reported to play an important role in the development of prostate cancer. The expression level of the target surface proteins can predict cancer risk. is a biomarker.

The fluorescent dye is a material characterized in that when it is in an excitation state by photoexcitation at a molecular wavelength, the absorbed energy is emitted as light of a specific wavelength and returned to a ground level. .

The fluorescent dye is Alexa555 (Alexa555), Alexa647 (Alexa647), body P TMR (bodipy TMR), Cy3, DY500, rhodamine red-X (rhodamine Red-X), spectrum orange (spectrum orange), fluorescein isothiocyanate (fluorescein isothiocyanate, FITC), tetramethylrhodamine isothiocyanate (TRITC), alexa fluor, texas red, tetramethylrhodamine, Casca It may be any one or more selected from the group consisting of cascade blue, DAPI (4',6-diamidino-2-phenylindole), coumarin, lucifer yellow and dansylamide.

The fluorescent dye according to the present invention has excitation and emission wavelengths of 546 nm and 576 nm when Alexa545 is 555 nm and 580 nm, Bodipy TMR is 540 nm and 580 nm, and Cy3 is 550 nm and 570 nm, DY500 is 500 nm and 575 nm, rhodamine Red-X is 580 nm and 590 nm, and spectrum orange is 550 nm and 590 nm.

The nucleic acid molecule that specifically binds to the target gene contained in the cell or its secretion is not particularly limited as long as it can specifically recognize and bind the target gene.

In the present invention, a 'target gene' refers to a substance capable of diagnosing a specific disease from a subject, more preferably a nucleic acid biomarker, and most preferably a miRNA. Since the amount of RNA in the extracellular endoplasmic reticulum is higher than that of DNA, miRNA can be detected more efficiently than conventional DNA technology.

The nucleic acid molecule may be a linear DNA or RNA having a sequence capable of complementary binding to a specific miRNA while present in a cell or secretion thereof, and the RNA may be mRNA, miRNA, snoRNA, snRNA, rRNA, tRNA, siRNA, It may be any one selected from the group consisting of hnRNA and shRNA. Preferably, it may be a molecular beacon in which a fluorescent material and a quencher are labeled at both ends of the nucleic acid that specifically binds to the target gene.

The molecular beacon is a nucleic acid region that complementarily binds to a target gene, which is a biomarker of a specific disease, present in cells or secretions thereof, a fluorescent substance bound to the end of the nucleic acid, and the other end of the nucleic acid. Including a quencher (quencher).

The molecular beacon is composed of a loop portion, which is a portion capable of binding to a target gene, and a stem portion, which forms a hairpin ring in an environment in which the target gene does not exist. The loop portion may vary depending on a specific miRNA type and whether the entire miRNA sequence or a portion is targeted, and the stem portion may vary depending on the efficiency of each molecular beacon.

Specifically, the molecular beacon may have complementary 5' and 3' sequences at both ends of the nucleic acid region to form a hairpin ring in an environment where a specific miRNA does not exist. Specifically, the molecular beacon may have complementary 5' and 3' sequences to form a hairpin structure at both ends of the main chain polynucleotide. The main chain polynucleotide may be composed of a sequence complementary enough to selectively hybridize to a target gene.

In the present invention, "complementary" means having a degree of complementarity capable of selectively hybridizing to a target gene of the present invention under certain specific hybridization or annealing conditions. Therefore, the term "complementary" has a meaning different from perfectly complementary, and the nucleic acid region of the present invention is miR-21 (hsa-miR-21), miR-574 (hsamiR-574), miR- 205 (hsa-miR-205), miR-92 (hsa-miR-92), miR-147 (hsa-miR-147), miR-141 (hsa-miR-141) or miR-547 (hsa-miR- 547), it may have one or more mismatched nucleotide sequences as long as it is capable of selectively hybridizing to the nucleotide sequence.

The nucleic acid region of the molecular beacon refers to natural or modified monomers or linear oligomers of linkages, capable of specifically hybridizing to a target gene, and may be naturally occurring or artificially synthesized.

The fluorescent material of the molecular beacon is selected from the group consisting of a Cyanine-based fluorescent molecule, a Rodamine-based fluorescent molecule, an Alexa-based fluorescent molecule, a FITC (fluorescein isothiocyanate) fluorescent molecule, a FAM (5-carboxy fluorescein) fluorescent molecule, and a Texas Red fluorescent molecule. It may be any one or more, preferably fluorescein (Fluorecein), fluorescein isothiocyanate (fluorescein isothiocyanate), Biodipy-FL (Biodipy-FL), Alexa Fluor Green (Alexa Fluor Green), R-Pico Erythrine (R-phycoerythrin), Texas Red (TexasRed), Phycoerythrin-Texas Red, Rhodamine, Alexa, Cyanine (Cy3, Cy3.5, Cy5) , Phycoerythrin-cyanine 5 (Phycoerythrinyanine5), phycoerythrin-cyanine 7 (Phycoerythrin-cyanine7), Peridinin-chlorophyll protein, Allophycocyanin, Allophycocyanin-Cyanine 7 (Allophycocyanin-cyanine7), o-phthalaldehyde, fluorescamine, BODIPY, coumarin and derivatives thereof may be at least one selected from the group consisting of, more preferably Cy3.

In the present invention, for more accurate detection, the fluorescent material of the molecular beacon is preferably a material different from the fluorescent dye of the antibody.

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

In the molecular beacon, in an environment where a target gene (preferably a specific miRNA) does not exist, both ends of the nucleic acid region of the molecular beacon interact closely with each other to form a bond, so that the nucleic acid is formed into a hairpin structure. . Due to this, since the fluorescent material and the quencher bound to both ends of the nucleic acid are close to each other so that FRET occurs, the molecular beacon does not normally emit fluorescence.

However, if there is a cell or a secretion thereof in which the target gene is present among the cells or secretions thereof, the molecular beacon and the target gene are hybridized, and the hairpin-type secondary structure of the molecular beacon is disrupted, and the structure is opened linearly As a result, the distance between the fluorescent material and the quencher bound to the end of the nucleic acid region of the molecular beacon is also increased. Therefore, as the FRET effect of the quencher, which had an effect on the fluorescent material, disappears, fluorescence is observed from the molecular beacon.

In the present invention, a super absorbent polymer (SAP) is a three-dimensional network structure in which polymer chains are crosslinked to control moisture content and physical properties, and has the ability to absorb moisture up to 300 times its own weight. It is a synthetic polymer with Each developer has different names such as SAM (Super Absorbency Material) and AGM (Absorbent Gel Material). The superabsorbent polymer as described above began to be put to practical use as a sanitary tool, and now, in addition to hygiene products such as paper diapers for children, a soil repair agent for gardening, a water-retaining material for civil engineering and construction, a sheet for seedlings, a freshness maintenance agent in the food distribution field, and It is widely used as a material for poultice, etc.

One of the most widely used superabsorbent polymers is polyacrylate, that is, a salt of polyacrylic acid. For example, a sodium salt of polyacrylic acid (crosslinked sodium polyacrylate) can be prepared by polymerizing acrylic acid mixed with sodium hydroxide in the presence of an initiator.

Other superabsorbent polymers include polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinylalcohol copolymer , may be any one or more selected from the group consisting of cross-linked polyethylene oxide (cross-linked polyethylene oxide) and a starch-grafted copolymer of polyacrylonitrile (polyacrylonitrile).

Another kind of super absorbent polymer that can be used in the present invention is alginate, that is, a salt of alginic acid. This material naturally exists as a viscous gum rich in the cell walls of brown algae, and commercial forms are derived from seaweed. Alginic acid is associated with (1-4)-linked β-D-mannuronate and its C-5 epimer α-L covalently linked together in different configurations and blocks. It is a linear copolymer with homopolymer blocks of -guluronate residues (C-5 epimer α-L-guluronate residues).

In the present invention, the superabsorbent polymer is not particularly limited as long as it is a commonly used superabsorbent polymer as described above, but may preferably have a water channel size of 1 to 5 nm average diameter, and the present invention In the example of , superabsorbent polymer beads with a diameter of 2 nm purchased from Jun Science (3006417364) were used as the superabsorbent polymer.

Cells or secretions thereof isolated and concentrated through the above method may be cells or secretions thereof having a disease-related biomarker, and the disease is a disease detectable through a biomarker present in the cells or secretions thereof, but is not particularly limited thereto. , specifically, a tumor (cancer).

The tumor is breast cancer, liver cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, gallbladder cancer cancer, head and neck cancer, kidney cancer, lung cancer (small or non-small cell), melanoma, ovarian cancer cancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penile cancer, skin cancer, soft-tissue sarcoma, Any one or more days selected from the group consisting of squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, bile duct cancer, and uterine cancer can

The cell secretion may refer to any substance that acts on a cell, separated from the cell, and is preferably an extracellular ER, and the extracellular ER may have a disease-related biomarker, specifically tumor-derived. It may be extracellular vesicles or tumor-derived exosomes. In order to analyze this, at least one or more, preferably at least two or more, of a wide range of biomarkers as described in the literature may be present.

In the present invention, the tumor-derived exosomes are breast cancer, liver cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer ( esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small or non-small cell), melanoma ( melanoma, ovarian cancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penile cancer, skin cancer, soft tissue sarcoma (soft-tissue sarcoma), squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, bile duct cancer, and uterine cancer It may be derived from any one or more selected from the group, but is not limited thereto.

According to another aspect of the present invention, a) in a biological sample comprising a cell or a secretion thereof, an antibody that specifically binds to a target surface protein included in the cell or secretion thereof, and a target gene included in the cell or secretion thereof adding a nucleic acid molecule that specifically binds to

b) separating and concentrating cells or secretions thereof by contacting the biological sample of step a) with a superabsorbent polymer; and

c) measuring the fluorescence intensity of the isolated and concentrated solution recovered from step b); provides a method for detecting cells or secretions thereof from a biological sample comprising a.

Steps a) and b) are described in detail in the 'Method for isolating and Concentrating Cells or Secretions from Biological Samples', so they are referred to and repeated content is omitted.

Finally, in step c), the presence or quantitative concentration of cells or secretions thereof from the biological sample was analyzed by measuring the fluorescence intensity of the isolated and concentrated solution recovered from step b).

By comparing the fluorescence intensity obtained from the biological sample with the fluorescence intensity of a control, information for diagnosing a specific disease may be provided.

For example, if there are cells or secretions thereof present in the biological sample, for example, disease-related cells or secretions thereof, including blood circulation cells, endoplasmic reticulum containing biomarkers of a specific disease, the nucleic acid molecule and the antibody are bound Capture disease-related cells or secretions thereof. Thereafter, through contact with the superabsorbent polymer, unreacted substances (antibodies or nucleic acid molecules) indicating a background signal are removed together with water molecules, and disease-related cells or secretions thereof to which the nucleic acid molecules and antibodies are bound remain at a high concentration. .

Therefore, when the fluorescence intensity is measured, fluorescence is emitted from the nucleic acid molecule and the antibody, and by measuring the fluorescence intensity, it is possible to detect whether 'cells having both the surface protein and the target gene or a secretion thereof' are present. Through this, it can be used for diagnosis of specific diseases.

According to another aspect of the present invention, the present invention provides an antibody that specifically binds to a target surface protein contained in the cell or its secretion, and a nucleic acid molecule that specifically binds to a target gene contained in the cell or its secretion. Phase 1 comprising; and a second phase comprising a superabsorbent resin; to provide a kit for separation and concentration of cells or secretions thereof, including.

An antibody that specifically binds to a target surface protein contained in the cell or its secretion, a nucleic acid molecule that specifically binds to a target gene contained in the cell or its secretion, and a superabsorbent resin are 'disease-related cells or Since it is described in detail in 'Method for Separating and Concentrating its Secretion', it is referred to and repeated content is omitted.

Preferably, the first phase and the second phase are individually packaged in separate containers. The first phase may be administered to capture the disease-related cells or secretions thereof, and the second phase may be administered to separate and concentrate the disease-related cells or secretions thereof. By measuring the fluorescence intensity of the concentrated solution through the kit, the presence or absence and quantitative concentration of disease-related cells or secretions thereof can be confirmed. In addition, information for diagnosing a disease can be provided by comparing the fluorescence intensity with a control.

According to the present invention, cells or secretions captured by the antibody and nucleic acid molecules are not absorbed by the superabsorbent resin, and unreacted substances and water molecules are removed through the superabsorbent polymer, so that the background signal is significantly reduced when measuring the fluorescence intensity and As it was confirmed that the signal was increased, it can be seen that cells having multiple biomarkers or secretions thereof from a biological sample can be measured more accurately. As a result, even a very small amount of cells or secretions thereof can be measured, problems of the prior art can be solved, and cells having multiple biomarkers or secretions thereof can be accurately measured to make a precise diagnosis of a specific disease.

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

(i) The present invention can reduce the background signal and amplify the fluorescence signal without a washing process by using a superabsorbent polymer (SAP) in the separation and concentration of a desired cell or secretion thereof.

(ii) The method of the present invention can be utilized for various studies using disease-related cells or secretions thereof by quickly and efficiently concentrating cells or secretions thereof to amplify fluorescence signals easily and efficiently.

(iii) The present method is a technology that can dramatically reduce the operation time, process, labor, and cost required for detection, particularly by rapidly detecting cells expressing specific disease markers or secretions thereof derived from various lesion tissues and cells. It can be very usefully applied to high-efficiency analysis for disease diagnosis, prognosis, and treatment response.

1 is a schematic diagram schematically illustrating the principle of a method for isolating and concentrating disease-related extracellular vesicles from a biological sample using a superabsorbent polymer. It can be seen that unreacted substances other than disease-related extracellular vesicles can be removed from a biological sample by a single process.
2 is a schematic diagram briefly showing the principle of a method for amplifying and detecting disease-related extracellular vesicles from a biological sample using a superabsorbent resin. As the above-mentioned unreacted substances are removed, it can be seen that the disease-related extracellular vesicle detection signal is amplified.
3 is a comparison of the size of the superabsorbent polymer increased when the initial superabsorbent polymer and PBS buffer were contacted.
4 is a graph showing the change in volume of the PBBS buffer according to the contact time when the superabsorbent polymer is in contact with the PBS buffer.
5 is a graph showing the change in volume of the PBBS buffer according to the contact time when in contact with the superabsorbent polymer of different capacities and the PBS buffer.
6 shows that the superabsorbent polymer was added to the first group (PBS), the second group (exosome), the third group (MB21), and the fourth group (exosome MB21), respectively (W/SAP) and the superabsorbent polymer was not added. It is a graph showing the fluorescence signal (signal) of Cy3 measured from a sample obtained from an untreated (W/O SAP). The fluorescent signal is due to the fluorescent substance (Cy3) of the molecular beacon, which indicates the presence of a nucleic acid biomarker (miR-21) in the extracellular endoplasmic reticulum.
7 shows that the superabsorbent polymer was added to the first group (PBS), the second group (exosome), the third group (MB21) and the fourth group (exosome MB21), respectively (W/ SAP) and the superabsorbent polymer was not added. It is a graph showing the fluorescence signal (signal) of Alexa647 measured from a sample obtained from a non-W/O SAP. The fluorescence signal is due to the fluorescent substance of CD63-Alexa647 (Alexa647), which indicates the presence of the CD63 surface protein in the extracellular endoplasmic reticulum.
8 is a miR-21 signal to background (S/B) for the sample obtained when the superabsorbent polymer was added to group 4 (exosome MB21) (W/SAP) and when the superabsorbent polymer was not added (W/O SAP). ) is a graph showing each calculated value. In this case, the fluorescence intensity measured in the sample of the third group (MB21) was used as the background signal.
9 is a CD63 signal to background (S/B) value for the sample obtained when the superabsorbent polymer was added to group 4 (exosome MB21) (W/SAP) and when the superabsorbent polymer was not added (W/O SAP) It is a graph showing the calculation of each. In this case, the fluorescence intensity measured in the sample of the third group (Alexa647) was used as the background signal.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Check the absorption rate of super absorbent polymer

The purpose of this study was to determine the absorption rate of a superabsorbent polymer (Jun Science 3006417364) having a particle size of 2 mm in the form of beads. First, superabsorbent polymers of different capacities were put in PBS (phosphate buffered saline) buffer, and absorption rates were calculated according to the contact time and the capacity of the superabsorbent polymer.

Specifically, 500 μl of PBS buffer was placed in a Micro Centrifuge Tube (SPL), one superabsorbent resin (19.2 mg) was added thereto, and then left for 80 minutes (contact time) ( FIGS. 3 and 4 ). The amount of the remaining solution was measured every 10 minutes from the start of the reaction using a pipette. The number of superabsorbent polymers (5, 10, 12) was varied and contacted with 500 μl of PBS buffer for 30 minutes, and the final remaining PBS buffer volume was measured (FIG. 5).

3 and 4, it was confirmed that as the contact time increased, the volume of the PBS buffer significantly decreased and the volume of the superabsorbent polymer increased.

As shown in FIG. 5 , it was confirmed that as the amount (number) of the superabsorbent polymer increased, the absorption rate increased significantly. Considering the detection time, 100 μl of the sample preferably contains 19.2 to 96 mg (1 to 5) of superabsorbent polymer, and most preferably 38.4 mg (2).

Isolation, Concentration and Detection of Disease-Related Extracellular Vesicles from HeLa Cells

1) To isolate, concentrate and detect cancer disease extracellular vesicles. To this end, to isolate extracellular vesicles from HeLa cells, an Exo-Quick exosome isolation kit (Invitrogen) was first used. HeLa cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Biowest, France) containing 10% FBS (fetal bovine serum, Biowest, France) and penicillin/streptomycin (Life Technologes, USA). The cells were maintained at 37° C. and 5% CO _{2 conditions in a 100 mm dish.} Put 10 ml of the cell culture solution in a 50 ml FALCON tube (SPL) and centrifuge (Centrifuge, Hanil Science Industry, Combi-514R) at 3000 G force for 15 minutes to remove cell debris. 10 ml of the supernatant of the cell lysate was collected, mixed with 2 ml of Exo-Quick solution (5 : 1 ratio), and then incubated at 4° C. for 16 hours. After centrifuging the supernatant of the cell lysate and the Exo-Quick mixture at 1500 G force for 30 minutes, the supernatant was removed and the pellet remaining at the bottom was recovered. 33 μl of nuclease free phosphate buffered saline (NFPBS) was added to the pellet and dissolved by pipetting. In the isolated extracellular ER, cancer disease extracellular ER and non-extracellular ER are mixed. From this, in order to diagnose a cancer disease, it is necessary to detect an extracellular ER (also called a disease-associated extracellular ER or target extracellular ER) having a plurality of biomarkers (CD63, miR-21) associated with cancer disease. In the example of the present invention, the following molecular beacons and fluorescently-labeled antibodies were used to detect multiple biomarkers.

2) Cy3 fluorescent material-labeled molecular beacon (IDT, 5'-Cy3-GCG CGT CAA CAT CAG TCT GAT AAG CTA CGC GC-BHQ2-3') and Alexa647 fluorescent material-labeled CD63 ( BD Bioscience) (hereinafter also referred to as 'CD63-Alexa647') was prepared. Then, the molecular beacon and CD63-Alexa647 were added to the extracellular vesicles obtained in step 1) according to the following reaction conditions. After putting each of the following four experimental groups in a Micro Centrifuge Tube, they were reacted at 37° C. for 1 hour, and 50 μl of each was recovered and fluorescence was measured.

Group 1 (PBS): only 500 μl of PBS solution was added

Group 2 (exosome): 500 μl of a solution in which the extracellular vesicles were mixed at a concentration ^{of 2 x 10 7 par/μl in PBS buffer was added.}

Group 3 (MB21, Alexa647): 1 μl of CD63-Alexa647 was added to 500 μl of PBS buffer in which 100 nM molecular beacon was dissolved

Group 4 (exosome, MB21, Alexa647): ^{Prepare 500 μl of a mixture of 2 x 10 7} par/μl concentration of extracellular vesicles and 100 nM molecular beacon in PBS buffer, and 1 μl of CD63-Alexa647 prepared by mixing

3) After dividing the four experimental groups, one was added with a super absorbent polymer (SAP bead) (W/ SAP), and the other was not added with SAP bead (W/O SAP), 30 minutes at 4°C The volume and fluorescence intensity of the final solution for a total of 8 samples reacted during the reaction were measured. At this time, 10 superabsorbent polymers (SAP beads) were added to 500 μl.

Analysis of the degree of fluorescence signal amplification according to the presence or absence of superabsorbent polymer

The first group (PBS), the second group (exosome), the third group (MB21, Alexa647) and the fourth group (exosome, MB21, Alexa647) were separated and concentrated samples by adding superabsorbent polymer (W/ SAP), respectively. ), it was confirmed that only about 50 μl of the final solution volume remained. In the sample without superabsorbent polymer (W/O SAP), 500 μl was present as it was.

Samples recovered from each group were placed in each well of a 384-well plate (corning), and the fluorescence intensity of each fluorescent substance was measured using varioskan flash (Thermo) ( FIGS. 6 and 7 ).

As shown in FIGS. 6 and 7 , the sample (W/SAP) separated and concentrated with the superabsorbent polymer significantly increased signal strength by 2 to 3 times or more compared to the sample (W/O SAP) that was not separated and concentrated with the superabsorbent polymer (W/O SAP). It can be seen that the strong

Signal to background (S/B) analysis according to the presence or absence of superabsorbent polymer

In the fourth group (exosome, MB21, Alexa647), samples separated and concentrated by adding superabsorbent resin (W/SAP) and samples without superabsorbent polymer (W/O SAP) were recovered, and The fluorescence intensity was measured. Thereafter, in order to obtain a signal to background (S/B) signal, a fluorescence signal measured from each sample was used as a signal. At this time, the fluorescence signal of the third group containing only the fluorescent substances (Cy3 and Alexa647) was used as a background signal, and the signal to background (S/B) was calculated by dividing the signal by the background signal ( FIGS. 8 and 9 ). .

As shown in FIGS. 8 and 9 , it was confirmed that the S/B of the sample (W/ SAP) in which the disease-related extracellular vesicles were isolated and concentrated with the superabsorbent resin was significantly increased. Through this, the increase in the signal is higher than the increase in the background signal, which means that unreacted substances representing the background signal are removed through the superabsorbent resin, and disease-related extracellular vesicles are isolated and purified. It can be seen that this is because

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

1) a combination of an antibody that specifically binds to a target surface protein contained in the extracellular ER and a nucleic acid molecule that specifically binds to a target gene contained in the extracellular ER in a biological sample containing the extracellular ER adding; and
2) contacting the biological sample of step 1) with a superabsorbent resin to remove antibodies and nucleic acid molecules that are not bound to the extracellular vesicles, thereby separating and concentrating the extracellular vesicles;
The superabsorbent polymer (SAP) is polyacrylate, polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, From the group consisting of polyvinylalcohol copolymer, cross-linked polyethylene oxide, starch-grafted copolymer of polyacrylonitrile and alginate Characterized by any one selected,
The size of the passageway of the superabsorbent polymer is characterized in that it has an average diameter of 1 to 5 nm,
A method for isolating and enriching extracellular vesicles from a biological sample.
delete delete delete According to claim 1,
The method, characterized in that the extracellular ER is a tumor-derived extracellular ER or a tumor-derived exosome. According to claim 1,
The antibody for detecting the target surface protein is selected from the group consisting of CD9, CD63, CD81, EpCAM (Epithelial cell adhesion molecule), EGFR (Epidermal growth factor receptor), Survivin and IGF-1R (Insulin-like growth factor 1 receptor) A method, characterized in that it is an antibody to any one antigen. According to claim 1,
The nucleic acid molecule that specifically binds to the target gene is a molecular beacon. 8. The method of claim 7,
The molecular beacon is a method, characterized in that the fluorescent material and quencher are labeled at both ends of the nucleic acid specifically binding to the target gene, respectively. 9. The method of claim 8,
The target gene is any one selected from the group consisting of miR-21, miR-574, miR-205, miR-92, miR-147, miR-141 and miR-547. 6. The method of claim 5,
The tumor-derived extracellular vesicles or tumor-derived exosomes are breast cancer, liver cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer ), esophageal cancer, gallbladder cancer, head and neck cancer, kidney cancer, lung cancer (small or non-small cell) , melanoma, ovarian cancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penile cancer, skin cancer ), soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, bile duct cancer and uterine cancer Cancer), a method characterized in that it is derived from any one or more selected from the group consisting of. a) a combination of an antibody that specifically binds to a target surface protein contained in the extracellular ER, and a nucleic acid molecule that specifically binds to a target gene contained in the extracellular ER, in a biological sample containing the extracellular ER adding a;
b) polyacrylate, polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinyl in the biological sample Alcohol copolymer (polyvinylalcohol copolymer), cross-linked polyethylene oxide (cross-linked polyethylene oxide), polyacrylonitrile (starch-grafted copolymer) of polyacrylonitrile (starch-grafted copolymer) and alginate (alginate) selected from the group consisting of contacting any one of the superabsorbent resins to remove antibodies and nucleic acid molecules not bound to the extracellular vesicles, thereby separating and concentrating the extracellular vesicles; and
In the method of detecting extracellular vesicles from a biological sample comprising; c) measuring the fluorescence intensity of the separated and concentrated solution,
The method of detecting extracellular vesicles, characterized in that the size of the passageway of the superabsorbent polymer has an average diameter of 1 to 5 nm.
a first phase comprising a combination of an antibody that specifically binds to a target surface protein contained in the extracellular endoplasmic reticulum, and a nucleic acid molecule that specifically binds to a target gene; and
Polyacrylate, polyacrylamide copolymer, ethylene maleic anhydride copolymer, cross-linked carboxymethylcellulose, polyvinylalcohol copolymer ), cross-linked polyethylene oxide, a starch-grafted copolymer of polyacrylonitrile, and alginate any one superabsorbent polymer selected from the group consisting of As a kit for isolation and enrichment of extracellular vesicles comprising a second phase comprising (SAP),
The size of the passageway of the superabsorbent polymer is characterized in that it has an average diameter of 1 to 5 nm,
Kits for isolation and enrichment of extracellular vesicles.

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