WO2019066501A1 - Analysis method for extracellular vesicles, using size exclusion chromatography, and use for same - Google Patents

Analysis method for extracellular vesicles, using size exclusion chromatography, and use for same Download PDF

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WO2019066501A1
WO2019066501A1 PCT/KR2018/011443 KR2018011443W WO2019066501A1 WO 2019066501 A1 WO2019066501 A1 WO 2019066501A1 KR 2018011443 W KR2018011443 W KR 2018011443W WO 2019066501 A1 WO2019066501 A1 WO 2019066501A1
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endoplasmic reticulum
extracellular endoplasmic
probe
analysis
extracellular
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PCT/KR2018/011443
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French (fr)
Korean (ko)
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고용송
이창진
박현택
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㈜로제타엑소좀
포항공과대학교 산학협력단
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Priority to EP18860890.5A priority Critical patent/EP3690434A4/en
Priority to CN201880075753.5A priority patent/CN111386458B/en
Priority to JP2020517365A priority patent/JP7298929B2/en
Priority to US16/651,940 priority patent/US11835502B2/en
Priority claimed from KR1020180115206A external-priority patent/KR102243415B1/en
Publication of WO2019066501A1 publication Critical patent/WO2019066501A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a method of analyzing an extracellular endoplasmic reticulum using size exclusion chromatography and a use thereof, more particularly, to a method of analyzing extracellular endoplasmic reticulum using size exclusion chromatography, And to a method for analyzing the extracellular endoplasmic reticulum contained in a sample.
  • Extracellular vesicles are a universal mechanism of cells and are nano-sized biological particles secreted from various types of cells in vivo or in vitro, and exist in body fluids such as blood, urine, saliva, tears, etc., Lipid bilayers, and membrane-structured vesicles of various sizes ranging from 20 to 1,000 nm.
  • extracellular endoplasmic reticulum are involved in many important functions in various life phenomena.
  • the extracellular endoplasmic reticulum derived from eukaryotic cells is involved in erythrocyte differentiation and regulation of immune response.
  • many functions relating to cancer progression, metastasis and angiogenesis are revealed in the cancer cell microenvironment, And has received high interest in the utilization of
  • the extracellular endoplasmic reticulum secreted from prokaryotic cells contains prokaryotic components similar to extracellular endoplasmic reticulum.
  • acute pulmonary inflammatory diseases are induced according to the pathway including systemic inflammation.
  • chronic inflammation of the local skin tissues can be induced to cause atopic dermatitis, one of the representative diseases of modern people.
  • extracellular endoplasmic reticulum from the prokaryotes has also been attracting much attention as it has been reported that the extracellular endoplasmic reticulum from bacterial cells is associated with various diseases including cancer.
  • the extracellular endoplasmic reticulum is composed of substances derived from parent cells such as proteins, lipids, nucleic acids and amino acids. In vivo, the endoplasmic reticulum plays a role of a carrier to transfer these substances. Therefore, proteins, lipids, amino acids and nucleic acids constituting the extracellular endoplasmic reticulum Analysis is an important basis for understanding the physiological and pathological characteristics of the parent cells. Therefore, the analysis of constituents of the extracellular endoplasmic reticulum present in various samples has received high interest in the basic and medical fields.
  • nucleic acid, growth hormone, and protein contained in the extracellular endoplasmic reticulum are protected by a phospholipid in the form of a cell membrane, so that they can perform more stable functions than a soluble form of growth factor and cytokine. And it is expected to be used for various purposes including the diagnosis and treatment of diseases by analyzing the substances contained in the extracellular endoplasmic reticulum.
  • the analysis of the constituents of the extracellular endoplasmic reticulum is based on purification through a complicated and inefficient ultracentrifugation technique
  • this ultracentrifugation method since the separation yield of the extracellular endoplasmic reticulum is low and the probe which is not bound to the extracellular endoplasmic reticulum is not efficiently removed, quantitative analysis of the body fluid exhibiting a relatively limited amount and high complexity It is almost impossible to do. Therefore, it is urgent to develop a new technique that is different from the conventional extracellular ER analysis method.
  • A mixing and reacting a sample containing a probe and an extracellular endoplasmic reticulum containing a binding moiety and a detectable signal moiety that specifically bind to a component of an extracellular endoplasmic reticulum, and (b) Injecting a sample into a size exclusion chromatography column and developing the sample; and (c) detecting an extracellular endoplasmic reticulum-probe complex and a free probe from the developed sample.
  • Another object of the present invention is to provide a method for preparing a recombinant vector comprising the steps of: (a) mixing and reacting a sample containing a probe and an extracellular endoplasmic reticulum containing a binding moiety and a detectable signal moiety, (C) separating the extracellular endoplasmic reticulum-probe complex from the size exclusion chromatography column, and (d) exposing the extracellular endoplasmic reticulum-probe complex to an extracellular endoplasmic reticulum complex And detecting the probe.
  • the present invention also provides a method for analyzing an extracellular endoplasmic reticulum.
  • the present invention has been made to solve the above problems, and it is an object of the present invention to provide an extracellular endoplasmic reticulum-probe complex by reacting with a sample using various substances specifically binding to components of the extracellular endoplasmic reticulum as a probe, And then analyzing the extracellular endoplasmic reticulum by detecting the probe.
  • SEC size exclusion chromatography
  • a porous stationary phase such as a gel, a matrix, or a bead
  • large molecules that can not pass through the hole of the column can not enter the hole,
  • the small molecules exit the column quickly, while the small molecules move relatively slowly through the hole in the column to exit the column.
  • This method is generally used for desalting for buffer exchange, separation for purification, or molecular weight measurement according to solute size.
  • an extracellular endoplasmic reticulum-probe complex is formed by reacting a probe that specifically binds to various samples including extracellular endoplasmic reticulum, and the endoplasmic reticulum-probe complex is passed through a size exclusion chromatography column so that the complex and the free probe can be rapidly and easily fractionated And analysis of the extracellular endoplasmic reticulum can be performed easily and efficiently by analyzing the separated eluate in the next step.
  • extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G.
  • microvesicles microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, , Membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, membrane blebs, epididymosomes, promininosome, texosome, or archeosome, but not limited thereto. It is not.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of mixing and reacting a sample containing a probe including a detectable signal portion and a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and an extracellular endoplasmic reticulum [ .
  • probe refers to a substance which specifically binds to components constituting the extracellular endoplasmic reticulum and which can be detected and analyzed using spectroscopic, physicochemical, quantum chemical, it means.
  • the extracellular endoplasmic reticulum is surrounded by a double lipid membrane and is composed of substances derived from parent cells such as proteins, lipids, nucleic acids and amino acids. Depending on the constituents, they may be coated on the membrane surface of the extracellular endoplasmic reticulum, the membrane of the extracellular endoplasmic reticulum, .
  • the binding portion of the probe specifically binds to at least one component selected from the group consisting of a membrane surface component of the extracellular endoplasmic reticulum, a membrane component of the extracellular endoplasmic reticulum and an inner component of the extracellular endoplasmic reticulum .
  • the probe of the present invention may be a protein, an antibody, an antibody-derived substance, a peptide, a nucleic acid, a nucleic acid-amino acid complex, an enzyme, an enzyme substrate, a chemical ligand, Compounds, but is not limited thereto.
  • the probe may be a substance that specifically binds to the extracellular endoplasmic reticulum component and is detectable at the detection step.
  • VPD450 or CFDA-SE which is one of substrates of esterase, an internal component of extracellular ER, was used as a probe.
  • the substrate specifically binds to an esterase in the extracellular matrix and can be converted into a fluorescent substance by an enzyme activity, so that fluorescent signals can be detected without a separate label.
  • the probe of the present invention may further comprise a detectable signal moiety in a substance that specifically binds to one or more of the components constituting the extracellular endoplasmic reticulum.
  • the signal portion of the probe may be selected from the group consisting of a fluorescent substance, an enzyme substrate, an enzyme, a protein, a peptide, a nucleic acid, a biotin, a metal and a radioisotope, but is not limited thereto.
  • a probe having a fluorescent label attached to an antibody recognizing the membrane surface protein of the extracellular endoplasmic reticulum was used.
  • sample includes a biological sample containing cell extracellular medium, a cell culture fluid, a tissue sample, and the like, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, Soil, and fermented food.
  • mammalian cell culture medium bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, Soil, and fermented food.
  • CSF yeast culture medium
  • CSF cerebrospinal fluid
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (b) of injecting the mixed sample into a size exclusion chromatography column and developing the same.
  • column of the present invention is a unit filled with a porous stationary phase used in size exclusion chromatography
  • the stationary phase refers to particles having various sizes of holes for fractionating substances according to the molecular weight.
  • the separation resolution depending on the molecular size of the molecules present in the sample varies depending on the size of the holes existing in the stationary phase. For example, if the pores present in the stationary phase are large, they are efficient for separating relatively large molecules and relatively small molecules are eluted without separation. On the other hand, if the size of the hole existing in the stationary phase is small, the degree of separation of large molecules is low, but it may be efficient to separate molecules having a certain size or smaller.
  • the size of the molecule to be separated and the size of the contaminant in the sample are taken into consideration, and a fixed phase having a hole having a size providing an optimum separation efficiency is selected.
  • the most widely used gels in size exclusion chromatography are Sepharose (GE Healthcare), Superose (GE Healthcare), Sephadex (Pharmacia), Bio-Gel P (Bio-Rad) and TSKgel® silica-based; Sigma).
  • a Sephacryl S500 stationary phase having pores having a size capable of separating the extracellular endoplasmic reticulum, which is a nanoparticle, from proteins of various sizes But is not limited thereto.
  • the size exclusion chromatography development method of the present invention may be a pump type, a rotary type, a gravity type, and the like, but is not limited thereto.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (c) of detecting an extracellular endoplasmic reticulum-probe complex and a free probe from the developed sample.
  • the detection step of the present invention can obtain the desired analysis results by simultaneously detecting the extracellular endoplasmic reticulum-probe complex isolated through the size exclusion chromatography as well as the free probe not bound to the extracellular endoplasmic reticulum.
  • the detecting step of the present invention may include quantifying the extracellular endoplasmic reticulum by detecting a light absorption chromatogram for a specific wavelength.
  • the specific wavelength may be selected from one or more values selected from the range of 200 nm to 800 nm.
  • the specific wavelength may be selected from among 330 nm to 450 nm, at least one wavelength, 230 nm, 260 nm, or 280 nm, but is not limited thereto.
  • the detecting step of the present invention may include detecting the signal portion of the probe to quantify the probe.
  • the step of detecting the signal portion of the probe may select a suitable detection and analysis method according to the type of the signal portion of the probe, and may perform a spectroscopic analysis (such as absorption, fluorescence, scattering, and radioactive), physicochemical analysis, quantum chemical analysis, Biotin analysis, and nucleic acid analysis, but the present invention is not limited thereto.
  • the probe detection step of the present invention includes a direct analysis or an indirect analysis depending on the nature of the signal portion of the probe. If direct spectroscopic analysis of the probe's wavelength is possible, the fluorescence signal, the extinction signal, the scatter signal, or the luminescent and radioactive signal of the probe can be detected and analyzed. If additional processing is required for the signal portion of the probe, it can be indirectly analyzed using biotin analysis, antibody analysis, enzyme analysis, and polymerase chain reaction (PCR) analysis.
  • PCR polymerase chain reaction
  • the present invention also provides a method for analyzing extracellular endoplasmic reticulum by separating extracellular endoplasmic reticulum-probe complex from a sample and analyzing the same.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of mixing and reacting a sample containing a probe including a detectable signal portion and a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and an extracellular endoplasmic reticulum [ And injecting the mixed sample into a size exclusion chromatography column and developing the mixture (step (b)).
  • the method of analyzing an extracellular endoplasmic reticulum of the present invention includes a step (c) of separating an extracellular endoplasmic reticulum-probe complex from the size exclusion chromatography column.
  • the extracellular endoplasmic reticulum-probe complex separating step of the present invention utilizes the fractional size of size exclusion chromatography.
  • the extracellular endoplasmic reticulum and other impurities contained in the sample are separated Time elution.
  • the extracellular endoplasmic reticulum has a molecular size greater than 1,000 kDa and is larger than free probes or other impurities Because it belongs, it dissolves relatively quickly.
  • Some of the probes mixed with the sample in the present invention react with the extracellular endoplasmic reticulum specifically to form a complex, while the remainder remain as a free probe that does not bind to the extracellular endoplasmic reticulum, and a relatively large extracellular endoplasmic reticulum-probe complex They dissolve and separate faster than free probes.
  • the elution time of each substance differs depending on the size and pore size of the porous stationary phase, the length of the column, the flow rate of the mobile phase, and elutes at a specific time under the same conditions.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (d) of detecting a probe in the separated extracellular endoplasmic reticulum-probe complex.
  • the detection step of the present invention quantitative analysis of the extracellular endoplasmic reticulum and the amount of the probe bound to the extracellular endoplasmic reticulum can be analyzed.
  • the specificity or affinity of the extracellular endoplasmic reticulum is analyzed according to the kind of the probe, Can be utilized.
  • the method for analyzing extracellular ER of the present invention uses the size-specific fractionation ability of size exclusion chromatography and the characteristics of a probe that specifically binds to extracellular ER, and is used to quantitatively analyze the extracellular ER contained in the sample,
  • the physicochemical characterization of the endoplasmic reticulum, the type and quantitative analysis of constituents contained in the extracellular endoplasmic reticulum, and the binding specificity or affinity analysis of the probe to the extracellular endoplasmic reticulum component can be quickly and easily performed.
  • the assay method of the present invention it is possible not only to accurately analyze the extracellular endoplasmic reticulum in the sample without purification or pretreatment of the sample, but also to analyze the constituents of the extracellular endoplasmic reticulum easily and accurately according to the type of the probe.
  • the diagnostic efficiency using the extracellular endoplasmic reticulum can be improved.
  • using the probe specificity and affinity analysis it can be used for extracellular endoplasmic reticulum-specific antibody screening, protein screening, and chemical screening.
  • FIG. 1 is a schematic diagram of a method for analyzing an extracellular ER according to the present invention.
  • FIG. 2 is a schematic view showing the isolation and isolation of the extracellular endoplasmic reticulum from the colorectal cancer cell line SW480 according to an embodiment of the present invention.
  • FIG. 3 is a UV (a) and fluorescence (b) chromatogram showing binding patterns of a fluorescent antibody-labeled mouse antibody (normal mouse IgG) and an extracellular endoplasmic reticulum in accordance with an embodiment of the present invention.
  • FIG. 4 is a UV (a) and fluorescence (b) chromatogram showing the binding pattern of CD63 antibody (anti-CD63 antibody) labeled with a fluorescent substance according to an embodiment of the present invention.
  • FIG. 5 is a UV (a) and fluorescence (b) chromatogram showing the binding pattern of CD81 antibody (anti-CD63 antibody) labeled with a fluorescent substance according to an embodiment of the present invention.
  • FIG. 6 is a fluorescence chromatogram showing the specificity of a fluorescently labeled CD63 antibody in a specimen extracellular endoplasmic reticulum according to an embodiment of the present invention.
  • FIG. 7 is a UV (a) and fluorescence (b) chromatogram showing the expression pattern of the CD81 extracellular ER marker in the extracellular endoplasmic reticulum of different origin according to an embodiment of the present invention.
  • TNFa Tumor necrosis factor alpha
  • FIG. 9 is a fluorescence chromatogram showing the extracellular endoplasmic reticulum without isolation of the extracellular endoplasmic reticulum from the colon cancer cell culture fluid using the fluorescence-labeled CD63 antibody according to an embodiment of the present invention.
  • FIG. 10 is a UV (a) and fluorescence (b) chromatogram showing the extracellular endoplasmic reticulum without the process of separating the extracellular endoplasmic reticulum from the colon cancer cell culture fluid using the fluorescently labeled CD81 antibody according to an embodiment of the present invention.
  • FIG. 11 is a graph showing the relationship between the concentration of nanoparticles (a) and the concentration of the extracellular endoplasmic reticulum in the sample without the separation of the extracellular endoplasmic reticulum from the culture medium of colon cancer cells cultured at different incubation times using fluorescently labeled CD81 antibody according to an embodiment of the present invention.
  • UV (b), and fluorescence (c) chromatograms are examples of fluorescence chromatograms.
  • FIG. 12 is a graph showing the results of analysis of the transmembrane VPD450 (violet proliferation dye 450) showing the fluorescence by the esterase activity and the extracellular endoplasmic reticulum using the esterase activity in the endoplasmic reticulum endoplasmic reticulum ) And fluorescence (b, c) chromatograms.
  • VPD450 violet proliferation dye 450
  • FIG. 13 is a graph showing the results of the isolation of extracellular endoplasmic reticulum from colon cancer cell culture using VPD450 (violet proliferation dye 450) showing fluorescence by the esterase activity according to an embodiment of the present invention and the esterase activity in the extracellular endoplasmic reticulum (B) and fluorescence (c) chromatograms of the extracellular endoplasmic reticulum in the sample.
  • VPD450 violet proliferation dye 450
  • FIG. 14 is a graph showing the results of fluorescence spectroscopy using a membrane-permeable VPD450 (violet proliferation dye 450) exhibiting fluorescence by an esterase activity according to an embodiment of the present invention and a fluorescent size- (b) chromatogram analysis.
  • VPD450 violet proliferation dye 450
  • FIG. 15 is a graph showing the fluorescence-labeled extracellular endoplasmic reticulum (ERCP) conjugated with a transmembrane carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) showing fluorescence by the esterase activity according to an embodiment of the present invention and a rotary size exclusion chromatography UV and fluorescence (b) chromatogram.
  • ERCP extracellular endoplasmic reticulum
  • CFDA-SE transmembrane carboxyfluorescein diacetate succinimidyl ester
  • FIG. 18 is a graph showing the results of human urine (b), human serum (c) reacted with biotin-cholesterol according to an embodiment of the present invention, and biotin-cholesterol- And the amount of the outer vesicle complex.
  • FIG. 19 is a fluorescence chromatogram of an extracellular endoplasmic reticulum reacted with DiI (lipophilic dye) according to an embodiment of the present invention by HPLC.
  • FIG. 20 is a fluorescence chromatogram of an extracellular endoplasmic reticulum derived from Escherichia coli reacted with DiI (lipophilic dye) according to an embodiment of the present invention by HPLC.
  • DiI lipophilic dye
  • the colon cancer cell line SW480 culture was centrifuged at 500 xg for 10 minutes and at 2,000 xg for 20 minutes to remove the precipitate.
  • the extracellular endoplasmic reticulum was added with polyethylene glycol solution (8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) for the first purification and precipitation of the extracellular endoplasmic reticulum present in the supernatant for 16 hours After refrigerated, the extracellular endoplasmic reticulum was centrifuged at 12,000 xg for 30 minutes and the precipitate was dissolved in HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4).
  • the purified extracellular endoplasmic reticulum was injected into a column packed with Sephacryl S500 (10 x 100 mm) using high performance liquid chromatography (HPLC) and subjected to molecular size exclusion chromatography
  • HPLC high performance liquid chromatography
  • the extracellular endoplasmic reticulum was purified and the process of separating the endoplasmic reticulum endoplasmic reticulum was shown in FIG. 2 (a).
  • the fractions of 10 regions were harvested from the surface of the sample after buoyancy density gradient ultracentrifugation of the second purification method and the distribution of extracellular ER marker (Alix, CD9, CD81, CD63) in each fraction was collected by Western blot This is shown in Fig. 2 (b).
  • the out-of-sample extracellular endoplasmic reticulum after the third purification method was observed using a transmission electron microscope (TEM), and the shape and size (about 50 to 200 nm) of the purified endoplasmic reticulum were confirmed 2 (c)).
  • TEM transmission electron microscope
  • Example 2 Analysis of specimen extracellular endoplasmic reticulum using pumped size exclusion chromatography and fluorescently labeled antibodies
  • the extracellular endoplasmic reticulum was detected at 6.5 minutes and the antibody was detected at 14.5 minutes from the absorbance chromatogram results.
  • the fluorescence band of the fluorescently labeled antibody was detected at 14.5 minutes in the fluorescence chromatogram, but the fluorescence band of the extracellular endoplasmic reticulum corresponding to the 6.5 minute fraction was not observed. This indicates that the sample extracellular endoplasmic reticulum and the mouse antibody did not undergo nonspecific binding under the conditions, and that the mixture of injected sample extracellular endoplasmic reticulum and mouse antibody was effectively separated by size exclusion chromatography and eluted .
  • the purified various amounts of the specimen extracellular endoplasmic reticulum and the fluorescently labeled anti-CD63 antibody (aCD63 antibody) recognizing the membrane surface protein CD63 of the extracellular endoplasmic reticulum were mixed and incubated at 37 ° C for 30 minutes, The column was filled with S500 and analyzed using 280 nm absorption spectrophotometer (FIG. 4 (a)) and fluorescence chromatogram (FIG. 4 (b)) while developing using an HPLC system.
  • the purified various amounts of the specimen extracellular endoplasmic reticulum and the fluorescence labeled anti-CD81 antibody (aCD81 antibody) recognizing another membrane protein CD81 of the extracellular endoplasmic reticulum were mixed and subjected to size exclusion chromatography under the same conditions as above
  • the absorption spectrophotometer (FIG. 5 (a)) and the fluorescence chromatogram (FIG. 5 (b)) were analyzed at 280 nm.
  • fluorescent bands appeared at the same detection time as the 280 nm absorbance band of the specimen extracellular endoplasmic reticulum detected at 6.5 minutes in the corresponding column, and the area of the detected bands was highly correlated with the injection amount of the extracellular endoplasmic reticulum there was.
  • the fluorescent band area of the fluorescently labeled free antibody detected at 14.5 minutes decreased in inverse proportion to the amount of injected specimen extracellular endoplasmic reticulum.
  • the antibody mixed in the specimen extracellular endoplasmic reticulum specifically recognizes the components (CD63, CD81) of the extracellular endoplasmic reticulum and binds to CD63 or CD81 respectively to form an " extracellular endoplasmic reticulum-antibody complex"
  • a small fluorescently labeled antibody (detected at 14.5 min) developed along with a macromolecular extracellular envelope, indicating that the fluorescent band was detected at the time of detection of the extracellular endoplasmic reticulum (6.5 min).
  • the area of the fluorescent band detected at the detection time of the fluorescently labeled free antibody reflects the amount of the free antibody except for the amount of the antibody bound to the extracellular endoplasmic reticulum in the total amount of the antibody mixed in the sample.
  • fluorescence labeled anti-CD63 antibody and label Anti-CD63 antibody was mixed and reacted. Specifically, the fluorescence labeled anti-CD63 antibody and the non-fluorescently labeled anti-CD63 antibody were mixed together in a ratio of 1:10 to the specimen extracellular endoplasmic reticulum, Fluorescence chromatograms were compared by size exclusion chromatography in the reacted groups.
  • the same amount of fluorescently labeled anti-CD81 antibody was mixed with the extracellular endoplasmic reticulum secreted from the same amount of different parental cells (SW480, HMEC1), and the expression pattern of CD81 protein in each extracellular endoplasmic reticulum was determined by 280 (Fig. 7 (a)) and a fluorescence chromatogram (Fig. 7 (b)).
  • the area of the 280 nm absorption band of each extracellular endoplasmic reticulum detected at 6.5 minutes in the column was the same, but the fluorescence band area of each of the extracellular endoplasmic reticulum and the anti-CD81 antibody complex was different at the same detection time (6.5 minutes) Respectively.
  • the extracellular endoplasmic reticulum of HMEC1 cells was significantly lower than that of SW480 colon cancer cells.
  • the fluorescence band area of 8.5 min of free antibody was higher in HMEC1 cell-derived samples. This is because the amount of extracellular endoplasmic reticulum per unit sample differs depending on the type of the parent cell. Therefore, in the analysis of various kinds of extracellular endoplasmic reticulum by the assay method of the present invention, It can be analyzed.
  • TNFa The effect of TNFa on the composition of extracellular endoplasmic reticulum secreted from colon cancer cells was analyzed using the method of the present invention. Specifically, in the culture of colon cancer cells, TNFa was divided into 24 hours (TNFa +) and untreated (TNFa), and the extracellular endoplasmic reticulum was purified by conventional methods.
  • the anti-CD81 antibody labeled with fluorescence (FITC) and the anti-ICAM1 antibody labeled with fluorescence (PE) were reacted to the extracellular endoplasmic reticulum purified in each group, and injected into the size exclusion chromatography column to develop a 280 nm absorbance chromatogram (Fig. 8 (a)) and a fluorescence chromatogram (Fig. 8 (b, c)).
  • the area (b) of the CD81 fluorescent band was similar to that of the 280 nm extinction band of each extracellular ER at 3.6 min in the corresponding column, while the fluorescence band of ICAM1 in the same detection time (3.6 min) (c) was significantly increased in the TNFa + group as compared to the TNFa group. From this, it was found that the amount of CD81 protein in the extracellular ER after treatment with TNFa was not changed but the amount of ICAM1 was greatly increased. From these results, it can be seen that physiological changes of cells caused by the cell state, environment or external factors lead to a change in constituents of the extracellular endoplasmic reticulum secreted from the cells even in the same kind of cells. I can confirm that I can.
  • this assay can be used to analyze not only the total amount of extracellular endoplasmic reticulum and the components of extracellular endoplasmic reticulum but also the specificity and affinity of antibodies and ligands to components of extracellular endoplasmic reticulum easily and rapidly .
  • Example 3 Analysis of extracellular endoplasmic reticulum in cell culture using pumped size exclusion chromatography and fluorescently labeled antibodies
  • SW480 colon cancer cells were cultured in RPMI culture medium for 24 hours to harvest the cell culture medium.
  • the RPMI culture medium in which the cells were not cultured was mixed with the fluorescently labeled anti-CD63 antibody, and the mixture of the colon cancer cell culture medium and the fluorescence-labeled anti-CD63 antibody was reacted at 37 ° C for 30 minutes and injected into the TSK6000 HPLC column The fluorescence chromatograms were analyzed while developing using an HPLC system.
  • the culture medium of the colon cancer cells and the fluorescently labeled anti-CD81 antibody were mixed and incubated at 37 ° C for 30 minutes. After the cells were injected into the Sepafrill S500 column and developed using an HPLC system, (FIG. 10 (a)) and a fluorescence chromatogram (FIG. 10 (b)) were analyzed.
  • the concentration of nanoparticles in the culture solution increased as the incubation time of the colon cancer cells increased.
  • size exclusion chromatography it was confirmed that 280 nm absorbance band and CD81 antibody fluorescence band were simultaneously increased at 3.6 minutes in which the extracellular endoplasmic reticulum eluted with increasing colon cancer cell culture time, And it was confirmed that they had a high correlation. From this, it can be seen that the method of the present invention can simultaneously analyze the amount and composition of the extracellular endoplasmic reticulum in the sample without further separation of the extracellular endoplasmic reticulum.
  • Example 4 Analysis of specimen extracellular endoplasmic reticulum using pumped size exclusion chromatography and transmembrane enzyme substrate
  • VPD450 which is a substance that is transmissive and is converted into a fluorescent substance by enzyme activity. Specifically, the purified endoplasmic reticulum endoplasmic reticulum, fluorescently labeled anti-CD81 antibody, and various concentrations of VPD450 were mixed and reacted at 37 ° C for 30 minutes. The resulting solution was injected into a Sepafrill S500 column and developed using an HPLC system. (Fig. 12 (a)) and a fluorescence chromatogram (Fig. 12 (b, c)).
  • the extracellular endoplasmic reticulum in the corresponding column was detected at 3.5 minutes through the 280 nm absorbance band.
  • the fluorescence band of the anti-CD81 antibody and the fluorescence band of VPD450 were simultaneously appear.
  • the extracellular endoplasmic reticulum expresses both the CD81 protein and the esterase.
  • the area of the fluorescent band of VPD450 was proportional to the concentration of VPD450. As the concentration of VPD450 increased, the fluorescent active product was accumulated in the extracellular endoplasmic reticulum.
  • the fluorescence product of VPD450 (late peak in FIG. 12 (c)) produced by natural hydrolysis was clearly distinguished by size exclusion chromatography method because of its small molecular size.
  • the area of the 280 nm extinction band of the specimen extracellular ER detected at 3.5 minutes in the column was constant regardless of the reaction time, while the fluorescence band was increased with the reaction time.
  • the esterase in the extracellular endoplasmic reticulum converts VPD450 introduced into the extracellular endoplasmic reticulum into a fluorescent substance in proportion to the reaction time, and the converted fluorescent substance accumulates inside the extracellular endoplasmic reticulum.
  • Example 6 Analysis of extracellular endoplasmic reticulum using rotary size exclusion chromatography and transmembrane enzyme substrate
  • the extracellular endoplasmic reticulum-fluorescent VPD450 complex was separated from a substance which did not react with the extracellular endoplasmic reticulum as shown in the schematic diagram shown in FIG. 14 (a) Respectively. Specifically, the sample extracellular endoplasmic reticulum and VPD450, which is a transmembrane substrate, were mixed and reacted, then loaded on a rotary Cefacill S500 column and harvested by centrifugation.
  • the eluted extracellular endoplasmic reticulum-fluorescent VPD450 complex was injected into a Sepafrill S500 column and analyzed using a HPLC system, while analyzing a 280 nm absorbance chromatogram and a fluorescence chromatogram (FIG. 14 (b)).
  • Fig. 15 (a) the reaction solution obtained by mixing CFDA-SE, another esterase substrate, with the extra-cellular endoplasmic reticulum was pre-treated according to the method of 6- System, the 280 nm absorption chromatogram and the fluorescence chromatogram (Fig. 15 (b)) were analyzed.
  • CFDA-SE also permeated the membrane according to the same mechanism as VPD450 and was activated by the activity of esterase in the extracellular endoplasmic reticulum It was found that the fluorescent substance accumulates inside the extracellular endoplasmic reticulum.
  • Example 7 Analysis of specimen extracellular endoplasmic reticulum using rotary size exclusion chromatography and biotinylated cholesterol
  • the different amounts of purified specimen extracellular endoplasmic reticulum and biotin-labeled cholesterol were mixed and reacted at 37 ° C. for 30 minutes.
  • biotin- To remove cholesterol each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate.
  • biotin-cholesterol single group or single extracellular ER group was loaded on the column in the same manner as above, and the eluate was harvested through rotation.
  • the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and then chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 16 (b)).
  • Example 8 Analysis of extracellular endoplasmic reticulum in colorectal cancer cell culture using rotary size exclusion chromatography and cholesterol probe
  • Example 7 it was proved that only the biotin-cholesterol-extracellular elastomer complex can be effectively separated through the preliminary rotary size exclusion chromatography in the mixed reaction product of the extracellular endoplasmic reticulum and biotin-cholesterol.
  • SW480 colon cancer cell culture medium and biotin-labeled cholesterol (biotin-cholesterol) were used. Specifically, as shown in the schematic diagram of Fig.
  • the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 17 (b)).
  • chemiluminescence was very low or not in the single group of biotin - cholesterol and in the culture of colon cancer cells, but high chemiluminescence was observed in the group in which the culture of colon cancer cells and biotin - cholesterol were mixed.
  • biotin-cholesterol molecule was inserted into the lipid bilayer of the extracellular endoplasmic reticulum in the cell culture medium and eluted from the rotary pretreatment size exclusion chromatography column with the extracellular endoplasmic reticulum.
  • Example 9 Analysis of extracellular endoplasmic reticulum in body fluid using rotary size exclusion chromatography and cholesterol probe
  • the extracellular endoplasmic reticulum in the cell culture could be analyzed without purification of the extracellular endoplasmic reticulum, and the method was applied to human body fluids.
  • human urine or cholesterol (biotin-cholesterol) labeled with human serum and biotin were mixed and reacted at 37 ° C for 30 minutes,
  • biotin-cholesterol biotin-cholesterol labeled with human serum and biotin
  • each mixture solution was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate.
  • a single biotin-cholesterol group or a single biological sample group was loaded onto a column in the same manner as above, and the eluate was harvested by rotation.
  • the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 18 (b, c)).
  • biotin-cholesterol monoclonal and colon cancer cells As a result, chemiluminescence was low or not in the single group of biotin-cholesterol monoclonal and colon cancer cells, but high chemiluminescence was observed in the group in which biosynthetic (urine, serum) and biotin-cholesterol were mixed and reacted there was.
  • the biotin-cholesterol molecule was inserted into the lipid bilayer of the extracellular endoplasmic reticulum in the biological sample and eluted from the rotary pre-size exclusion chromatography column along with the extracellular endoplasmic reticulum to measure the total amount of extracellular endoplasmic reticulum present in a variety of body fluids It can be used.
  • Example 10 Analysis of specimen extracellular endoplasmic reticulum using rotational size exclusion chromatography and lipophilic probe (DiI)
  • Example 11 Analysis of E. coli-derived extracellular endoplasmic reticulum using rotational size exclusion chromatography and lipophilic probe (DiI)
  • Example 10 The same probe as in Example 10 was applied to the E. coli-derived extracellular endoplasmic reticulum and analyzed. Specifically, as shown in the schematic diagram of FIG. 20 (a), a group obtained by mixing fluorescently labeled lipophilic DiI single group or E. coli-derived extracellular endoplasmic reticulum with fluorescently labeled lipophilic DiI was reacted at 37 ° C for 30 minutes, To remove the fluorescence-labeled lipophilic DiI not bound to the endoplasmic reticulum, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. Thereafter, the eluate was injected into a Cefacill S500 column, and a 280 nm absorption chromatogram and a fluorescence chromatogram (FIG. 20 (b)) were analyzed while developing using an HPLC system.
  • FIG. 20 (a) a group obtained by mixing fluorescent
  • fluorescently labeled lipophilic DiI having a small molecular weight was not eluted in the rotary size exclusion chromatography column and fluorescence band was not detected.
  • a fluorescence band having a high elution time of 3.5 minutes was confirmed.
  • fluorescently labeled lipophilic DiI having a small molecular weight was inserted into the lipid bilayer of the E. coli-derived extracellular endoplasmic reticulum and eluted together with the E. coli-derived endoplasmic reticulum endoplasmic reticulum in the rotary size exclusion chromatography column.
  • the bacterial-derived extracellular endoplasmic reticulum was also composed of a lipid bilayer, and this analysis method can be used to analyze the extracellular endoplasmic reticulum as well as the total amount of extracellular endoplasmic reticulum.

Abstract

The analysis method for extracellular vesicles, according to the present invention, uses the size-specific separation ability of size exclusion chromatography and the properties of a probe that specifically binds with extracellular vesicles, and by using same is capable of the rapid and easy analysis of the quantity of extracellular vesicles included in a sample, analysis of the physicochemical properties of the extracellular vesicles, analysis of the kind and quantity of the components included in the extracellular vesicles, and analysis of the binding properties or affinity of the probe with respect to the components of the extracellular vesicles. In addition, using the analysis method of the present invention not only enables accurate analysis of extracellular vesicles in a sample, without a sample purification or pre-processing step, but also enables accurate and simple analysis of the components of extracellular vesicles, according to the kind of probe, and thus can improve the efficiency of diagnosis using extracellular vesicles. Also, analysis of the properties or affinity of the probe can be applied to, for example, extracellular vesicle-specific antibody screening, protein screening and chemical-substance screening.

Description

크기 배제 크로마토그래피를 이용한 세포밖 소포체의 분석 방법 및 이의 용도METHODS FOR ANALYZING EXTRACELLULAR ERATORS USING SCALE EXCEPTION CHROMATOGRAPHY
본 발명은 크기 배제 크로마토그래피를 이용한 세포밖 소포체의 분석 방법 및 이의 용도에 관한 것으로서, 보다 구체적으로 본 발명은 크기 배제 크로마토그래피의 크기별 분획능과 세포밖 소포체에 특이적으로 결합하는 탐침을 이용하여 시료 중 포함된 세포밖 소포체를 분석하는 방법에 관한 것이다.The present invention relates to a method of analyzing an extracellular endoplasmic reticulum using size exclusion chromatography and a use thereof, more particularly, to a method of analyzing extracellular endoplasmic reticulum using size exclusion chromatography, And to a method for analyzing the extracellular endoplasmic reticulum contained in a sample.
세포밖 소포체(extracellular vesicles)는 세포의 보편적인 기작으로, 생체 내 또는 시험관 내의 여러 종류의 세포로부터 분비되는 나노 크기의 생체 입자로서 혈액, 소변, 침, 눈물 등과 같은 체액에 존재하고 세포에서 유래한 지질이중층을 포함하며, 20 ~ 1,000 nm 범위의 다양한 크기를 갖는 막 구조의 소포체이다. Extracellular vesicles are a universal mechanism of cells and are nano-sized biological particles secreted from various types of cells in vivo or in vitro, and exist in body fluids such as blood, urine, saliva, tears, etc., Lipid bilayers, and membrane-structured vesicles of various sizes ranging from 20 to 1,000 nm.
이들 세포밖 소포체는 다양한 생명 현상에서 여러 중요한 기능에 관여한다. 진핵세포에서 유래한 세포밖 소포체의 경우, 적혈구 분화, 면역반응 조절 등에 관여하며, 특히 암세포 미세환경에서는 암의 진행, 전이, 혈관형성 등에 관련된 많은 기능들이 밝혀짐으로써 암을 포함한 다양한 질병의 진단 마커로의 활용에 있어 높은 관심을 받고 있다.These extracellular endoplasmic reticulum are involved in many important functions in various life phenomena. The extracellular endoplasmic reticulum derived from eukaryotic cells is involved in erythrocyte differentiation and regulation of immune response. In particular, many functions relating to cancer progression, metastasis and angiogenesis are revealed in the cancer cell microenvironment, And has received high interest in the utilization of
원핵세포로부터 분비되는 세포밖 소포체는 진핵세포의 세포밖 소포체와 유사하게 원핵세포의 구성물을 함유하고 있으며, 인체에서는 전신염증을 비롯, 유입 경로에 따라 급성 폐염증 질환을 유도하고, 피부의 경우, 국소 피부 조직의 염증반응을 만성적으로 유도하여 현대인의 대표 질병 중 하나인 아토피성 피부염(atopic dermatitis)의 원인이 될 수 있음이 보고되었다. 또한 인체에서 박테리아 유래 세포밖 소포체가 암을 비롯한 다양한 질환과 연관성이 있다는 점이 보고됨에 따라 원핵세포 유래 세포밖 소포체 또한 높은 관심을 받고 있다. The extracellular endoplasmic reticulum secreted from prokaryotic cells contains prokaryotic components similar to extracellular endoplasmic reticulum. In the human body, acute pulmonary inflammatory diseases are induced according to the pathway including systemic inflammation. In the case of skin, It has been reported that chronic inflammation of the local skin tissues can be induced to cause atopic dermatitis, one of the representative diseases of modern people. In addition, extracellular endoplasmic reticulum from the prokaryotes has also been attracting much attention as it has been reported that the extracellular endoplasmic reticulum from bacterial cells is associated with various diseases including cancer.
세포밖 소포체는 단백질, 지질, 핵산, 아미노산 등 모세포로부터 유래한 물질로 구성되어 있으며, 생체에서는 이러한 물질들을 전달하는 운송체 역할을 하기 때문에 세포밖 소포체를 구성하는 단백질, 지질, 아미노산, 핵산 등을 분석함으로써 모세포의 생리적, 병리적 특성을 알 수 있는 중요한 근거가 된다. 따라서 다양한 시료에 존재하는 세포밖 소포체의 구성 성분 분석은 기초 및 의학 분야에서 높은 관심을 받고 있다.The extracellular endoplasmic reticulum is composed of substances derived from parent cells such as proteins, lipids, nucleic acids and amino acids. In vivo, the endoplasmic reticulum plays a role of a carrier to transfer these substances. Therefore, proteins, lipids, amino acids and nucleic acids constituting the extracellular endoplasmic reticulum Analysis is an important basis for understanding the physiological and pathological characteristics of the parent cells. Therefore, the analysis of constituents of the extracellular endoplasmic reticulum present in various samples has received high interest in the basic and medical fields.
또한, 세포밖 소포체에 포함되어 있는 핵산, 성장호르몬, 단백질 등은 세포막 형태의 인지질에 의해 보호되고 있어, 가용성 형태의 성장인자 및 사이토카인보다 안정적인 기능을 수행할 수 있다는 점이 알려지면서, 세포밖 소포체의 중요성이 점차 증대되고 있으며, 세포밖 소포체에 포함된 물질을 분석하여 질병의 진단, 치료를 포함한 다양한 용도로의 활용 가능성이 기대되고 있다. It is known that nucleic acid, growth hormone, and protein contained in the extracellular endoplasmic reticulum are protected by a phospholipid in the form of a cell membrane, so that they can perform more stable functions than a soluble form of growth factor and cytokine. And it is expected to be used for various purposes including the diagnosis and treatment of diseases by analyzing the substances contained in the extracellular endoplasmic reticulum.
최근 비 침습적 액체 생검(liquid biopsy)을 질병 진단에 활용하는 방안이 다각도로 전개되고 있고, 더 나아가 체액 내 세포밖 소포체를 활용하여 새로운 질병 진단 마커를 발굴하고 이를 이용해 진단 방법을 개발하려는 노력들이 시도되고 있다. 진단 방법 개발에 있어서 핵심요소는 탐침을 이용하여 소량의 시료에서 빠르고 정확하게 대상 물질을 정량하는 기술 개발에 있다. 따라서 생물학적 시료에 존재하는 세포밖 소포체의 구성 성분을 탐침을 이용해 분석하는 방법이 매우 중요한데, 이러한 세포밖 소포체의 구성 성분 분석은 단계가 복잡하고 효율이 떨어지는 초원심분리 기술을 통한 정제에 기반하여 이루어지고 있으며 이러한 초원심분리법에서는 세포밖 소포체 분리 수율이 낮고 세포밖 소포체와 결합하지 않은 탐침의 제거가 효율적으로 이루어지지 않기 때문에, 상대적으로 제한된 양과 높은 복잡성을 나타내는 체액의 경우 상기 방법으로 정량적인 분석을 하기란 거의 불가능하다. 따라서 통상의 세포밖 소포체 분석법과는 차별화되는 신속하고 과정이 단순한 신규한 기술개발이 시급하다. Recently, there have been various attempts to utilize liquid biopsy for the diagnosis of diseases. Furthermore, attempts have been made to develop new diagnostic methods using the extracellular endoplasmic reticulum in the body fluids. . A key element in the development of diagnostic methods is the development of techniques for rapidly and accurately quantifying target substances in a small sample using probes. Therefore, it is very important to analyze the constituents of the extracellular endoplasmic reticulum present in the biological sample using a probe. The analysis of the constituents of the extracellular endoplasmic reticulum is based on purification through a complicated and inefficient ultracentrifugation technique In this ultracentrifugation method, since the separation yield of the extracellular endoplasmic reticulum is low and the probe which is not bound to the extracellular endoplasmic reticulum is not efficiently removed, quantitative analysis of the body fluid exhibiting a relatively limited amount and high complexity It is almost impossible to do. Therefore, it is urgent to develop a new technique that is different from the conventional extracellular ER analysis method.
본 발명의 목적은 (a) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계, (b) 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계 및 (c) 상기 전개된 시료로부터 세포밖 소포체-탐침 복합체 및 자유 탐침을 검출하는 단계를 포함하는 세포밖 소포체의 분석 방법을 제공하는 것이다.(A) mixing and reacting a sample containing a probe and an extracellular endoplasmic reticulum containing a binding moiety and a detectable signal moiety that specifically bind to a component of an extracellular endoplasmic reticulum, and (b) Injecting a sample into a size exclusion chromatography column and developing the sample; and (c) detecting an extracellular endoplasmic reticulum-probe complex and a free probe from the developed sample.
본 발명의 또 다른 목적은 (a) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계, (b) 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계 및 (c) 상기 크기 배제 크로마토그래피 컬럼으로부터 세포밖 소포체-탐침 복합체를 분리하는 단계 및 (d) 상기 분리된 세포밖 소포체-탐침 복합체에서 탐침을 검출하는 단계를 포함하는 세포밖 소포체의 분석 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing a recombinant vector comprising the steps of: (a) mixing and reacting a sample containing a probe and an extracellular endoplasmic reticulum containing a binding moiety and a detectable signal moiety, (C) separating the extracellular endoplasmic reticulum-probe complex from the size exclusion chromatography column, and (d) exposing the extracellular endoplasmic reticulum-probe complex to an extracellular endoplasmic reticulum complex And detecting the probe. The present invention also provides a method for analyzing an extracellular endoplasmic reticulum.
본 발명은 상술한 문제점을 해결하기 위한 것으로, 세포밖 소포체의 구성성분과 특이적으로 결합하는 다양한 물질을 탐침으로 사용하여 시료와 반응시킴으로써 세포밖 소포체-탐침 복합체를 형성하고, 이를 크기 배제 크로마토그래피로 전개시킨 후 탐침을 검출함으로써 세포밖 소포체를 분석하는 방법을 제공한다.Disclosure of the Invention The present invention has been made to solve the above problems, and it is an object of the present invention to provide an extracellular endoplasmic reticulum-probe complex by reacting with a sample using various substances specifically binding to components of the extracellular endoplasmic reticulum as a probe, And then analyzing the extracellular endoplasmic reticulum by detecting the probe.
본 발명에서 "크기 배제 크로마토그래피(size exclusion chromatography, SEC)"라 함은 다양한 크기의 용질이 다공성 매트릭스를 통과하는 속도(투과도)를 기반으로 혼합물을 분리하는 기술을 의미한다. 즉 분석대상 시료를 젤, 매트릭스, 구슬(bead)과 같은 다공성 정지상(stationary phase)이 채워진 컬럼을 통과시키면, 컬럼의 구멍을 통과할 수 없는 큰 분자들은 구멍에 들어가지 못하고 주변의 빈 공간을 통해 빠르게 컬럼을 빠져 나오는 반면, 작은 분자들은 컬럼의 구멍을 통해 나오면서 상대적으로 천천히 이동하여 컬럼을 빠져 나오는 원리를 이용한 것이다. 이 방법은 일반적으로 완충액 교환(buffer　exchange)을 위한 탈염화(desalting), 정제를 위한 분리 또는 용질 크기에 따른 분자량 측정에 사용된다.The term " size exclusion chromatography (SEC) " in the present invention refers to a technique for separating a mixture based on the rate (permeability) at which various sizes of solute pass through the porous matrix. That is, when the sample to be analyzed is passed through a column filled with a porous stationary phase such as a gel, a matrix, or a bead, large molecules that can not pass through the hole of the column can not enter the hole, The small molecules exit the column quickly, while the small molecules move relatively slowly through the hole in the column to exit the column. This method is generally used for desalting for buffer exchange, separation for purification, or molecular weight measurement according to solute size.
본 발명에서는 세포밖 소포체를 포함하는 다양한 시료에 특이적으로 결합하는 탐침을 반응시켜 세포밖 소포체-탐침 복합체를 형성하고, 이를 크기 배제 크로마토그래피 컬럼에 통과시킴으로써, 상기 복합체와 자유 탐침을 빠르고 쉽게 분획할 수 있으며, 다음 단계에서 이렇게 분리된 용출물을 분석함으로써 세포밖 소포체의 정량 분석 및 구성성분 분석을 용이하고 효율적으로 할 수 있다.In the present invention, an extracellular endoplasmic reticulum-probe complex is formed by reacting a probe that specifically binds to various samples including extracellular endoplasmic reticulum, and the endoplasmic reticulum-probe complex is passed through a size exclusion chromatography column so that the complex and the free probe can be rapidly and easily fractionated And analysis of the extracellular endoplasmic reticulum can be performed easily and efficiently by analyzing the separated eluate in the next step.
본 발명의 용어 "세포밖 소포체"는 고세균(Archaea), 원핵생물(Prokarya) 또는 진핵생물(Eukarya)의 세포로부터 유래한 생체 나노입자를 통칭하며, 세포밖 소포체(exosome), 아그로좀(argosomes), 덱소좀(dexosomes), 엑토좀(ectosomes), 엑소베지클(exovesicle), 온코좀(oncosome), 프로미노좀(prominosome), 프로스타좀(prostasome), 톨레로좀(tolerosome), 미세입자(microparticle), 미세소포(microvesicle), 나노소포(nanovesicle), 수포성 소포(blebbing vesicle), 출아성 소포(budding vesicle), 세포밖 소포체-유사 소포(exosome-like vesicle), 매트릭스 소포(matrix vesicle), 막 소포(membrane vesicle), 탈피성 소포(shedding vesicle), 막 입자(membrane particle), 탈피성 미세소포(shedding microvesicle), 막 수포(membrane bleb), 에피디디모좀(epididimosome), 프로미니노좀(promininosome), 텍소좀(texosome) 또는 아키오좀(archeosome)을 포함할 수 있으나, 이에 한정되는 것은 아니다.The term " extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G. microvesicles, microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, , Membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, membrane blebs, epididymosomes, promininosome, texosome, or archeosome, but not limited thereto. It is not.
본 발명에 따른 세포밖 소포체 분석 방법을 도 1에 모식적으로 나타내었다.The method for analyzing extracellular ER according to the present invention is schematically shown in Fig.
본 발명의 세포밖 소포체 분석 방법은 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계[(a) 단계]를 포함한다.The method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of mixing and reacting a sample containing a probe including a detectable signal portion and a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and an extracellular endoplasmic reticulum [ .
본 발명의 용어 "탐침(probe)"은 세포밖 소포체를 구성하는 성분과 특이적으로 결합함과 동시에 분광학적, 물리화학적, 양자화학적, 효소학적 분석 방법 등을 이용하여 검출 및 분석이 가능한 물질을 의미한다.The term " probe " of the present invention refers to a substance which specifically binds to components constituting the extracellular endoplasmic reticulum and which can be detected and analyzed using spectroscopic, physicochemical, quantum chemical, it means.
본 발명에 있어서 상기 탐침은 i) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 단일 물질 또는 ii) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부를 포함하는 물질에 분석 가능한 하나 이상의 신호부를 포함하는 물질이 결합된 복합 물질일 수 있다.In the present invention, the probe comprises i) a single substance comprising a binding moiety and a detectable signal moiety that specifically bind to a component of an extracellular endoplasmic reticulum or ii) a binding moiety that specifically binds to a component of an extracellular endoplasmic reticulum Lt; RTI ID = 0.0 > analyte < / RTI >
세포밖 소포체는 이중지질막으로 둘러싸여 있고, 단백질, 지질, 핵산, 아미노산 등 모세포로부터 유래한 물질로 구성되어 있으며, 이들은 성분에 따라 세포밖 소포체의 막 표면, 세포밖 소포체의 막 또는 세포밖 소포체의 내부에 분포한다. 본 발명에 있어서 상기 탐침의 결합부는 상기와 같은 세포밖 소포체의 막 표면 성분, 세포밖 소포체의 막 성분 및 세포밖 소포체의 내부 성분으로 이루어진 군에서 선택되는 하나 이상의 성분과 특이적으로 결합하는 특징을 갖는다. The extracellular endoplasmic reticulum is surrounded by a double lipid membrane and is composed of substances derived from parent cells such as proteins, lipids, nucleic acids and amino acids. Depending on the constituents, they may be coated on the membrane surface of the extracellular endoplasmic reticulum, the membrane of the extracellular endoplasmic reticulum, . In the present invention, the binding portion of the probe specifically binds to at least one component selected from the group consisting of a membrane surface component of the extracellular endoplasmic reticulum, a membrane component of the extracellular endoplasmic reticulum and an inner component of the extracellular endoplasmic reticulum .
본 발명의 상기 탐침은 세포밖 소포체를 구성하는 성분 중 하나 이상의 물질과 특이적으로 결합하는 단백질, 항체, 항체 유래 물질, 펩티드, 핵산, 핵산-아미노산 복합체, 효소, 효소 기질, 화학적 리간드 및 이들의 화합물로 이루어진 군에서 선택될 수 있으나, 이에 한정되지 않는다. 상기 탐침은 세포밖 소포체 구성성분과 특이적으로 결합함과 동시에 검출 단계에서 검출 가능한 물질일 수 있다. 본 발명의 일실시예에 따르면 세포밖 소포체 내부 성분인 에스테르분해효소(esterase)의 기질 중 하나인 VPD450 또는 CFDA-SE를 탐침으로 사용하였다. 상기 기질은 세포밖 소포체 중 에스테르분해효소와 특이적으로 결합함과 동시에, 효소 활성에 의해 형광물질로 변환될 수 있어 별도의 표지 없이 형광 신호 검출이 가능하다.The probe of the present invention may be a protein, an antibody, an antibody-derived substance, a peptide, a nucleic acid, a nucleic acid-amino acid complex, an enzyme, an enzyme substrate, a chemical ligand, Compounds, but is not limited thereto. The probe may be a substance that specifically binds to the extracellular endoplasmic reticulum component and is detectable at the detection step. According to one embodiment of the present invention, VPD450 or CFDA-SE, which is one of substrates of esterase, an internal component of extracellular ER, was used as a probe. The substrate specifically binds to an esterase in the extracellular matrix and can be converted into a fluorescent substance by an enzyme activity, so that fluorescent signals can be detected without a separate label.
본 발명의 상기 탐침은 세포밖 소포체를 구성하는 성분 중 하나 이상의 물질과 특이적으로 결합하는 물질에 검출 가능한 신호부를 추가로 포함할 수 있다. 상기 탐침의 신호부는 형광 물질, 효소 기질, 효소, 단백질, 펩티드, 핵산, 비오틴, 금속 및 방사성동위원소로 이루어진 군에서 하나 이상이 선택될 수 있으나, 이에 한정되지 않는다. 본 발명의 일실시예에 따르면 세포밖 소포체의 막 표면 단백질을 인식하는 항체에 형광 표지를 부착한 탐침을 사용하였다.The probe of the present invention may further comprise a detectable signal moiety in a substance that specifically binds to one or more of the components constituting the extracellular endoplasmic reticulum. The signal portion of the probe may be selected from the group consisting of a fluorescent substance, an enzyme substrate, an enzyme, a protein, a peptide, a nucleic acid, a biotin, a metal and a radioisotope, but is not limited thereto. According to one embodiment of the present invention, a probe having a fluorescent label attached to an antibody recognizing the membrane surface protein of the extracellular endoplasmic reticulum was used.
본 발명의 용어 "시료"는 세포밖 소포체를 포함하는 생체 시료 또는 세포 배양액, 조직 시료 등을 포함하는 것으로서, 구체적으로 포유동물 세포 배양 배지, 박테리아 세포 배양 배지, 효모 배양 배지, 조직 추출물, 암 조직, 혈청, 혈장, 침, 눈물, 안방수, 땀, 소변, 대변, 뇌척수액(CSF, cerebrospinal fluid), 복수(ascite), 양수(amniotic fluid), 정액, 유(milk), 먼지, 담수, 해수, 토양 및 발효식품으로 이루어진 군에서 하나 이상이 선택될 수 있으나 이에 한정되지 않는다.The term " sample " of the present invention includes a biological sample containing cell extracellular medium, a cell culture fluid, a tissue sample, and the like, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, Soil, and fermented food. However, the present invention is not limited thereto.
본 발명의 세포밖 소포체 분석 방법은 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계[(b) 단계]를 포함한다.The method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (b) of injecting the mixed sample into a size exclusion chromatography column and developing the same.
본 발명의 용어 "컬럼"은 크기 배제 크로마토그래피에 사용되는 다공성 고정상이 채워진 단위이며, 상기 고정상은 분자량에 따라 물질을 분획하기 위한 다양한 크기의 구멍이 있는 입자를 의미한다. 상기 고정상에 존재하는 구멍의 크기에 따라 시료 내 존재하는 분자들의 분자 크기에 따른 분리 해상능이 변하게 된다. 예로, 고정상에 존재하는 구멍이 큰 경우는 상대적으로 큰 분자들의 분리에 효율적이고 상대적으로 작은 분자는 분리되지 않고 용출된다. 반면, 고정상에 존재하는 구멍의 크기가 작은 경우 큰 분자들의 분리 정도는 낮게 용출되지만, 일정 크기 이상과 이하의 분자를 분리하는 데는 효율적일 수 있다. 따라서 통상적으로는 분리하고자 하는 분자의 크기와 시료 내 오염물질의 크기를 고려하여 최적의 분리 효율을 제공하는 크기의 구멍을 가진 고정상을 선택하게 된다. 크기 배제 크로마토그래피에서 가장 널리 사용되는 젤은 세파로즈(Sepharose; GE Healthcare), 수퍼로즈(Superose; GE Healthcare), 세파덱스(Sephadex;　Pharmacia),　Bio-Gel　P(Bio-Rad)와 TSKgel® (silica-based; Sigma) 등의 계열이며, 본 발명의 일실시예에서는 나노 입자인 세포밖 소포체를 다양한 크기의 단백질과 분리할 수 있는 크기의 구멍을 가진 세파크릴(Sephacryl) S500 고정상을 사용하였지만, 이에 한정되는 것은 아니다. 본 발명의 크기 배제 크로마토그래피 전개 방식은 펌프식, 회전식, 중력식 등이 있으나, 이에 한정되지 않는다.The term " column " of the present invention is a unit filled with a porous stationary phase used in size exclusion chromatography, and the stationary phase refers to particles having various sizes of holes for fractionating substances according to the molecular weight. The separation resolution depending on the molecular size of the molecules present in the sample varies depending on the size of the holes existing in the stationary phase. For example, if the pores present in the stationary phase are large, they are efficient for separating relatively large molecules and relatively small molecules are eluted without separation. On the other hand, if the size of the hole existing in the stationary phase is small, the degree of separation of large molecules is low, but it may be efficient to separate molecules having a certain size or smaller. Thus, typically, the size of the molecule to be separated and the size of the contaminant in the sample are taken into consideration, and a fixed phase having a hole having a size providing an optimum separation efficiency is selected. The most widely used gels in size exclusion chromatography are Sepharose (GE Healthcare), Superose (GE Healthcare), Sephadex (Pharmacia), Bio-Gel P (Bio-Rad) and TSKgel® silica-based; Sigma). In one embodiment of the present invention, a Sephacryl S500 stationary phase having pores having a size capable of separating the extracellular endoplasmic reticulum, which is a nanoparticle, from proteins of various sizes, But is not limited thereto. The size exclusion chromatography development method of the present invention may be a pump type, a rotary type, a gravity type, and the like, but is not limited thereto.
본 발명의 세포밖 소포체 분석 방법은 상기 전개된 시료로부터 세포밖 소포체-탐침 복합체 및 자유 탐침을 검출하는 단계[(c) 단계]를 포함한다.The method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (c) of detecting an extracellular endoplasmic reticulum-probe complex and a free probe from the developed sample.
본 발명의 상기 검출 단계는 상기 크기 배제 크로마토그래피를 통해 분리된 세포밖 소포체-탐침 복합체뿐만 아니라 세포밖 소포체와 결합하지 않은 자유 탐침을 동시에 검출함으로써 목적하는 분석 결과를 얻을 수 있다.The detection step of the present invention can obtain the desired analysis results by simultaneously detecting the extracellular endoplasmic reticulum-probe complex isolated through the size exclusion chromatography as well as the free probe not bound to the extracellular endoplasmic reticulum.
본 발명의 상기 검출 단계는 특정 파장에 대한 흡광 크로마토그램을 검출함으로써 세포밖 소포체를 정량하는 단계를 포함할 수 있다. 본 발명에 있어서, 상기 특정 파장은 200 nm ~ 800 nm 범위에서 선택되는 하나 이상의 값이 선택될 수 있다. 본 발명에서 상기 특정 파장은 330 내지 450 nm 범위 중 하나 이상의 파장, 230 nm, 260 nm 또는 280 nm의 파장 중에서 선택될 수 있으나, 이에 한정되지 않는다.The detecting step of the present invention may include quantifying the extracellular endoplasmic reticulum by detecting a light absorption chromatogram for a specific wavelength. In the present invention, the specific wavelength may be selected from one or more values selected from the range of 200 nm to 800 nm. In the present invention, the specific wavelength may be selected from among 330 nm to 450 nm, at least one wavelength, 230 nm, 260 nm, or 280 nm, but is not limited thereto.
본 발명의 상기 검출 단계는 탐침의 신호부를 검출하여 탐침을 정량하는 단계를 포함할 수 있다. 구체적으로 상기 탐침의 신호부를 검출하는 단계는 탐침의 신호부 종류에 따라 적합한 검출 및 분석 방법을 선택할 수 있으며, 분광학적 분석(흡광, 형광, 산란, 방사성), 물리화학적 분석, 양자화학적 분석, 효소학적 분석, 비오틴 분석 및 핵산 분석으로 이루어진 군에서 선택될 수 있으나, 이에 한정되지 않는다. The detecting step of the present invention may include detecting the signal portion of the probe to quantify the probe. Specifically, the step of detecting the signal portion of the probe may select a suitable detection and analysis method according to the type of the signal portion of the probe, and may perform a spectroscopic analysis (such as absorption, fluorescence, scattering, and radioactive), physicochemical analysis, quantum chemical analysis, Biotin analysis, and nucleic acid analysis, but the present invention is not limited thereto.
본 발명의 탐침 검출 단계는 탐침의 신호부의 성질에 따라 직접적 분석 또는 간접적 분석을 포함한다. 탐침이 가지는 빛의 파장에 따른 직접적인 분광학적 분석이 가능한 경우, 탐침의 형광 신호, 흡광 신호, 산란 신호, 또는 발광 및 방사성 신호를 검출하여 분석할 수 있다. 탐침의 신호부에 부가적인 처리가 필요한 경우, 비오틴 분석, 항체 분석, 효소 분석, 중합효소연쇄반응(Polymerase chain reaction; PCR) 분석 등을 이용하여 간접적으로 분석할 수 있다.The probe detection step of the present invention includes a direct analysis or an indirect analysis depending on the nature of the signal portion of the probe. If direct spectroscopic analysis of the probe's wavelength is possible, the fluorescence signal, the extinction signal, the scatter signal, or the luminescent and radioactive signal of the probe can be detected and analyzed. If additional processing is required for the signal portion of the probe, it can be indirectly analyzed using biotin analysis, antibody analysis, enzyme analysis, and polymerase chain reaction (PCR) analysis.
본 발명은 또한 시료로부터 세포밖 소포체-탐침 복합체를 분리한 후 이를 분석함으로써 세포밖 소포체를 분석하는 방법을 제공한다. The present invention also provides a method for analyzing extracellular endoplasmic reticulum by separating extracellular endoplasmic reticulum-probe complex from a sample and analyzing the same.
본 발명의 세포밖 소포체 분석 방법은 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계[(a) 단계] 및 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계[(b) 단계]를 포함한다.The method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of mixing and reacting a sample containing a probe including a detectable signal portion and a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and an extracellular endoplasmic reticulum [ And injecting the mixed sample into a size exclusion chromatography column and developing the mixture (step (b)).
본 발명의 탐침, 시료 또는 크기 배제 크로마토그래피에 관한 상세한 설명은 상기한 바와 같다.Details of the probe, sample or size exclusion chromatography of the present invention are as described above.
본 발명의 세포밖 소포체 분석 방법은 상기 크기 배제 크로마토그래피 컬럼으로부터 세포밖 소포체-탐침 복합체를 분리하는 단계[(c) 단계]를 포함한다.The method of analyzing an extracellular endoplasmic reticulum of the present invention includes a step (c) of separating an extracellular endoplasmic reticulum-probe complex from the size exclusion chromatography column.
본 발명의 세포밖 소포체-탐침 복합체 분리 단계는 크기 배제 크로마토그래피의 크기별 분획능을 이용한 것으로서, 시료를 크기 배제 크로마토그래피 컬럼에 통과시키면 시료 중 포함된 세포밖 소포체와 기타 불순물이 크기가 큰 순서대로 시간 순으로 용출된다. 나노 입자인 세포밖 소포체를 다양한 크기의 단백질과 분리할 수 있는 크기의 구멍을 가진 고정상을 사용하면, 세포밖 소포체는 분자의 크기가 1,000 kDa 이상으로 자유 탐침 또는 다른 불순물에 비해 크기가 큰 입자에 속하기 때문에 상대적으로 빠르게 용출된다. 본 발명에서 시료에 혼합시킨 탐침 중 일부는 세포밖 소포체와 특이적으로 반응하여 복합체를 형성하고 나머지는 세포밖 소포체와 결합하지 않는 자유 탐침으로 남으며, 상대적으로 크기가 큰 세포밖 소포체-탐침 복합체가 자유 탐침에 비해 빠르게 용출되어 분리된다. 각 물질의 용출 시간은 다공성 정지상의 크기 및 구멍 크기, 컬럼의 길이, 이동상의 유속 등에 따라 상이하며, 동일한 조건에서는 특정 시간에 용출된다.The extracellular endoplasmic reticulum-probe complex separating step of the present invention utilizes the fractional size of size exclusion chromatography. When the sample is passed through a size exclusion chromatography column, the extracellular endoplasmic reticulum and other impurities contained in the sample are separated Time elution. Using a stationary phase with pores sized to separate nanoparticulate extracellular endoplasmic reticulum from proteins of various sizes, the extracellular endoplasmic reticulum has a molecular size greater than 1,000 kDa and is larger than free probes or other impurities Because it belongs, it dissolves relatively quickly. Some of the probes mixed with the sample in the present invention react with the extracellular endoplasmic reticulum specifically to form a complex, while the remainder remain as a free probe that does not bind to the extracellular endoplasmic reticulum, and a relatively large extracellular endoplasmic reticulum-probe complex They dissolve and separate faster than free probes. The elution time of each substance differs depending on the size and pore size of the porous stationary phase, the length of the column, the flow rate of the mobile phase, and elutes at a specific time under the same conditions.
본 발명의 세포밖 소포체 분석 방법은 상기 분리된 세포밖 소포체-탐침 복합체에서 탐침을 검출하는 단계[(d) 단계]를 포함한다.The method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (d) of detecting a probe in the separated extracellular endoplasmic reticulum-probe complex.
본 발명의 상기 검출 단계에 관한 상세한 설명은 상기한 바와 같다.The details of the detection step of the present invention are as described above.
본 발명의 상기 검출 단계에서는 세포밖 소포체의 정량 분석, 세포밖 소포체와 결합한 탐침의 양을 분석할 수 있으며, 탐침의 종류에 따라 세포밖 소포체에 대한 특이성 또는 친화도를 분석하여 탐침의 성능 평가에 활용할 수 있다.In the detection step of the present invention, quantitative analysis of the extracellular endoplasmic reticulum and the amount of the probe bound to the extracellular endoplasmic reticulum can be analyzed. The specificity or affinity of the extracellular endoplasmic reticulum is analyzed according to the kind of the probe, Can be utilized.
본 발명의 세포밖 소포체 분석 방법은 크기 배제 크로마토그래피의 크기별 분획능과 세포밖 소포체에 특이적으로 결합하는 탐침의 특성을 이용한 것으로서, 이를 이용하여 시료 중 포함된 세포밖 소포체의 정량 분석, 세포밖 소포체의 물리화학적 특성 분석, 세포밖 소포체에 포함된 구성성분의 종류 및 정량 분석, 세포밖 소포체 구성 성분에 대한 탐침의 결합 특이성 또는 친화도 분석을 빠르고 용이하게 할 수 있다. 또한 본 발명의 분석 방법을 이용하면 시료의 정제 또는 전처리 과정 없이 시료 중 세포밖 소포체를 정확하게 분석할 수 있을 뿐만 아니라, 탐침의 종류에 따라 세포밖 소포체의 구성성분을 간단하고 정확하게 분석할 수 있기 때문에 세포밖 소포체를 이용한 진단 효율을 향상시킬 수 있다. 또한 탐침의 특이성 및 친화도 분석을 이용하여 세포밖 소포체 특이적 항체 스크리닝, 단백질 스크리닝, 화학물질 스크리닝 등에 활용할 수 있다.The method for analyzing extracellular ER of the present invention uses the size-specific fractionation ability of size exclusion chromatography and the characteristics of a probe that specifically binds to extracellular ER, and is used to quantitatively analyze the extracellular ER contained in the sample, The physicochemical characterization of the endoplasmic reticulum, the type and quantitative analysis of constituents contained in the extracellular endoplasmic reticulum, and the binding specificity or affinity analysis of the probe to the extracellular endoplasmic reticulum component can be quickly and easily performed. In addition, by using the assay method of the present invention, it is possible not only to accurately analyze the extracellular endoplasmic reticulum in the sample without purification or pretreatment of the sample, but also to analyze the constituents of the extracellular endoplasmic reticulum easily and accurately according to the type of the probe The diagnostic efficiency using the extracellular endoplasmic reticulum can be improved. In addition, using the probe specificity and affinity analysis, it can be used for extracellular endoplasmic reticulum-specific antibody screening, protein screening, and chemical screening.
도 1은 본 발명에 따른 세포밖 소포체 분석 방법에 대한 모식도이다.1 is a schematic diagram of a method for analyzing an extracellular ER according to the present invention.
도 2는 본 발명의 일실시예에 따른 대장암 세포주 SW480으로부터 표본 세포밖 소포체의 분리 모식도 및 분리한 결과이다.FIG. 2 is a schematic view showing the isolation and isolation of the extracellular endoplasmic reticulum from the colorectal cancer cell line SW480 according to an embodiment of the present invention.
도 3은 본 발명의 일실시예에 따른 형광물질로 표지된 마우스 항체 (Normal mouse IgG)와 표본 세포밖 소포체의 결합 양상을 확인한 UV(a) 및 형광(b) 크로마토그램이다. FIG. 3 is a UV (a) and fluorescence (b) chromatogram showing binding patterns of a fluorescent antibody-labeled mouse antibody (normal mouse IgG) and an extracellular endoplasmic reticulum in accordance with an embodiment of the present invention.
도 4는 본 발명의 일실시예에 따른 형광물질로 표지된CD63항체(anti-CD63 antibody)와 표본 세포밖 소포체의 결합 양상을 확인한 UV(a) 및 형광(b) 크로마토그램이다.FIG. 4 is a UV (a) and fluorescence (b) chromatogram showing the binding pattern of CD63 antibody (anti-CD63 antibody) labeled with a fluorescent substance according to an embodiment of the present invention.
도 5는 본 발명의 일실시예에 따른 형광물질로 표지된CD81항체(anti-CD63 antibody)와 표본 세포밖 소포체의 결합 양상을 확인한 UV(a) 및 형광(b) 크로마토그램이다.FIG. 5 is a UV (a) and fluorescence (b) chromatogram showing the binding pattern of CD81 antibody (anti-CD63 antibody) labeled with a fluorescent substance according to an embodiment of the present invention.
도 6은 본 발명의 일실시예에 따른 표본 세포밖 소포체에서 형광 표지된 CD63 항체의 특이성을 확인한 형광 크로마토그램이다.FIG. 6 is a fluorescence chromatogram showing the specificity of a fluorescently labeled CD63 antibody in a specimen extracellular endoplasmic reticulum according to an embodiment of the present invention.
도 7은 본 발명의 일실시예에 따른 기원이 다른 세포밖 소포체에서 CD81 세포밖 소포체 마커의 발현 양상을 확인한 UV(a) 및 형광(b) 크로마토그램이다.FIG. 7 is a UV (a) and fluorescence (b) chromatogram showing the expression pattern of the CD81 extracellular ER marker in the extracellular endoplasmic reticulum of different origin according to an embodiment of the present invention.
도 8은 본 발명의 일실시예에 따른 TNFa(Tumor necrosis factor alpha) 가 대장암세포에서 분비되는 세포밖 소포체의 ICAM1 단백질 발현에 미치는 영향을 분석한 UV (a) 및 형광 (b, c) 크로마토그램이다.8 is a graph showing the effect of TNFa (Tumor necrosis factor alpha) on the expression of ICAM1 protein in extracellular endoplasmic reticulum secreted from colon cancer cells according to one embodiment of the present invention. to be.
도 9는 본 발명의 일실시예에 따른 형광이 표지된 CD63 항체를 이용하여 대장암 세포 배양액으로부터 세포밖 소포체의 분리 과정 없이 세포밖 소포체를 확인한 형광 크로마토그램이다.FIG. 9 is a fluorescence chromatogram showing the extracellular endoplasmic reticulum without isolation of the extracellular endoplasmic reticulum from the colon cancer cell culture fluid using the fluorescence-labeled CD63 antibody according to an embodiment of the present invention.
도 10은 본 발명의 일실시예에 따른 형광이 표지된 CD81 항체를 이용하여 대장암 세포 배양액으로부터 세포밖 소포체의 분리 과정 없이 세포밖 소포체를 확인한 UV(a) 및 형광(b) 크로마토그램이다.FIG. 10 is a UV (a) and fluorescence (b) chromatogram showing the extracellular endoplasmic reticulum without the process of separating the extracellular endoplasmic reticulum from the colon cancer cell culture fluid using the fluorescently labeled CD81 antibody according to an embodiment of the present invention.
도 11은 본 발명의 일실시예에 따른 형광이 표지된 CD81 항체를 이용하여 배양 시간을 달리한 대장암 세포 배양액으로부터 세포밖 소포체의 분리 과정 없이 시료내 세포밖 소포체를 나노입자농도(a) 및 UV(b), 형광(c) 크로마토그램으로 분석한 결과이다.FIG. 11 is a graph showing the relationship between the concentration of nanoparticles (a) and the concentration of the extracellular endoplasmic reticulum in the sample without the separation of the extracellular endoplasmic reticulum from the culture medium of colon cancer cells cultured at different incubation times using fluorescently labeled CD81 antibody according to an embodiment of the present invention. UV (b), and fluorescence (c) chromatograms.
도 12는 본 발명의 일실시예에 따른 에스테르분해효소 활성에 의하여 형광을 나타내는 막투과성VPD450(violet proliferation dye450)과 표본 세포밖 소포체 내 에스테르분해효소 활성을 이용한 세포밖 소포체의 분석을 나타낸 UV(a) 및 형광(b, c) 크로마토그램이다.FIG. 12 is a graph showing the results of analysis of the transmembrane VPD450 (violet proliferation dye 450) showing the fluorescence by the esterase activity and the extracellular endoplasmic reticulum using the esterase activity in the endoplasmic reticulum endoplasmic reticulum ) And fluorescence (b, c) chromatograms.
도 13은 본 발명의 일실시예에 따른 에스테르분해효소 활성에 의하여 형광을 나타내는 막투과성VPD450(violet proliferation dye450)과 세포밖 소포체 내 에스테르분해효소 활성을 이용하여 대장암 세포 배양액으로부터 세포밖 소포체의 분리 과정 없이 시료내 세포밖 소포체를 UV(b) 및 형광(c) 크로마토그램으로 분석한 결과이다.FIG. 13 is a graph showing the results of the isolation of extracellular endoplasmic reticulum from colon cancer cell culture using VPD450 (violet proliferation dye 450) showing fluorescence by the esterase activity according to an embodiment of the present invention and the esterase activity in the extracellular endoplasmic reticulum (B) and fluorescence (c) chromatograms of the extracellular endoplasmic reticulum in the sample.
도 14는 본 발명의 일실시예에 따른 에스테르분해효소 활성에 의하여 형광을 나타내는 막투과성VPD450(violet proliferation dye450)과 회전식 크기 배제 크로마토그래피를 이용하여 형광물질이 결합된 표본 세포밖 소포체를 UV 및 형광(b) 크로마토그램으로 분석한 결과이다.FIG. 14 is a graph showing the results of fluorescence spectroscopy using a membrane-permeable VPD450 (violet proliferation dye 450) exhibiting fluorescence by an esterase activity according to an embodiment of the present invention and a fluorescent size- (b) chromatogram analysis.
도 15는 본 발명의 일실시예에 따른 에스테르분해효소 활성에 의하여 형광을 나타내는 막투과성CFDA-SE(carboxyfluorescein diacetate succinimidyl ester)　와 회전식 크기 배제 크로마토그래피를 이용하여 형광물질이 결합된 표본 세포밖 소포체를 UV 및 형광(b) 크로마토그램으로 분석한 결과이다.FIG. 15 is a graph showing the fluorescence-labeled extracellular endoplasmic reticulum (ERCP) conjugated with a transmembrane carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) showing fluorescence by the esterase activity according to an embodiment of the present invention and a rotary size exclusion chromatography UV and fluorescence (b) chromatogram.
도 16은 본 발명의 일실시예에 따른 비오틴-콜레스테롤(Biotin-cholesterol)과 반응시킨 표본 세포밖 소포체를 회전식 크기 배제 크로마토그래피를 이용하여 분리한 비오틴-콜레스테롤-세포밖 소포체 복합체의 양을 분석한 결과이다.FIG. 16 is a graph showing the amount of biotin-cholesterol-extracellular < RTI ID = 0.0 > extracellular < / RTI > endoplasmic reticulum complexes isolated from a sample extracellular endoplasmic reticulum reacted with biotin-cholesterol according to an embodiment of the present invention Results.
도 17은 본 발명의 일실시예에 따른 비오틴-콜레스테롤(Biotin-cholesterol)과 반응시킨 대장암 세포 배양액을 회전식 크기 배제 크로마토그래피를 이용하여 분리한 비오틴-콜레스테롤-세포밖 소포체 복합체의 양을 분석한 결과이다.17 is a graph showing the amount of a biotin-cholesterol-extracellular < RTI ID = 0.0 > extracellular < / RTI > elastomer complex isolated from a colon cancer cell culture solution reacted with biotin-cholesterol according to an embodiment of the present invention using a rotary size exclusion chromatography Results.
도 18은 본 발명의 일실시예에 따른 비오틴-콜레스테롤(Biotin-cholesterol)과 반응시킨, 인간 오줌(b), 인간 혈청(c)을 회전식 크기 배제 크로마토그래피를 이용하여 분리한 비오틴-콜레스테롤-세포밖 소포체 복합체의 양을 분석한 결과이다.FIG. 18 is a graph showing the results of human urine (b), human serum (c) reacted with biotin-cholesterol according to an embodiment of the present invention, and biotin-cholesterol- And the amount of the outer vesicle complex.
도 19는 본 발명의 일실시예에 따른 DiI(Lipophilic dye)과 반응시킨 표본 세포밖 소포체를 HPLC를 이용하여 분석한 형광 크로마토그램이다.FIG. 19 is a fluorescence chromatogram of an extracellular endoplasmic reticulum reacted with DiI (lipophilic dye) according to an embodiment of the present invention by HPLC.
도 20은 본 발명의 일실시예에 따른 DiI(Lipophilic dye)과 반응시킨 대장균 유래 세포밖 소포체를 HPLC를 이용하여 분석한 형광 크로마토그램이다.FIG. 20 is a fluorescence chromatogram of an extracellular endoplasmic reticulum derived from Escherichia coli reacted with DiI (lipophilic dye) according to an embodiment of the present invention by HPLC.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
실시예 1. 표본 세포밖 소포체의 정제 및 분석Example 1. Purification and analysis of extracellular endoplasmic reticulum
대장암 세포주 SW480 배양액을 500 xg에서 10분, 2,000 xg에서 20분 간 원심분리하여 침전물을 제거하였다. 상기 상층액에 존재하는 세포밖 소포체를 1차 정제 및 침전하기 위하여, 세포밖 소포체를 폴리에틸렌글리콜 용액(8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH7.4)을 첨가하여 16시간 동안 냉장 보관한 후, 12,000 xg에서 30분간 원심분리하여 침전된 세포밖 소포체를 수확 후 HEPES 완충용액(HEPES-buffered saline, 20 mM HEPES, 150 mM NaCl, pH7.4)에 침전물을 녹였다.The colon cancer cell line SW480 culture was centrifuged at 500 xg for 10 minutes and at 2,000 xg for 20 minutes to remove the precipitate. The extracellular endoplasmic reticulum was added with polyethylene glycol solution (8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) for the first purification and precipitation of the extracellular endoplasmic reticulum present in the supernatant for 16 hours After refrigerated, the extracellular endoplasmic reticulum was centrifuged at 12,000 xg for 30 minutes and the precipitate was dissolved in HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4).
밀도 및 부력을 이용하여 세포밖 소포체를 2차 정제하기 위하여 상기 시료에 대하여 30% - 20% - 5% 옵티프랩(Optiprep) 부력 밀도 구배 초원심분리를 200,000 xg에서 2 시간 동안 수행하였다. 초원심분리 후 세포밖 소포체와 상등 밀도(1.08 ~ 1.12 g/ml) 영역을 수확하였다. 상기 정제된 세포밖 소포체를 3차 정제하기 위하여, 고성능 액체 크로마토그래피(HPLC)를 이용하여 세파크릴(Sephacryl) S500으로 충전된 컬럼(10 x 100 mm)에 주입하고 분자 크기 배제 크로마토그래피를 통해 최종 세포밖 소포체 정제하였고 본 표본 세포밖 소포체 분리 과정을 도 2(a)에 나타내었다. 20% - 5% Optiprep buoyancy density gradient ultracentrifugation was performed on the samples for 2 hours at 200,000 xg for secondary purification of the extracellular endoplasmic reticulum using density and buoyancy. After ultracentrifugation, the extracellular endoplasmic reticulum was harvested at an equal density (1.08 ~ 1.12 g / ml). The purified extracellular endoplasmic reticulum was injected into a column packed with Sephacryl S500 (10 x 100 mm) using high performance liquid chromatography (HPLC) and subjected to molecular size exclusion chromatography The extracellular endoplasmic reticulum was purified and the process of separating the endoplasmic reticulum endoplasmic reticulum was shown in FIG. 2 (a).
상기 2차 정제 방법의 부력 밀도 구배 초원심분리 후 얻은 시료의 표면으로부터 10개의 영역의 분획물을 수확하였고, 각 분획물에서 세포밖 소포체 마커 (Alix, CD9, CD81, CD63)들의 분포를 웨스턴블럿을 통하여 확인한 후 이를 도 2(b)에 나타내었다. 상기 3차 정제 방법을 따라 최종 분리한 표본 세포밖 소포체를 투과 전자 현미경(TEM)을 이용하여 관찰한 결과 정제한 표본 세포밖 소포체의 모양 및 크기(약 50 ~ 200 nm)를 확인할 수 있었다(도 2(c)). The fractions of 10 regions were harvested from the surface of the sample after buoyancy density gradient ultracentrifugation of the second purification method and the distribution of extracellular ER marker (Alix, CD9, CD81, CD63) in each fraction was collected by Western blot This is shown in Fig. 2 (b). The out-of-sample extracellular endoplasmic reticulum after the third purification method was observed using a transmission electron microscope (TEM), and the shape and size (about 50 to 200 nm) of the purified endoplasmic reticulum were confirmed 2 (c)).
실시예 2. 펌프식 크기 배제 크로마토그래피와 형광으로 표지된 항체를 이용한 표본 세포밖 소포체의 분석Example 2. Analysis of specimen extracellular endoplasmic reticulum using pumped size exclusion chromatography and fluorescently labeled antibodies
크기 배제 크로마토그래피의 크기별 분획능과 세포밖 소포체의 구성 성분을 인식하는 다양한 탐침의 양을 정량함으로써 세포밖 소포체의 구성성분의 양을 분석할 수 있음을 규명하기 위해, 하기와 같은 실험을 수행하였다.The following experiments were carried out in order to determine the amount of components of the extracellular endoplasmic reticulum by quantifying the size of the size-exclusion chromatography fraction and the amount of various probes recognizing the constituents of the extracellular endoplasmic reticulum .
2-1. 형광 표지된 마우스 항체(normal mouse IgG)2-1. Fluorescently labeled mouse antibodies (normal mouse IgG)
정제된 표본 세포밖 소포체와 형광 표지된 마우스 항체(normal mouse IgG)를 혼합하여 37℃에서 30분 간 반응시킨 후 세파크릴 S500으로 채워진 컬럼에 주입하고 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램(도 3(a))과 형광 크로마토그램(도 3(b))을 분석하였다. Purified specimen extracellular endoplasmic reticulum and fluorescent mouse antibody (normal mouse IgG) were mixed and incubated at 37 ° C for 30 minutes. The reaction mixture was injected into a column packed with Sephacryl 500 and analyzed using a 280 nm absorption spectrophotometer 3 (a)) and a fluorescence chromatogram (Fig. 3 (b)) were analyzed.
그 결과, 흡광 크로마토그램 결과로부터 표본 세포밖 소포체가 6.5 분에 검출되고, 14.5분에 항체가 검출되는 것을 확인할 수 있었다. 그러나 형광 크로마토그램에서 14.5분에 형광 표지된 항체의 형광 밴드는 검출되었으나, 6.5분 분획에 해당하는 표본 세포밖 소포체의 형광 밴드가 나타나지 않았다. 이는 표본 세포밖 소포체와 마우스 항체가 해당 조건에서 비특이적 결합을 하지 않음을 의미하며, 또한 함께 주입된 표본 세포밖 소포체와 마우스 항체의 혼합물은 크기 배제 크로마토그래피에 의하여 효과적으로 분리되어 용출되었다는 것을 알 수 있었다.As a result, the extracellular endoplasmic reticulum was detected at 6.5 minutes and the antibody was detected at 14.5 minutes from the absorbance chromatogram results. However, the fluorescence band of the fluorescently labeled antibody was detected at 14.5 minutes in the fluorescence chromatogram, but the fluorescence band of the extracellular endoplasmic reticulum corresponding to the 6.5 minute fraction was not observed. This indicates that the sample extracellular endoplasmic reticulum and the mouse antibody did not undergo nonspecific binding under the conditions, and that the mixture of injected sample extracellular endoplasmic reticulum and mouse antibody was effectively separated by size exclusion chromatography and eluted .
2-2. 형광 표지된 항CD63 및 항CD81 항체(aCD63 and aCD81 antibody)2-2. Fluorescently labeled anti-CD63 and anti-CD81 antibodies (aCD63 and aCD81 antibody)
상기 정제된 다양한 양의 표본 세포밖 소포체와 세포밖 소포체의 막 단백질(membrane surface protein) CD63을 인식하는 형광 표지된 항CD63 항체(aCD63 antibody)를 혼합하여 37℃에서 30분 간 반응시킨 후 세파크릴 S500으로 채워진 컬럼에 주입하고 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램(도 4(a))과 형광 크로마토그램(도 4(b))을 분석하였다. The purified various amounts of the specimen extracellular endoplasmic reticulum and the fluorescently labeled anti-CD63 antibody (aCD63 antibody) recognizing the membrane surface protein CD63 of the extracellular endoplasmic reticulum were mixed and incubated at 37 ° C for 30 minutes, The column was filled with S500 and analyzed using 280 nm absorption spectrophotometer (FIG. 4 (a)) and fluorescence chromatogram (FIG. 4 (b)) while developing using an HPLC system.
또한, 상기 정제된 다양한 양의 표본 세포밖 소포체와 세포밖 소포체의 또 다른 막 단백질 CD81을 인식하는 형광 표지된 항CD81 항체(aCD81 antibody)를 혼합하여 상기와 동일한 조건으로 크기 배제 크로마토그래피를 전개하면서 280 nm 흡광 크로마토그램(도 5(a))과 형광 크로마토그램(도 5(b))을 분석하였다. Further, the purified various amounts of the specimen extracellular endoplasmic reticulum and the fluorescence labeled anti-CD81 antibody (aCD81 antibody) recognizing another membrane protein CD81 of the extracellular endoplasmic reticulum were mixed and subjected to size exclusion chromatography under the same conditions as above The absorption spectrophotometer (FIG. 5 (a)) and the fluorescence chromatogram (FIG. 5 (b)) were analyzed at 280 nm.
그 결과, 해당 컬럼에서 6.5 분에 검출되는 표본 세포밖 소포체의 280 nm 흡광 밴드와 동일한 검출 시간에 형광 밴드가 나타나고 검출된 밴드의 면적은 표본 세포밖 소포체의 주입량과 높은 상관관계를 보이는 것을 확인할 수 있었다. 반면, 14.5분에 검출되는 형광 표지된 자유 항체의 형광 밴드 면적이 주입된 표본 세포밖 소포체의 양에 반비례하여 감소하는 것을 알 수 있었다. 이는 표본 세포밖 소포체에 혼합한 항체가 세포밖 소포체의 구성성분(CD63, CD81)을 특이적으로 인식하여 각각 CD63 또는 CD81과 결합함으로써 "세포밖 소포체-항체 복합체"를 형성하고, 상대적으로 분자량이 작은 형광 표지된 항체(14.5분에 검출)가 거대 분자인 세포밖 소포체와 함께 전개되어 세포밖 소포체의 검출 시간(6.5분)에 형광 밴드가 검출되었음을 뜻한다. 또한 형광 표지된 자유 항체의 검출 시간에 검출되는 형광 밴드의 면적은 시료에 혼합한 항체의 총량에서 세포밖 소포체와 결합한 항체의 양을 제외한 자유 항체의 양을 반영함을 알 수 있다. As a result, fluorescent bands appeared at the same detection time as the 280 nm absorbance band of the specimen extracellular endoplasmic reticulum detected at 6.5 minutes in the corresponding column, and the area of the detected bands was highly correlated with the injection amount of the extracellular endoplasmic reticulum there was. On the other hand, the fluorescent band area of the fluorescently labeled free antibody detected at 14.5 minutes decreased in inverse proportion to the amount of injected specimen extracellular endoplasmic reticulum. This is because the antibody mixed in the specimen extracellular endoplasmic reticulum specifically recognizes the components (CD63, CD81) of the extracellular endoplasmic reticulum and binds to CD63 or CD81 respectively to form an " extracellular endoplasmic reticulum-antibody complex " A small fluorescently labeled antibody (detected at 14.5 min) developed along with a macromolecular extracellular envelope, indicating that the fluorescent band was detected at the time of detection of the extracellular endoplasmic reticulum (6.5 min). In addition, the area of the fluorescent band detected at the detection time of the fluorescently labeled free antibody reflects the amount of the free antibody except for the amount of the antibody bound to the extracellular endoplasmic reticulum in the total amount of the antibody mixed in the sample.
2-3. 항CD63 항체 경쟁적 결합2-3. Anti-CD63 antibody competitive binding
상기에서 형광 표지된 항CD63 항체가 표본 세포밖 소포체에 존재하는 CD63을 특이적으로 인식하여 복합체를 형성할 때 형광 표지가 특이적 결합에 미치는지 여부를 확인하기 위해, 형광 표지된 항CD63 항체와 표지 없는 항CD63 항체를 혼합하여 반응시켰다. 구체적으로, 상기 표본 세포밖 소포체에 형광 표지된 항 CD63 항체를 혼합한 군과, 표본 세포밖 소포체에 형광 표지된 항 CD63 항체와 형광 표지되지 않은 항 CD63 항체를 1:10의 비율로 함께 혼합하여 반응시킨 군에서 크기 배제 크로마토그래피를 통한 형광 크로마토그램을 비교하였다. In order to confirm whether the fluorescently labeled anti-CD63 antibody specifically binds to the fluorescent label when the complex is formed by recognizing CD63 specifically present in the extracellular endoplasmic reticulum, fluorescence labeled anti-CD63 antibody and label Anti-CD63 antibody was mixed and reacted. Specifically, the fluorescence labeled anti-CD63 antibody and the non-fluorescently labeled anti-CD63 antibody were mixed together in a ratio of 1:10 to the specimen extracellular endoplasmic reticulum, Fluorescence chromatograms were compared by size exclusion chromatography in the reacted groups.
그 결과, 상기 형광 표지되지 않은 과량의 항 CD63항체를 추가로 첨가한 군에서 세포밖 소포체-항체 복합체의 형광 밴드가 크게 감소한 것을 확인할 수 있었다(도 6). 이는 과량의 형광 표지되지 않은 항 CD63항체가 세포밖 소포체의 CD63과 결합하여 형광 표지된 항 CD63 항체의 결합을 방해함으로써 세포밖 소포체에 결합된 형광 표지된 항 CD63 항체의 양이 크게 줄었음을 의미하고, 이는 세포밖 소포체와 항 CD63 항체의 결합이 매우 특이적이며 형광 표지가 항 CD63 항체의 기능에 영향이 없음을 알 수 있었다.As a result, it was confirmed that the fluorescence band of the extracellular endoplasmic reticulum-antibody complex was greatly reduced in the group to which the above-mentioned non-fluorescent-labeled excessive amount of the anti-CD63 antibody was added (FIG. 6). This indicates that the excess of non-fluorescently labeled anti-CD63 antibody binds to CD63 of the extracellular vesicle and interferes with the binding of the fluorescently labeled anti-CD63 antibody, thereby greatly reducing the amount of fluorescently labeled anti-CD63 antibody bound to the extracellular endoplasmic reticulum , Suggesting that the binding of the extracellular ER and anti - CD63 antibody is highly specific and that the fluorescent label does not affect the function of the anti - CD63 antibody.
2-4. 서로 다른 모세포에서 분비된 세포밖 소포체와 형광 표지된 항CD81 항체2-4. The extracellular endoplasmic reticulum and fluorescence-labeled anti-CD81 antibody secreted from different blastocysts
동량의 서로 다른 모세포(SW480, HMEC1)에서 분비된 세포밖 소포체에 동량의 형광 표지된 항 CD81 항체를 혼합하여, 각 세포밖 소포체에서 CD81 단백질의 발현 양상을 상기 크기 배제 크로마토그래피 방법을 이용하여 280 nm 흡광 크로마토그램(도 7(a))과 형광 크로마토그램(도 7(b))으로 분석하였다. The same amount of fluorescently labeled anti-CD81 antibody was mixed with the extracellular endoplasmic reticulum secreted from the same amount of different parental cells (SW480, HMEC1), and the expression pattern of CD81 protein in each extracellular endoplasmic reticulum was determined by 280 (Fig. 7 (a)) and a fluorescence chromatogram (Fig. 7 (b)).
그 결과, 해당 컬럼에서 6.5 분에 검출되는 각 세포밖 소포체의 280 nm 흡광 밴드의 면적이 동일한 반면, 동일 검출 시간(6.5분)에서 각 세포밖 소포체와 항 CD81 항체 복합체의 형광 밴드의 면적이 상이하게 나타났다. SW480 대장암 세포 유래 세포밖 소포체에 비하여 HMEC1 세포 유래 세포밖 소포체에서 크게 낮았고, 반면 8.5 분의 자유 항체의 형광 밴드 면적은 HMEC1 세포 유래 시료에서 더 높게 나타남을 관찰할 수 있었다. 이는 모세포의 종류에 따라 단위 시료당 세포밖 소포체의 양이 상이하기 때문이며, 이로부터 본 발명의 분석법에 의하여 여러 종류의 세포밖 소포체의 분석에 있어 단위 세포밖 소포체 당 각 구성 성분의 상대량을 빠르게 분석할 수 있음을 알 수 있었다.As a result, the area of the 280 nm absorption band of each extracellular endoplasmic reticulum detected at 6.5 minutes in the column was the same, but the fluorescence band area of each of the extracellular endoplasmic reticulum and the anti-CD81 antibody complex was different at the same detection time (6.5 minutes) Respectively. In contrast, the extracellular endoplasmic reticulum of HMEC1 cells was significantly lower than that of SW480 colon cancer cells. On the other hand, the fluorescence band area of 8.5 min of free antibody was higher in HMEC1 cell-derived samples. This is because the amount of extracellular endoplasmic reticulum per unit sample differs depending on the type of the parent cell. Therefore, in the analysis of various kinds of extracellular endoplasmic reticulum by the assay method of the present invention, It can be analyzed.
2-5. TNFa 처리 유무에 따른 세포밖 소포체 성분 변화2-5. Changes in extracellular ER components with and without TNFa treatment
본 발명의 방법을 이용하여 TNFa가 대장암 세포로부터 분비되는 세포밖 소포체의 구성 성분 변화에 미치는 영향을 분석하였다. 구체적으로 대장암 세포 배양 시 TNFa를 24시간 처리한 군(TNFa+)과 처리하지 않은 군(TNFa-)으로 나누고, 각 배양액에서 세포밖 소포체를 종래의 방법으로 정제하였다. 각 군에서 정제한 세포밖 소포체에 형광(FITC) 표지된 항 CD81 항체와 형광(PE) 표지된 항 ICAM1 항체를 혼합하여 반응시키고, 상기 크기 배제 크로마토그래피 컬럼에 주입하여 전개하면서 280 nm 흡광 크로마토그램(도 8(a)) 및 형광 크로마토그램(도 8(b, c))을 분석하였다. The effect of TNFa on the composition of extracellular endoplasmic reticulum secreted from colon cancer cells was analyzed using the method of the present invention. Specifically, in the culture of colon cancer cells, TNFa was divided into 24 hours (TNFa +) and untreated (TNFa), and the extracellular endoplasmic reticulum was purified by conventional methods. The anti-CD81 antibody labeled with fluorescence (FITC) and the anti-ICAM1 antibody labeled with fluorescence (PE) were reacted to the extracellular endoplasmic reticulum purified in each group, and injected into the size exclusion chromatography column to develop a 280 nm absorbance chromatogram (Fig. 8 (a)) and a fluorescence chromatogram (Fig. 8 (b, c)).
그 결과, 해당 컬럼에서 3.6 분에 검출되는 각 세포밖 소포체의 280 nm 흡광 밴드의 면적(a)과 CD81 형광 밴드의 면적(b)이 유사한 반면, 동일 검출 시간(3.6분)에 ICAM1의 형광 밴드(c)가 TNFa+ 군에서TNFa- 군에 비하여 크게 증가하는 것을 확인할 수 있었다. 이로부터 TNFa의 처리 후 세포밖 소포체 중의 CD81 단백질의 양은 변화가 거의 없었으나, ICAM1의 양은 크게 증가한다는 사실을 알 수 있었다. 이로부터 동종의 세포라 하더라도 세포의 상태, 환경 또는 외부 인자에 의해 야기된 세포의 생리적 변화가 이로부터 분비된 세포밖 소포체의 구성 성분 변화를 이끌어 냄을 알 수 있었으며, 본 발명의 방법으로 쉽게 분석할 수 있다는 점을 확인할 수 있었다. As a result, the area (b) of the CD81 fluorescent band was similar to that of the 280 nm extinction band of each extracellular ER at 3.6 min in the corresponding column, while the fluorescence band of ICAM1 in the same detection time (3.6 min) (c) was significantly increased in the TNFa + group as compared to the TNFa group. From this, it was found that the amount of CD81 protein in the extracellular ER after treatment with TNFa was not changed but the amount of ICAM1 was greatly increased. From these results, it can be seen that physiological changes of cells caused by the cell state, environment or external factors lead to a change in constituents of the extracellular endoplasmic reticulum secreted from the cells even in the same kind of cells. I can confirm that I can.
종합하여, 본 분석 방법을 통하여 간편하고 신속하게 시료 내 세포밖 소포체의 총량 및 세포밖 소포체의 성분 분석뿐 아니라, 세포밖 소포체의 구성 성분에 대한 항체 및 리간드의 특이성 및 친화도 분석에도 활용될 수 있음을 알 수 있다.In addition, this assay can be used to analyze not only the total amount of extracellular endoplasmic reticulum and the components of extracellular endoplasmic reticulum but also the specificity and affinity of antibodies and ligands to components of extracellular endoplasmic reticulum easily and rapidly .
실시예 3. 펌프식 크기 배제 크로마토그래피와 형광으로 표지된 항체를 이용한 세포 배양액 내 세포밖 소포체의 분석Example 3. Analysis of extracellular endoplasmic reticulum in cell culture using pumped size exclusion chromatography and fluorescently labeled antibodies
3-1. 형광 표지된 항C63 항체3-1. Fluorescently labeled anti-C63 antibody
본 발명의 분석 방법을 이용하여 세포 배양액에서 세포밖 소포체의 양 또는 구성 성분을 분석하기 위하여, SW480 대장암 세포를 RPMI 배양 배지에서 24시간 배양하여 세포 배양액을 수확하였다. 세포를 배양하지 않은 RPMI 배양 배지와 형광 표지된 항 CD63 항체를 혼합한 군, 대장암 세포 배양액과 형광 표지된 항 CD63 항체를 혼합한 군 각각을 37℃에서 30분 간 반응시키고 TSK6000 HPLC컬럼에 주입한 후 HPLC 시스템을 이용해 전개하면서 형광 크로마토그램을 분석하였다. In order to analyze the amount or components of extracellular ER in cell culture using the assay method of the present invention, SW480 colon cancer cells were cultured in RPMI culture medium for 24 hours to harvest the cell culture medium. The RPMI culture medium in which the cells were not cultured was mixed with the fluorescently labeled anti-CD63 antibody, and the mixture of the colon cancer cell culture medium and the fluorescence-labeled anti-CD63 antibody was reacted at 37 ° C for 30 minutes and injected into the TSK6000 HPLC column The fluorescence chromatograms were analyzed while developing using an HPLC system.
그 결과, 도 9에 나타난 바와 같이 RPMI 배양 배지와 형광 표지된 항 CD63 항체를 혼합한 군에서는 22 분에 검출되는 자유 항체의 형광 밴드만 검출된 반면, 대장암 세포 배양액과 형광 표지된 항 CD63 항체를 혼합한 군에서는 22 분에 검출되는 자유 항체의 형광 밴드와 더불어 17분에 검출되는 형광 밴드가 추가로 검출되었다. 또한, 대장암 세포 배양액이 포함된 시료에서 22 분에 검출된 형광 밴드의 면적이 RPMI 배양 배지와 형광 표지된 항 CD63 항체를 혼합한 군의 형광 밴드 면적의 약 50% 임을 확인할 수 있었다. 이는 형광 표지된 항 CD63 항체의 약 50% 가량이 대장암 세포 배양액 내 세포밖 소포체의 CD63 단백질과 특이적으로 결함하여 "세포밖 소포체-항체 복합체"를 형성하여 세포밖 소포체와 함께 전개되었기 때문에 17분의 형광 밴드가 형성된 것이다. 따라서 상기 분석 방법을 이용하면 세포 배양액으로부터 세포밖 소포체를 별도로 정제하지 않고도 세포 배양액 내 세포밖 소포체의 성분을 분석할 수 있음을 알 수 있었다. As a result, as shown in Fig. 9, only the fluorescent band of the free antibody detected at 22 minutes was detected in the RPMI culture medium and the fluorescence-labeled anti-CD63 antibody mixed group, whereas the fluorescent-labeled anti-CD63 antibody Was detected in addition to the fluorescent band of free antibody detected at 22 minutes and fluorescence band detected at 17 minutes. In addition, it was confirmed that the area of the fluorescent band detected at 22 minutes in the sample containing the culture medium of the colon cancer cell was about 50% of the fluorescence band area of the mixture of the RPMI culture medium and the fluorescently labeled anti-CD63 antibody. This is because about 50% of the fluorescently labeled anti-CD63 antibody specifically fused with the CD63 protein of the extracellular endoplasmic reticulum in the culture medium of the cell culture to form an " extracellular endoplasmicant-antibody complex " Minute fluorescent bands are formed. Therefore, it was found that the above-described analytical method can analyze the components of the extracellular endoplasmic reticulum in the cell culture fluid without separately purifying the extracellular endoplasmic reticulum from the cell culture fluid.
3-2. 형광 표지된 항CD81 항체3-2. Fluorescently labeled anti-CD81 antibody
상기 3-1과 유사하게, 상기 대장암 세포의 배양액과 형광 표지된 항 CD81항체를 혼합하여 37℃에서 30분 간 반응시키고 세파크릴 S500 컬럼에 주입한 후 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램(도 10(a)) 및 형광 크로마토그램(도 10(b))을 분석하였다.Similar to the above-mentioned 3-1, the culture medium of the colon cancer cells and the fluorescently labeled anti-CD81 antibody were mixed and incubated at 37 ° C for 30 minutes. After the cells were injected into the Sepafrill S500 column and developed using an HPLC system, (FIG. 10 (a)) and a fluorescence chromatogram (FIG. 10 (b)) were analyzed.
그 결과, 해당 컬럼에서 3.5분에 280 nm 흡광 밴드와 형광 밴드가 동시에 검출되었다. 이는 CD81 단백질을 발현하는 세포밖 소포체임을 알 수 있었고, 별도의 세포밖 소포체 정제 과정 없이 시료내 세포밖 소포체 구성 성분을 분석할 수 있음을 확인하였다.As a result, a 280 nm absorbance band and a fluorescence band were simultaneously detected in the corresponding column at 3.5 minutes. It was found that this was an extracellular vesicle expressing the CD81 protein and that the extracellular vesicle components in the sample can be analyzed without separate extracellular vesicle purification.
3-3. 배양 시간에 따른 세포밖 소포체 분석3-3. Analysis of extracellular endoplasmic reticulum by incubation time
세포가 자라는 동안 분비되는 세포밖 소포체의 양상을 분석하기 위하여, 배양 시간을 달리한 대장암 세포 배양액에 각각 동량의 형광 표지된 항 CD81항체를 혼합하여 37℃에서 30분 간 반응시키고 세파크릴 S500 컬럼에 주입한 후 HPLC 시스템을 이용해 전개하였다. 각 세포 배양액에 대해 나노 입자 농도 분석(도 11(a)), 상기 크기 배제 크로마토그래피를 통한 280 nm 흡광 크로마토그램(도 11(b)) 및 형광 크로마토그램(도 11(c))을 분석하였다.In order to analyze the state of the extracellular endoplasmic reticulum secreted during cell growth, the same amount of fluorescently labeled anti-CD81 antibody was added to each culture medium of colon cancer cells at different incubation times, and the cells were incubated at 37 ° C for 30 minutes. And developed using an HPLC system. 11 (a)), a 280 nm absorbance chromatogram (FIG. 11 (b)) and a fluorescence chromatogram (FIG. 11 (c)) by the size exclusion chromatography were analyzed for each cell culture medium .
그 결과, 나노 입자 농도 분석을 통하여 대장암 세포의 배양 시간이 증가함에 따라 배양액 내 나노 입자 농도가 증가되는 것을 알 수 있었다. 아울러 크기 배제 크로마토그래피 결과, 대장암 세포 배양 시간이 증가함에 따라 세포밖 소포체가 용출되는 3.6분에 280 nm 흡광 밴드 및 CD81 항체 형광 밴드가 동시에 증가하는 것을 확인하였고, 이는 배양액 내 나노 입자 농도 증가와 높은 상관관계를 가짐을 확인할 수 있었다. 이로부터 본 발명의 방법을 이용하면 세포밖 소포체의 추가 분리 과정 없이 시료 내 세포밖 소포체의 상대량 및 구성 성분의 분석을 동시에 할 수 있음을 알 수 있었다. As a result, it was found that the concentration of nanoparticles in the culture solution increased as the incubation time of the colon cancer cells increased. As a result of size exclusion chromatography, it was confirmed that 280 nm absorbance band and CD81 antibody fluorescence band were simultaneously increased at 3.6 minutes in which the extracellular endoplasmic reticulum eluted with increasing colon cancer cell culture time, And it was confirmed that they had a high correlation. From this, it can be seen that the method of the present invention can simultaneously analyze the amount and composition of the extracellular endoplasmic reticulum in the sample without further separation of the extracellular endoplasmic reticulum.
실시예 4. 펌프식 크기 배제 크로마토그래피와 막 투과성 효소 기질을 이용한 표본 세포밖 소포체의 분석Example 4. Analysis of specimen extracellular endoplasmic reticulum using pumped size exclusion chromatography and transmembrane enzyme substrate
본 발명의 방법에 있어서 세포밖 소포체의 내부 성분을 인식하는 탐침 또는 효소를 사용함으로써 세포밖 소포체 구성성분 정량 분석의 가능성을 확인하기 위해 세포밖 소포체 내부에 존재하는 효소 중 하나인 에스테르분해효소(esterase)의기질로서 막 투과성이 있고 효소 활성에 의해 형광물질로 변환되는 물질인 VPD450을 사용하였다. 구체적으로 상기 정제된 표본 세포밖 소포체와 형광 표지된 항 CD81항체 및 다양한 농도의 VPD450을 혼합하여 37℃에서 30분 간 반응시키고 세파크릴 S500 컬럼에 주입한 후 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램(도 12(a)) 및 형광 크로마토그램(도 12(b, c))을 분석하였다.In order to confirm the possibility of quantitative analysis of extracellular vesicle components by using a probe or an enzyme which recognizes the internal components of the extracellular endoplasmic reticulum in the method of the present invention, esterase ), VPD450, which is a substance that is transmissive and is converted into a fluorescent substance by enzyme activity, is used. Specifically, the purified endoplasmic reticulum endoplasmic reticulum, fluorescently labeled anti-CD81 antibody, and various concentrations of VPD450 were mixed and reacted at 37 ° C for 30 minutes. The resulting solution was injected into a Sepafrill S500 column and developed using an HPLC system. (Fig. 12 (a)) and a fluorescence chromatogram (Fig. 12 (b, c)).
그 결과, 280 nm 흡광 밴드를 통해 해당 컬럼에서 표본 세포밖 소포체는 3.5분에 검출되는 것을 알 수 있었으며, 형광 크로마토그램 결과에서 같은 용출시간 대에 항 CD81 항체의 형광 밴드 및 VPD450의 형광 밴드가 동시에 나타났다. 이로써, 상기 표본 세포밖 소포체는 CD81 단백질과 에스테르분해효소를 모두 발현함을 알 수 있었다. 더불어 VPD450의 형광 밴드의 면적은 VPD450의 농도에 비례하였으며, VPD450 농도가 증가함에 따라 형광을 띄는 효소 활성 산물이 세포밖 소포체 내에 누적됨을 알 수 있었다. 한편, 자연적인 가수분해에 의하여 생성된 VPD450의 형광 산물(도 12(c) 중 늦게 나오는 피크)은 분자의 크기가 작아 크기 배제 크로마토그래피 방법을 통하여 명확하게 구분됨을 알 수 있었다. As a result, it was found that the extracellular endoplasmic reticulum in the corresponding column was detected at 3.5 minutes through the 280 nm absorbance band. In the fluorescence chromatogram results, the fluorescence band of the anti-CD81 antibody and the fluorescence band of VPD450 were simultaneously appear. As a result, it was found that the extracellular endoplasmic reticulum expresses both the CD81 protein and the esterase. In addition, the area of the fluorescent band of VPD450 was proportional to the concentration of VPD450. As the concentration of VPD450 increased, the fluorescent active product was accumulated in the extracellular endoplasmic reticulum. On the other hand, the fluorescence product of VPD450 (late peak in FIG. 12 (c)) produced by natural hydrolysis was clearly distinguished by size exclusion chromatography method because of its small molecular size.
실 room 시예 5. 펌프식 크기 배제 크로마토그래피와 막투과성 효소 기질을 이용한 세포 배양액 내 세포밖 소포체의 분석5. Analysis of extracellular endoplasmic reticulum in cell culture using pump-type size exclusion chromatography and membrane permeable enzyme substrate
상기 실시예 4에서 증명된 세포밖 소포체의 내부에 존재하는 효소에 대한 기질을 이용하여 세포밖 소포체를 분석하는 방법이 생물학적 시료에서 세포밖 소포체의 추가 정제 없이도 가능함을 증명하고자 세포 배양액을 이용하여 실험하였다. 구체적으로 SW480대장암 세포 배양액을 상기 막 투과성 VPD450과 혼합하여 37℃에서 다양한 시간 동안 반응시킨 후 세파크릴 S500 컬럼에 주입 후 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램(도 13(a)) 및 형광 크로마토그램(도 13(b))을 분석하였다. In order to demonstrate that the method of analyzing the extracellular endoplasmic reticulum using a substrate for an enzyme present inside the extracellular endoplasmic reticulum as described in Example 4 is possible without further purification of the extracellular endoplasmic reticulum in the biological sample, Respectively. Specifically, SW480 colon cancer cell culture fluid was mixed with the above-mentioned membrane permeable VPD450, and reacted at 37 DEG C for various times. After injection into a CEPACRYL S500 column, a 280 nm absorbance chromatogram (FIG. 13 The fluorescence chromatogram (Fig. 13 (b)) was analyzed.
그 결과, 해당 컬럼에서 3.5분에 검출되는 표본 세포밖 소포체의 280 nm 흡광 밴드의 면적은 반응 시간에 관계없이 일정한 반면 형광 밴드는 반응 시간에 따라 증가하였다. 이는 세포밖 소포체 내부의 에스테르분해효소가 반응 시간에 비례하여 세포밖 소포체의 내부로 유입된 VPD450을 형광 물질로 전환하고, 전환된 형광물질이 세포밖 소포체 내부에 축적되기 때문이다. 상기 방법을 활용하여 기질의 양 또는 반응시간을 고정하여 시료를 분석하면 세포밖 소포체의 정제 없이도 생물학적 시료에서 세포밖 소포체의 총량 분석에 활용할 수 있음을 알 수 있었다.As a result, the area of the 280 nm extinction band of the specimen extracellular ER detected at 3.5 minutes in the column was constant regardless of the reaction time, while the fluorescence band was increased with the reaction time. This is because the esterase in the extracellular endoplasmic reticulum converts VPD450 introduced into the extracellular endoplasmic reticulum into a fluorescent substance in proportion to the reaction time, and the converted fluorescent substance accumulates inside the extracellular endoplasmic reticulum. Using the above method, it was found that the analysis of the sample by fixing the amount of the substrate or the reaction time can be used for the total amount of the extracellular endoplasmic reticulum in the biological sample without purification of the extracellular endoplasmic reticulum.
실시예 6. 회전식 크기 배제 크로마토그래피와 막투과성 효소 기질을 이용한 표본 세포밖 소포체의 분석Example 6. Analysis of extracellular endoplasmic reticulum using rotary size exclusion chromatography and transmembrane enzyme substrate
6-1. 표본 세포밖 소포체-형광VPD450 복합체6-1. Specimen extracellular endoplasmic reticulum-fluorescent VPD450 complex
상기 막 투과성 효소 기질을 이용한 세포밖 소포체의 분석 방법에 있어서, 도 14(a)에 나타낸 모식도와 같이 세포밖 소포체와 반응하지 않은 물질로부터 세포밖 소포체-형광VPD450 복합체를 분리한 후, 분리된 복합체를 분석하였다. 구체적으로, 표본 세포밖 소포체와 막투과성 기질인 VPD450을 혼합하여 반응시킨 후, 회전식 세파크릴 S500 컬럼에 로딩하고 원심분리를 통하여 용출액을 수확하였다. 상기 용출된 세포밖 소포체-형광VPD450 복합체를 세파크릴 S500 컬럼에 주입 후 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램 및 형광 크로마토그램(도 14(b))을 분석하였다. In the method of analyzing the extracellular endoplasmic reticulum using the above-mentioned transmembrane enzyme substrate, the extracellular endoplasmic reticulum-fluorescent VPD450 complex was separated from a substance which did not react with the extracellular endoplasmic reticulum as shown in the schematic diagram shown in FIG. 14 (a) Respectively. Specifically, the sample extracellular endoplasmic reticulum and VPD450, which is a transmembrane substrate, were mixed and reacted, then loaded on a rotary Cefacill S500 column and harvested by centrifugation. The eluted extracellular endoplasmic reticulum-fluorescent VPD450 complex was injected into a Sepafrill S500 column and analyzed using a HPLC system, while analyzing a 280 nm absorbance chromatogram and a fluorescence chromatogram (FIG. 14 (b)).
그 결과, VPD450과 반응시키지 않은 표본 세포밖 소포체는 3.5 분에 280 nm 흡광 밴드만 검출되고 동일 시간에 형광 밴드는 검출 되지 않았으나, 상기 회전식 크기 배제 크로마토그래피를 이용하여 전처리한 시료는 3.5분에 280 nm 흡광 밴드와 강한 VPD450 형광 밴드가 검출되는 것을 확인하였다. 이로부터 세포밖 소포체와 반응하지 않은 분자량이 작은 물질들이 전처리를 통하여 효과적으로 제거됨을 알 수 있었다.As a result, only the extinction band of 280 nm was detected at 3.5 minutes and the fluorescence band was not detected at the same time. However, the sample pretreated with the above-mentioned rotary size exclusion chromatography was 280 minutes at 3.5 minutes nm absorbance band and a strong VPD450 fluorescent band were detected. From these results, it was found that substances with low molecular weight that did not react with the extracellular endoplasmic reticulum were effectively removed through pretreatment.
6-2. 표본 세포밖 소포체-CFDA-SE 복합체6-2. Specimen extracellular ER-CFDA-SE complex
도 15(a)의 모식도에서 나타낸 바와 같이, 다른 에스테르분해효소 기질인 CFDA-SE를 표본 세포밖 소포체와 혼합한 반응 용액을 상기 6-의 방법에 따라 전처리한 후 세파크릴 S500 컬럼에 주입하고 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램 및 형광 크로마토그램(도 15(b))을 분석하였다. As shown in the schematic diagram of Fig. 15 (a), the reaction solution obtained by mixing CFDA-SE, another esterase substrate, with the extra-cellular endoplasmic reticulum was pre-treated according to the method of 6- System, the 280 nm absorption chromatogram and the fluorescence chromatogram (Fig. 15 (b)) were analyzed.
그 결과, 해당 컬럼에서 3.5분에 280 nm 흡광 밴드가 검출되었으며 동일한 검출 시간에 형광 밴드가 검출됨으로써 CFDA-SE 또한 VPD450과 동일한 기전에 따라 막을 투과하고 세포밖 소포체 내부의 에스테르분해효소의 활성에 의하여 형광물질로 세포밖 소포체의 내부에 축적됨을 알 수 있었다. As a result, a 280 nm absorbance band was detected in the corresponding column at 3.5 min, and a fluorescence band was detected at the same detection time. As a result, CFDA-SE also permeated the membrane according to the same mechanism as VPD450 and was activated by the activity of esterase in the extracellular endoplasmic reticulum It was found that the fluorescent substance accumulates inside the extracellular endoplasmic reticulum.
따라서 상기 세포밖 소포체를 분리하는 단계를 포함하는 방법을 이용하여, 시료 내 세포밖 소포체의 총량을 분석할 수 있을 뿐 아니라, 정제된 탐침-세포밖 소포체 복합체를 제공하여 세포밖 소포체를 이용한 다양한 추적 연구에 활용할 수 있다.Therefore, it is possible to analyze the total amount of extracellular endoplasmic reticulum in the sample by using the method including the step of isolating the extracellular endoplasmic reticulum, and also to provide a purified probe-extracellular endoplasmic reticulum complex, It can be used for research.
실시예 7. 회전식 크기 배제 크로마토그래피와 콜레스테롤 탐침(biotinylated cholesterol)을 이용한 표본 세포밖 소포체의 분석Example 7. Analysis of specimen extracellular endoplasmic reticulum using rotary size exclusion chromatography and biotinylated cholesterol
크기 배제 크로마토그래피 분석 방법을 이용해 세포밖 소포체의 구성 성분 중 지질이중층과 결합하거나 삽입(transmembrane)되는 탐침의 양을 정량함으로써 세포밖 소포체의 총량을 분석할 수 있음을 규명하기 위해, 탐침으로서 비오틴이 표지된 콜레스테롤(Biotin-cholesterol) 사용하였다.To confirm that the total amount of extracellular endoplasmic reticulum can be analyzed by quantitative determination of the amount of probe that binds or transmembrane with the lipid bilayer in the components of the extracellular endoplasmic reticulum using size exclusion chromatography analysis, Labeled cholesterol (Biotin-cholesterol) was used.
구체적으로, 도 16(a)에 나타낸 모식도와 같이 서로 다른 양의 정제된 표본 세포밖 소포체와 비오틴이 표지된 콜레스테롤을 혼합하여 37℃에서 30분 간 반응시킨 후 세포밖 소포체와 결합하지 않은 비오틴-콜레스테롤을 제거하기 위하여 상기 각 혼합 용액을 회전식 세파크릴 S500 컬럼에 로딩하고 700 xg 5 분간 원심분리하여 용출액을 수확하였다. 대조군으로 비오틴-콜레스테롤 단일 군 또는 표본 세포밖 소포체 단일 군을 상기와 동일한 방법으로 컬럼에 로딩한 후 회전을 통하여 용출액을 수확하였다. 상기 용출액 내 비오틴의 양을 정량하기 위하여 용출액을 96-well 플레이트(microplate)에 담은 후 시료 내 물질을 플레이트에 흡착시켜 고정하였다. 이 후 스트렙타비딘-퍼옥시다아제(streptavidin-peroxidase)와 반응시키고 세척한 후 플레이트에 잔존하는 퍼옥시다아제 효소 활성에 따른 화학 발광(chemiluminescence)을 측정하였다(도 16 (b)). Specifically, as shown in the schematic diagram of FIG. 16 (a), the different amounts of purified specimen extracellular endoplasmic reticulum and biotin-labeled cholesterol were mixed and reacted at 37 ° C. for 30 minutes. Then, biotin- To remove cholesterol, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. As a control group, biotin-cholesterol single group or single extracellular ER group was loaded on the column in the same manner as above, and the eluate was harvested through rotation. In order to quantify the amount of biotin in the eluate, the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and then chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 16 (b)).
그 결과, 비오틴-콜레스테롤 단일 군 또는 표본 세포밖 소포체 단일 군에서는 화학 발광이 거의 없었다. 이는 분자량이 작은 비오틴-콜레스테롤은 회전식 크기 배제 크로마토그래피 컬럼으로부터 용출되지 않았음을 의미하고, 분자량이 큰 세포밖 소포체의 경우 상기 도 15의 결과와 같이 회전식 크기 배제 크로마토그래피 컬럼으로부터 용출되지만, 용출된 표본 세포밖 소포체 자체에는 스트렙타비딘-퍼옥시다아제와 결합할 수 있는 비오틴이 존재 하지 않음을 알 수 있다. 반면, 상기 표본 세포밖 소포체와 비오틴-콜레스테롤을 혼합하여 반응시킨 군에서는 높은 화학 발광이 측정되었으며 상기 발광의 세기는 표본 세포밖 소포체의 양에 비례하여 증가하였다. 이는 분자량이 상대적으로 작은 비오틴-콜레스테롤이 세포밖 소포체를 구성하는 지질이중층에 삽입되어 분자량이 큰 세포밖 소포체와 함께 회전식 크기 배제 크로마토그래피 컬럼으로부터 용출될 수 있고, 비오틴-콜레스테롤의 지질이중충에 삽입되는 정도는 세포밖 소포체의 양에 비례함을 확인할 수 있었다. As a result, there was almost no chemiluminescence in the biotin-cholesterol single group or the single extracellular extracellular group. This means that biotin-cholesterol having a small molecular weight was not eluted from the rotary size exclusion chromatography column. In the case of the extracellular endoplasmic reticulum having a large molecular weight, elution was carried out from the rotary size exclusion chromatography column as shown in Fig. 15, It can be seen that biotin capable of binding to streptavidin-peroxidase is not present in the specimen extracellular endoplasmic reticulum itself. On the other hand, high chemiluminescence was measured in the group in which the specimen extracellular endoplasmic reticulum and biotin-cholesterol were mixed and the intensity of the luminescence was increased in proportion to the amount of extracellular ER. This is because the biotin-cholesterol having a relatively small molecular weight can be inserted into the lipid bilayer constituting the extracellular endoplasmic reticulum and can be eluted from the rotary size exclusion chromatography column together with the extracellular endoplasmic reticulum having a large molecular weight and inserted into the double lipid bilayer of biotin-cholesterol The amount of extracellular ER was proportional to the amount of extracellular ER.
실시예 8. 회전식 크기 배제 크로마토그래피와 콜레스테롤 탐침을 이용한 대장암 세포 배양액 내 세포밖 소포체의 분석Example 8. Analysis of extracellular endoplasmic reticulum in colorectal cancer cell culture using rotary size exclusion chromatography and cholesterol probe
상기 실시예 7에서 표본 세포밖 소포체와 비오틴-콜레스테롤의 혼합 반응물에서 회전식 크기 배제 크로마토그래피 전처리를 통하여 비오틴-콜레스테롤-세포밖 소포체 복합체만을 효과적으로 분리할 수 있음을 증명하였다. 상기 방법을 통하여 세포밖 소포체의 정제 없이 세포 배양액 내 세포밖 소포체의 총량을 분석 할 수 있는지를 확인하기 위하여, SW480 대장암 세포 배양액과 비오틴이 표지된 콜레스테롤(Biotin-cholesterol)을 사용하였다. 구체적으로, 도 17(a)에 나타낸 모식도와 같이 서로 다른 농도의 SW480 대장암 세포 배양액과 비오틴-콜레스테롤을 혼합하여 37℃에서 30분 간 반응시켰다. 다음으로 시료 내 세포밖 소포체와 결합하지 않은 비오틴-콜레스테롤을 제거하기 위하여 상기 각 혼합 용액을 회전식 세파크릴 S500 컬럼에 로딩하고 700 xg 5 분간 원심분리하여 용출액을 수확하였다. 대조군으로 비오틴-콜레스테롤 단일 군 또는 대장암 세포 배양액 단일 군을 상기와 동일한 방법으로 컬럼에 로딩한 후 회전을 통하여 용출액을 수확하였다. 상기 용출액 내 비오틴의 양을 정량하기 위하여 용출액을 96-well 플레이트 (microplate)에 담은 후 시료 내 물질을 플레이트에 흡착시켜 고정하였다. 이 후 스트렙타비딘-퍼옥시다아제(streptavidin-peroxidase)와 반응시키고 세척한 후 플레이트에 잔존하는 퍼옥시다아제 효소 활성에 따른 화학 발광(chemiluminescence)을 측정하였다(도 17(b)). In Example 7, it was proved that only the biotin-cholesterol-extracellular elastomer complex can be effectively separated through the preliminary rotary size exclusion chromatography in the mixed reaction product of the extracellular endoplasmic reticulum and biotin-cholesterol. In order to confirm whether the total amount of extracellular endoplasmic reticulum in the cell culture solution can be analyzed without purification of the extracellular endoplasmic reticulum by the above method, SW480 colon cancer cell culture medium and biotin-labeled cholesterol (biotin-cholesterol) were used. Specifically, as shown in the schematic diagram of Fig. 17 (a), cultures of SW480 colon cancer cells at different concentrations and biotin-cholesterol were mixed and reacted at 37 DEG C for 30 minutes. Next, to remove biotin-cholesterol not bound to the extracellular endoplasmic reticulum in the sample, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. As a control group, a single biotin-cholesterol group or a single culture group of colon cancer cells was loaded onto a column in the same manner as above, and the eluate was harvested through rotation. In order to quantify the amount of biotin in the eluate, the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 17 (b)).
그 결과, 비오틴-콜레스테롤 단일 군 및 대장암 세포 배양액 단일 군에서는 화학 발광이 매우 낮거나 없었으나, 대장암 세포 배양액과 비오틴-콜레스테롤을 혼합하여 반응시킨 군에서는 높은 화학 발광을 관찰할 수 있었다. 이는 비오틴-콜레스테롤 분자가 세포 배양액 내 세포밖 소포체의 지질이중층에 삽입되어 세포밖 소포체와 함께 회전식 전처리 크기 배제 크로마토그래피 컬럼으로부터 용출 되었다는 점을 증명한다.As a result, chemiluminescence was very low or not in the single group of biotin - cholesterol and in the culture of colon cancer cells, but high chemiluminescence was observed in the group in which the culture of colon cancer cells and biotin - cholesterol were mixed. This demonstrates that the biotin-cholesterol molecule was inserted into the lipid bilayer of the extracellular endoplasmic reticulum in the cell culture medium and eluted from the rotary pretreatment size exclusion chromatography column with the extracellular endoplasmic reticulum.
실시예 9. 회전식 크기 배제 크로마토그래피와 콜레스테롤 탐침을 이용한 체액 내 세포밖 소포체의 분석Example 9. Analysis of extracellular endoplasmic reticulum in body fluid using rotary size exclusion chromatography and cholesterol probe
상기 실시예 8에서 확인한 바와 같이 세포밖 소포체의 정제 없이 세포 배양액 내 세포밖 소포체를 분석할 수 있었고, 상기 방법을 인간 체액에 적용하였다. 구체적으로, 도 18(a)의 모식도에 나타낸 바와 같이 인간 오줌 또는 인간 혈청과 비오틴이 표지된 콜레스테롤(Biotin-cholesterol)을 혼합하여 각 군을 37℃에서 30분 간 반응시킨 후 시료 내 세포밖 소포체와 결합하지 않은 비오틴-콜레스테롤을 제거하기 위하여 상기 각 혼합 용액을 회전식 세파크릴 S500 컬럼에 로딩하고 700 xg 5 분간 원심분리하여 용출액을 수확하였다. 대조 군으로 비오틴-콜레스테롤 단일 군 또는 각 생물학적 시료 단일 군을 상기와 동일한 방법으로 컬럼에 로딩한 후 회전을 통하여 용출액을 수확하였다. 상기 용출액 내 비오틴의 양을 정량하기 위하여 용출액을 96-well 플레이트 (microplate)에 담은 후 시료내 물질을 플레이트에 흡착시켜 고정하였다. 이 후 스트렙타비딘-퍼옥시다아제(streptavidin-peroxidase)와 반응시키고 세척한 후 플레이트에 잔존하는 퍼옥시다아제 효소 활성에 따른 화학 발광(chemiluminescence)을 측정하였다(도 18(b, c)). As confirmed in Example 8 above, the extracellular endoplasmic reticulum in the cell culture could be analyzed without purification of the extracellular endoplasmic reticulum, and the method was applied to human body fluids. Specifically, as shown in the schematic diagram of Fig. 18 (a), human urine or cholesterol (biotin-cholesterol) labeled with human serum and biotin were mixed and reacted at 37 ° C for 30 minutes, To remove biotin-cholesterol that was not bound with the above-mentioned each mixture solution, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. As a control group, a single biotin-cholesterol group or a single biological sample group was loaded onto a column in the same manner as above, and the eluate was harvested by rotation. In order to quantify the amount of biotin in the eluate, the eluate was immersed in a 96-well microplate and adsorbed on the plate. Thereafter, the plate was reacted with streptavidin-peroxidase, washed, and chemiluminescence was measured according to the peroxidase enzyme activity remaining on the plate (FIG. 18 (b, c)).
그 결과, 비오틴-콜레스테롤 단일 군 및 대장암 세포 배양액 단일 군에서는 화학 발광이 매우 낮거나 없었으나, 생물학적 시료(오줌, 혈청)와 비오틴-콜레스테롤을 혼합하여 반응시킨 군에서는 높은 화학 발광을 관찰할 수 있었다. 이는 비오틴-콜레스테롤 분자가 생물학적 시료 내 세포밖 소포체의 지질이중층에 삽입되어 세포밖 소포체와 함께 회전식 전처리 크기 배제 크로마토그래피 컬럼으로부터 용출되었으며, 이를 통해 여러 다양한 체액 내 존재하는 세포밖 소포체의 총량을 측정하는데 활용할 수 있음을 알 수 있었다.As a result, chemiluminescence was low or not in the single group of biotin-cholesterol monoclonal and colon cancer cells, but high chemiluminescence was observed in the group in which biosynthetic (urine, serum) and biotin-cholesterol were mixed and reacted there was. The biotin-cholesterol molecule was inserted into the lipid bilayer of the extracellular endoplasmic reticulum in the biological sample and eluted from the rotary pre-size exclusion chromatography column along with the extracellular endoplasmic reticulum to measure the total amount of extracellular endoplasmic reticulum present in a variety of body fluids It can be used.
실시예 10. 회전식 크기 배제 크로마토그래피와 친유성 탐침 (DiI)을 이용한 표본 세포밖 소포체의 분석Example 10. Analysis of specimen extracellular endoplasmic reticulum using rotational size exclusion chromatography and lipophilic probe (DiI)
세포밖 소포체의 구성 성분 중 지질이중층과 결합하거나 삽입되는 탐침으로서 콜레스테롤 대신 형광 표지된 친유성 DiI를 사용하여 추가 실험을 실시하였다. 구체적으로, 도 19(a)에 나타낸 모식도와 같이 형광 표지된 친유성 DiI 단일 군 또는 표본 세포밖 소포체와 형광 표지된 친유성 DiI를 혼합한 군을 37℃에서 30분 간 반응시킨 후 세포밖 소포체와 결합하지 않은 형광 표지된 친유성 DiI를 제거하기 위하여 상기 각 혼합 용액을 회전식 세파크릴 S500 컬럼에 로딩하고 700 xg 5 분간 원심분리하여 용출액을 수확하였다. 이 후 상기 용출액을 세파크릴 S500 컬럼에 주입하고 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램 및 형광 크로마토그램(도 19(b))을 분석하였다.Further experiments were carried out using fluorescently labeled lipophilic DiI instead of cholesterol as a probe that binds or is inserted into the lipid bilayer in the constituents of the extracellular endoplasmic reticulum. Specifically, as shown in the schematic diagram shown in FIG. 19 (a), a mixture of fluorescently labeled lipophilic DiI single group or extracellular extracellular medium and fluorescently labeled lipophilic DiI was reacted at 37 ° C for 30 minutes, To remove the fluorescence-labeled lipophilic DiI that did not bind to each of the above mixtures, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. Thereafter, the eluate was injected into a CEPACRYL S500 column, and a 280 nm absorption chromatogram and a fluorescence chromatogram (FIG. 19 (b)) were analyzed while developing using an HPLC system.
그 결과, 분자량이 작은 형광 표지된 친유성 DiI는 회전식 크기 배제 크로마토그래피 컬럼에서 용출되지 못하여 형광 밴드가 검출되지 않는 반면, 형광 표지된 친유성 DiI와 표본 세포밖 소포체를 혼합한 군에서는 세포밖 소포체의 용출 시간인 3.5분에서 높은 형광 밴드를 확인할 수 있었다. 따라서 분자량이 작은 형광 표지된 친유성 DiI가 세포밖 소포체의 지질이중층에 삽입되어 회전식 크기 배제 크로마토그래피 컬럼에서 세포밖 소포체와 함께 용출되는 것을 알 수 있었다.As a result, fluorescently labeled lipophilic DiI having a small molecular weight could not be eluted in a rotary size exclusion chromatography column and fluorescence band was not detected. On the other hand, in the group in which fluorescence labeled lipophilic DiI and specimen extracellular vesicle were mixed, The fluorescence band was confirmed to be 3.5 minutes. Thus, a fluorescently labeled lipophilic DiI with a small molecular weight was inserted into the lipid bilayer of the extracellular endoplasmic reticulum and eluted with the extracellular endoplasmic reticulum in the rotary size exclusion chromatography column.
실시예 11. 회전식 크기 배제 크로마토그래피와 친유성 탐침(DiI)을 이용한 대장균 유래 세포밖 소포체의 분석Example 11. Analysis of E. coli-derived extracellular endoplasmic reticulum using rotational size exclusion chromatography and lipophilic probe (DiI)
상기 실시예 10과 동일한 탐침을 대장균 유래 세포밖 소포체에 적용하여 분석하였다. 구체적으로, 도 20(a)에 나타낸 모식도와 같이 형광 표지된 친유성 DiI 단일 군 또는 대장균 유래 세포밖 소포체와 형광 표지된 친유성 DiI를 혼합한 군을 37℃에서 30분 간 반응시킨 후 세포밖 소포체와 결합하지 않은 형광 표지된 친유성 DiI를 제거하기 위하여 상기 각 혼합 용액을 회전식 세파크릴 S500 컬럼에 로딩하고 700 xg 5 분간 원심분리하여 용출액을 수확하였다. 이 후 상기 용출액을 세파크릴 S500 컬럼에 주입하고 HPLC 시스템을 이용해 전개하면서 280 nm 흡광 크로마토그램 및 형광 크로마토그램(도 20(b))을 분석하였다. The same probe as in Example 10 was applied to the E. coli-derived extracellular endoplasmic reticulum and analyzed. Specifically, as shown in the schematic diagram of FIG. 20 (a), a group obtained by mixing fluorescently labeled lipophilic DiI single group or E. coli-derived extracellular endoplasmic reticulum with fluorescently labeled lipophilic DiI was reacted at 37 ° C for 30 minutes, To remove the fluorescence-labeled lipophilic DiI not bound to the endoplasmic reticulum, each of the above mixed solutions was loaded on a rotary Cefacill S500 column and centrifuged at 700 xg for 5 minutes to harvest the eluate. Thereafter, the eluate was injected into a Cefacill S500 column, and a 280 nm absorption chromatogram and a fluorescence chromatogram (FIG. 20 (b)) were analyzed while developing using an HPLC system.
그 결과, 분자량이 작은 형광 표지된 친유성 DiI는 회전식 크기 배제 크로마토그래피 컬럼에서 용출되지 못하여 형광 밴드가 검출되지 않는 반면, 형광 표지된 친유성 DiI와 대장균 유래 세포밖 소포체를 혼합한 군에서는 세포밖 소포체의 용출 시간인 3.5분에서 높은 형광 밴드를 확인할 수 있었다. 따라서 분자량이 작은 형광 표지된 친유성 DiI가 대장균 유래 세포밖 소포체의 지질이중층에 삽입되어 회전식 크기 배제 크로마토그래피 컬럼에서 대장균 유래 세포밖 소포체와 함께 용출되는 것을 알 수 있었다.As a result, fluorescently labeled lipophilic DiI having a small molecular weight was not eluted in the rotary size exclusion chromatography column and fluorescence band was not detected. On the other hand, in the group in which fluorescently labeled lipophilic DiI and E. coli-derived extracellular endoplasmic reticulum were mixed, A fluorescence band having a high elution time of 3.5 minutes was confirmed. Thus, it was found that fluorescently labeled lipophilic DiI having a small molecular weight was inserted into the lipid bilayer of the E. coli-derived extracellular endoplasmic reticulum and eluted together with the E. coli-derived endoplasmic reticulum endoplasmic reticulum in the rotary size exclusion chromatography column.
이로부터 박테리아 유래 세포밖 소포체 역시 지질이중층으로 구성되어 있음을 확인하였으며, 본 분석 방법은 세포밖 소포체의 총량 뿐 아니라 세포밖 소포체의 특성 분석에도 활용될 수 있음을 시사한다.From this, it was confirmed that the bacterial-derived extracellular endoplasmic reticulum was also composed of a lipid bilayer, and this analysis method can be used to analyze the extracellular endoplasmic reticulum as well as the total amount of extracellular endoplasmic reticulum.

Claims (20)

  1. (a) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계;(a) mixing and reacting a sample comprising a probe comprising a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and a detectable signal portion, and an extracellular endoplasmic reticulum;
    (b) 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계; 및(b) injecting the mixed sample into a size exclusion chromatography column and developing the same; And
    (c) 상기 전개된 시료로부터 세포밖 소포체-탐침 복합체 및 자유 탐침을 검출하는 단계를 포함하는 (c) detecting an extracellular endoplasmic reticulum-probe complex and a free probe from the expanded sample
    세포밖 소포체의 분석 방법.Method of analysis of extracellular endoplasmic reticulum.
  2. 제1항에 있어서,The method according to claim 1,
    상기 탐침은 The probe
    세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 단일 물질; 또는 A single substance comprising a binding moiety and a detectable signal moiety that specifically bind to a component of an extracellular endoplasmic reticulum; or
    세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부를 포함하는 물질에 분석 가능한 하나 이상의 신호부를 포함하는 물질이 결합된 복합 물질인 것인The compound comprising at least one signal portion capable of analyzing a substance including a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum
    세포밖 소포체의 분석 방법.Method of analysis of extracellular endoplasmic reticulum.
  3. 제1항에 있어서,The method according to claim 1,
    상기 탐침의 결합부는 세포밖 소포체의 막 표면 성분, 세포밖 소포체의 막 성분 및 세포밖 소포체의 내부 성분으로 이루어진 군에서 선택되는 하나 이상의 성분과 특이적으로 결합하는 것인 세포밖 소포체의 분석 방법.Wherein the binding portion of the probe specifically binds to at least one component selected from the group consisting of a membrane surface component of an extracellular endoplasmic reticulum, a membrane component of an extracellular endoplasmic reticulum, and an inner component of an extracellular endoplasmic reticulum.
  4. 제1항에 있어서,The method according to claim 1,
    상기 탐침은 단백질, 항체, 항체 유래 물질, 펩티드, 핵산, 핵산-아미노산 복합체, 효소, 효소 기질, 화학적 리간드 및 이들의 화합물로 이루어진 군에서 선택되는 하나 이상인 세포밖 소포체의 분석 방법.Wherein the probe is at least one selected from the group consisting of a protein, an antibody, an antibody-derived substance, a peptide, a nucleic acid, a nucleic acid-amino acid complex, an enzyme, an enzyme substrate, a chemical ligand and a compound thereof.
  5. 제1항에 있어서,The method according to claim 1,
    상기 탐침은 형광 물질, 효소 기질, 효소, 단백질, 펩티드, 핵산, 비오틴, 금속 및 방사성동위원소로 이루어진 군에서 선택되는 하나 이상의 신호부를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the probe comprises at least one signal portion selected from the group consisting of a fluorescent material, an enzyme substrate, an enzyme, a protein, a peptide, a nucleic acid, a biotin, a metal and a radioactive isotope.
  6. 제1항에 있어서,The method according to claim 1,
    상기 검출 단계는 특정 파장에 대한 흡광 크로마토그램을 검출함으로써 세포밖 소포체를 정량하는 단계를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein said detecting step comprises quantifying the extracellular endoplasmic reticulum by detecting an absorbance chromatogram for a particular wavelength.
  7. 제6항에 있어서,The method according to claim 6,
    상기 특정 파장은 200 nm ~ 800 nm 범위에서 선택되는 하나 이상의 값인 세포밖 소포체의 분석 방법.Wherein the specific wavelength is one or more values selected from the range of 200 nm to 800 nm.
  8. 제1항에 있어서,The method according to claim 1,
    상기 검출 단계는 탐침의 신호부를 검출하여 탐침을 정량하는 단계를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the detecting step comprises detecting the signal portion of the probe to quantify the probe.
  9. 제8항에 있어서,9. The method of claim 8,
    상기 탐침의 신호부를 검출하는 단계는 분광학적 분석, 물리화학적 분석, 양자화학적 분석, 효소학적 분석, 비오틴 분석 및 핵산 분석으로 이루어진 군에서 선택되는 하나 이상의 방법을 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the step of detecting the signal portion of the probe comprises one or more methods selected from the group consisting of spectroscopic analysis, physicochemical analysis, quantum chemical analysis, enzymatic analysis, biotin analysis and nucleic acid analysis. .
  10. 제1항에 있어서,The method according to claim 1,
    상기 시료는 포유동물 세포 배양 배지, 박테리아 세포 배양 배지, 효모 배양 배지, 조직 추출물, 암 조직, 혈청, 혈장, 침, 눈물, 안방수, 땀, 소변, 대변, 뇌척수액(CSF, cerebrospinal fluid), 복수(ascite), 양수(amniotic fluid), 정액, 유(milk), 먼지, 담수, 해수, 토양 및 발효식품으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 세포밖 소포체의 분석 방법.The sample can be used in a mammalian cell culture medium, a bacterial cell culture medium, a yeast culture medium, a tissue extract, a cancer tissue, a serum, plasma, a saliva, a tear, an aqueous solution, sweat, urine, feces, cerebrospinal fluid (CSF) wherein the at least one selected from the group consisting of ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermented food.
  11. (a) 세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 탐침 및 세포밖 소포체를 포함하는 시료를 혼합하여 반응시키는 단계;(a) mixing and reacting a sample comprising a probe comprising a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum and a detectable signal portion, and an extracellular endoplasmic reticulum;
    (b) 상기 혼합 시료를 크기 배제 크로마토그래피 컬럼에 주입하여 전개시키는 단계; 및(b) injecting the mixed sample into a size exclusion chromatography column and developing the same; And
    (c) 상기 크기 배제 크로마토그래피 컬럼으로부터 세포밖 소포체-탐침 복합체를 분리하는 단계; 및(c) separating the extracellular endoplasmic reticulum-probe complex from the size exclusion chromatography column; And
    (d) 상기 분리된 세포밖 소포체-탐침 복합체에서 탐침을 검출하는 단계를 포함하는 세포밖 소포체의 분석 방법.(d) detecting the probe in the isolated extracellular endoplasmic reticulum-probe complex.
  12. 제11항에 있어서,12. The method of claim 11,
    상기 탐침은 The probe
    세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부 및 검출 가능한 신호부를 포함하는 단일 물질; 또는 A single substance comprising a binding moiety and a detectable signal moiety that specifically bind to a component of an extracellular endoplasmic reticulum; or
    세포밖 소포체의 구성성분과 특이적으로 결합하는 결합부를 포함하는 물질에 분석 가능한 신호부를 포함하는 물질이 결합된 복합 물질인 것인Wherein the substance is a complex substance in which a substance including an analytical signal portion is bound to a substance including a binding portion that specifically binds to a component of an extracellular endoplasmic reticulum
    세포밖 소포체의 분석 방법.Method of analysis of extracellular endoplasmic reticulum.
  13. 제11항에 있어서,12. The method of claim 11,
    상기 탐침의 결합부는 세포밖 소포체의 막 표면 성분, 세포밖 소포체의 막 성분 및 세포밖 소포체의 내부 성분으로 이루어진 군에서 선택되는 하나 이상의 성분과 특이적으로 결합하는 것인 세포밖 소포체의 분석 방법.Wherein the binding portion of the probe specifically binds to at least one component selected from the group consisting of a membrane surface component of an extracellular endoplasmic reticulum, a membrane component of an extracellular endoplasmic reticulum, and an inner component of an extracellular endoplasmic reticulum.
  14. 제11항에 있어서,12. The method of claim 11,
    상기 탐침은 단백질, 항체, 항체 유래 물질, 펩티드, 핵산, 핵산-아미노산 복합체, 효소, 효소 기질, 화학적 리간드 및 이들의 화합물로 이루어진 군에서 선택되는 하나 이상인 세포밖 소포체의 분석 방법.Wherein the probe is at least one selected from the group consisting of a protein, an antibody, an antibody-derived substance, a peptide, a nucleic acid, a nucleic acid-amino acid complex, an enzyme, an enzyme substrate, a chemical ligand and a compound thereof.
  15. 제11항에 있어서,12. The method of claim 11,
    상기 탐침은 형광 물질, 효소 기질, 효소, 단백질, 펩티드, 핵산, 비오틴, 금속 및 방사성동위원소로 이루어진 군에서 선택되는 하나 이상의 신호부를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the probe comprises at least one signal portion selected from the group consisting of a fluorescent material, an enzyme substrate, an enzyme, a protein, a peptide, a nucleic acid, a biotin, a metal and a radioactive isotope.
  16. 제11항에 있어서,12. The method of claim 11,
    상기 검출 단계는 특정 파장에 대한 흡광 크로마토그램을 검출함으로써 세포밖 소포체를 정량하는 단계를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein said detecting step comprises quantifying the extracellular endoplasmic reticulum by detecting an absorbance chromatogram for a particular wavelength.
  17. 제16항에 있어서,17. The method of claim 16,
    상기 특정 파장은 200 nm ~ 800 nm 범위에서 선택되는 하나 이상의 값인 세포밖 소포체의 분석 방법.Wherein the specific wavelength is one or more values selected from the range of 200 nm to 800 nm.
  18. 제11항에 있어서,12. The method of claim 11,
    상기 검출 단계는 탐침의 신호부를 검출하여 탐침을 정량하는 단계를 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the detecting step comprises detecting the signal portion of the probe to quantify the probe.
  19. 제18항에 있어서,19. The method of claim 18,
    상기 탐침의 신호부를 검출하는 단계는 분광학적 분석, 물리화학적 분석, 양자화학적 분석, 효소학적 분석, 비오틴 분석, 방사선 분석 및 핵산 분석으로 이루어진 군에서 선택되는 하나 이상의 방법을 포함하는 것인 세포밖 소포체의 분석 방법.Wherein the step of detecting the signal portion of the probe comprises one or more methods selected from the group consisting of spectroscopic analysis, physicochemical analysis, quantum chemical analysis, enzymatic analysis, biotin analysis, radiation analysis and nucleic acid analysis. .
  20. 제1항에 있어서,The method according to claim 1,
    상기 시료는 포유동물 세포 배양 배지, 박테리아 세포 배양 배지, 효모 배양 배지, 조직 추출물, 암 조직, 혈청, 혈장, 침, 눈물, 안방수, 땀, 소변, 대변, 뇌척수액(CSF, cerebrospinal fluid), 복수(ascite), 양수(amniotic fluid), 정액, 유(milk), 먼지, 담수, 해수, 토양 및 발효식품으로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 하는 세포밖 소포체의 분석 방법.The sample can be used in a mammalian cell culture medium, a bacterial cell culture medium, a yeast culture medium, a tissue extract, a cancer tissue, a serum, plasma, a saliva, a tear, an aqueous solution, sweat, urine, feces, cerebrospinal fluid (CSF) wherein the at least one selected from the group consisting of ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermented food.
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