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WO2019066501A1 - Procédé d'analyse pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique, et son utilisation - Google Patents

Procédé d'analyse pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique, et son utilisation 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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Korean (ko)
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고용송
이창진
박현택
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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Priority to JP2020517365A priority Critical patent/JP7298929B2/ja
Priority to CN201880075753.5A priority patent/CN111386458B/zh
Priority to EP18860890.5A priority patent/EP3690434A4/fr
Priority to US16/651,940 priority patent/US11835502B2/en
Priority claimed from KR1020180115206A external-priority patent/KR102243415B1/ko
Publication of WO2019066501A1 publication Critical patent/WO2019066501A1/fr
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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.

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Abstract

Le procédé d'analyse pour vésicules extracellulaires, selon la présente invention, utilise la capacité de séparation en fonction de la taille de la chromatographie d'exclusion stérique et les propriétés d'une sonde qui se lie spécifiquement à des vésicules extracellulaires, et en utilisant celles-ci est capable d'analyser rapidement et facilement la quantité de vésicules extracellulaires présentes dans un échantillon, d'analyser les propriétés physico-chimiques des vésicules extracellulaires, d'analyser le type et la quantité des constituants présents dans les vésicules extracellulaires, et d'analyser les propriétés de liaison ou d'affinité de la sonde par rapport aux constituants des vésicules extracellulaires. De plus, l'utilisation du procédé d'analyse selon la présente invention permet non seulement une analyse précise des vésicules extracellulaires dans un échantillon, sans étape de purification ni de prétraitement d'échantillon, mais permet également une analyse précise et simple des constituants des vésicules extracellulaires, en fonction du type de sonde, et peut ainsi améliorer l'efficacité de diagnostic à l'aide de vésicules extracellulaires. En outre, l'analyse des propriétés ou de l'affinité de la sonde peut être appliquée, par exemple, au criblage d'anticorps spécifiques aux vésicules extracellulaires, au criblage de protéines et au criblage de substances chimiques.
PCT/KR2018/011443 2017-09-27 2018-09-27 Procédé d'analyse pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique, et son utilisation Ceased WO2019066501A1 (fr)

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CN201880075753.5A CN111386458B (zh) 2017-09-27 2018-09-27 利用尺寸排阻色谱法的细胞外囊泡的分析方法及其用途
EP18860890.5A EP3690434A4 (fr) 2017-09-27 2018-09-27 Procédé d'analyse pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique, et son utilisation
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