WO2019066485A1 - Purity analysis method for extracellular vesicles, using size exclusion chromatography - Google Patents

Abstract

The present invention relates to a purity analysis method for extracellular vesicles, using size exclusion chromatography, and more specifically the present invention relates to a method of using the size-specific separation ability of size exclusion chromatography and spectroscopy, that is single or in which two or more kinds are combined, in order to quantify, from various samples, the volume of extracellular vesicles and various contaminants, simultaneously, and thereby analyze the purity, total amount and yield of extracellular vesicles. The purity analysis method for extracellular vesicles, according to the present invention, has the benefit of being capable of effective analysis whilst preserving the form or properties of the extracellular vesicles. Also, the purity analysis method for extracellular vesicles, according to the present invention, makes it possible to rapidly determine, simultaneously, the purity, total amount and yield of extracellular vesicles that have been separated using a conventional method, and can be utilized in, for example, disease diagnosis, disease treatment, multi-omics research and research into the properties of extracellular vesicles which use separated extracellular vesicles. In addition, the purity analysis method according to the present invention can also be applied to the development of a new separation method.

Description

Method for analyzing purity of extracellular endoplasmic reticulum using size exclusion chromatography

The present invention relates to a method for analyzing the purity of an extracellular endoplasmic reticulum using size exclusion chromatography, and more particularly, to a method for analyzing the purity of extracellular endoplasmic reticulum by using the size-fractionating ability of size exclusion chromatography and the spectroscopic characteristics of the sample And the like.

Extracellular vesicles are nano-sized (20-1000 nm) biological particles secreted naturally by all cells and have a cell-derived lipid bilayer membrane structure. In animals, blood, saliva, Tears, nasal mucus, sweat, urine, feces, cerebrospinal fluid, ascites, amniotic fluid, bronchoalveolar lavage (CSF) fluid, pleural effusion, semen, milk, and the like, as well as various cancers or normal tissues.

These extracellular ERs are composed of various physiologically active substances such as proteins, lipids, nucleic acids and amino acids derived from the parent cells, and not only contain the physiological and pathological characteristics of the parent cells, but also bind to other cells and tissues to produce physiological activities It acts as a carrier while transferring substances to recipient cells and tissues. As the various physiological and pathological functions of the extracellular endoplasmic reticulum have been identified, studies on the components and functions of the extracellular endoplasmic reticulum have been actively conducted in a variety of fields including the life field, and development of therapeutic agents for treating various diseases and diseases This is happening at the same time.

Therefore, it is essential to effectively separate and purify the extracellular endoplasmic reticulum from various types of samples in order to study the constituents and functions of the extracellular endoplasmic reticulum and to develop a therapeutic agent therefor. Therefore, various separation methods are being used, It is being actively developed.

Conventional extracellular ER separation techniques include ultrafast centrifugation, density gradient, size exclusion, polymer-based precipitation, salt-based precipitation based precipitation, organic solvent-based precipitation, ion exchange chromatography, affinity chromatography, and aqueous two-phase system. Among them, ultrafast centrifugation is the most widely used.

In the development of new biomarkers based on the extracellular endoplasmic reticulum and the development of therapeutic agents based on extracellular endoplasmic reticulum, the purity analysis of the extracellular endoplasmic reticulum is the basis of the field in the future and is recognized as very important. However, the above-described methods for separating the extracellular endoplasmic reticulum and for evaluating the purity of the separated extracellular endoplasmic reticulum depend on an indirect method such as a total amount of nanoparticles / a total amount of proteins.

Such a purity analysis method requires not only analyzing the concentration of nanoparticles in the sample and quantifying the protein independently but also using the nanoparticle tracking analysis (NTA) or the nanopore (Nanopore) (TRPS). However, there is a problem in that the concentration of nanoparticles is measured differently depending on the sensitivity of the assay and the setting of the equipment, even though the same sample is analyzed. In addition, for the purpose of the above purity analysis, since the method of measuring the sum of the extracellular endoplasmic reticulum and the contaminant in the sample is limited to the quantitative determination of the substance of the class of protein, when the non-protein material is contaminated, The purity can be evaluated to be high. On the other hand, when the protein is not detected during the protein determination of the sample, the purity is evaluated to be low when the substance generating the protein detection signal is contaminated with the sample. In addition, since basic information such as the absorbance and the molecular weight of the contaminants in the sample can not be known, analysis of the contaminants in the sample has many problems such as additional cost and time.

Similarly, in the case of the purity analysis method of the extracellular endoplasmic reticulum sample through the recently introduced size exclusion chromatography, since the ultraviolet absorbance (254 nm) is recorded and analyzed for protein or nucleic acid measurement while the sample is developed, There is a problem that additional nanoparticle analysis for distinguishing extracellular endoplasmic reticulum and contaminant band or analysis for specifying extracellular endoplasmic reticulum (e.g., Western blot analysis) can not be performed and contamination other than the above-mentioned specific substance can not be detected.

Therefore, it is urgent to develop a direct, simple and rapid method of analyzing the extracellular vesicle purity which is different from the conventional method.

The object of the present invention can be achieved by a method for preparing a sample, comprising the steps of: (a) injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column, (b) detecting an absorbance chromatogram for a specific wavelength of the developed sample, ) Separating the extinction band of the extracellular endoplasmic reticulum from the extinction band of the impurity from the extinction chromatogram of step (b), (d) determining the extinction band area of the extracellular endoplasmic reticulum from the extinction chromatogram of step (b) And calculating the purity of the extracellular endoplasmic reticulum from the extinction band area value of the extracellular endoplasmic reticulum and the extinction band area value of the impurity. will be.

It is still another object of the present invention to provide a method for screening a sample, comprising the steps of (a) injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column, (b) detecting a light absorption chromatogram for the first wavelength of the developed sample (c) detecting a light absorbance chromatogram for a second wavelength different from the first wavelength of the developed sample; and (d) detecting an extinction band area of an extracellular vesicle from the respective extinction chromatogram or an extinction band And calculating an area of the extracellular vesicle.

It is still another object of the present invention to provide a method for preparing a sample, which comprises the steps of: (a) injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column, (b) detecting an absorbance chromatogram for a specific wavelength of the developed sample, (c) calculating the extinction band area of the extracellular endoplasmic reticulum from the absorbance chromatogram of step (b); and (e) calculating the total amount of extracellular endoplasmic reticulum from the extinction band area of the extracellular endoplasmic reticulum And to provide a quantitative analysis method of the outer endoplasmic reticulum.

Disclosure of Invention Technical Problem [9] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of separating extracellular endoplasmic reticulum and impurities from a sample, A method for analyzing the purity of extracellular endoplasmic reticulum quickly and accurately by combining a spectroscopic analysis method is provided.

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 in the stationary phase 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.

In the present invention, by passing various samples containing extracellular endoplasmic reticulum to a size exclusion chromatography column, it is possible to rapidly and easily fractionate various molecules contained in the sample, and in the next step, the spectroscopic characteristics of the separated eluate are analyzed Analysis of the extracellular endoplasmic reticulum can be performed easily and efficiently.

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.

The present invention provides a method for analyzing the purity of an extracellular endoplasmic reticulum.

The purity analysis method of the extracellular endoplasmic reticulum according to the present invention is schematically shown in Fig.

The purity analysis method of the extracellular endoplasmic reticulum of the present invention includes a step (a) of injecting a sample containing extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the same.

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. Therefore, it is important to choose a stationary phase with holes sized to separate nanoparticles, extracellular endoplasmic reticulum, from proteins of various sizes. Gels most commonly used in size exclusion chromatography is Sepharose (Sepharose; GE Healthcare), Super Rose (Superose; GE Healthcare), Sephadex (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 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, , Serum, plasma, saliva, tears, sweat, urine, feces, CSF, ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermentation One or more of which may be selected from the group consisting of foods.

The method for analyzing purity of an extracellular endoplasmic reticulum of the present invention includes a step (b) of detecting the absorbance chromatogram for a specific wavelength of the developed sample.

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 four or more wavelengths including at least one of wavelengths in the range of 330 to 450 nm, 230 nm, 260 nm, and 280 nm, but is not limited thereto.

The present invention can distinguish fractions of the size exclusion chromatography as well as quantitatively analyze each substance using the spectroscopic property that the substance exhibits absorbance at a specific wavelength depending on the substance.

The method for analyzing purity of an extracellular endoplasmic reticulum of the present invention comprises the steps of: (a) separating an extinction band of an extracellular endoplasmic reticulum from an extinction band of an impurity from the absorbance chromatogram of the step (b) And calculating an absorption band area of the impurity (step (d)).

The term " impurity " or " contaminant " of the present invention refers to a substance other than the extracellular endoplasmic reticulum contained in the sample, and includes proteins, lipids, nucleic acids (RNAs, DNAs), peptides, metabolites, compounds and carbohydrates.

When the sample is passed through a size exclusion chromatography column as described above, the extracellular endoplasmic reticulum and other impurities contained in the sample are eluted in a time sequence. In general, extracellular ERs are relatively large in size because they are larger in size than other impurities and have a molecular size of 1,000 kDa or more. 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. In an embodiment of the present invention, a column (10 x 100 mm) packed with Sephacryl S500 was connected to an HPLC system and 50 - 100 mL of sample was injected and the column was developed at a flow rate of 1.0 mL / min, Ultraviolet / visible light absorbance chromatograms of various wavelengths (230 nm, 260 nm, 280 nm and 450 nm) were recorded and analyzed. In the column chromatography under the above conditions, the extracellular endoplasmic reticulum was released in about 3.3 minutes, and albumin (BSA) was eluted in about 7.8 minutes.

By using the fact that extracellular ER and other impurities are eluted at a certain time when size exclusion chromatography is performed under specific conditions, the extinction band of the extracellular ER and the extinction band of the impurity are determined from the extinction chromatogram of various wavelengths And the area of each absorption band can be calculated.

In the present invention, the extinction band of the extracellular endoplasmic reticulum is an extinction band satisfying all of the following conditions:

i) a peak having absorbance at 230 nm, 260 nm and 280 nm; And

ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; And

iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.

Size exclusion chromatography Although the elution time of the fractions varies depending on the conditions of the chromatography column, a column packed with a stationary phase having pores having a size capable of separating the extracellular endoplasmic reticulum, which is a nanoparticle, from various sizes of proteins, The peaks of the extracellular endoplasmic reticulum can be distinguished by using the feature that the peaks observed at the same elution time are the same regardless of the range of wavelengths used for the absorbance measurement for the fractions.

The extinction band area (A _EV ) of the extracellular endoplasmic reticulum of the present invention can be obtained by calculating the corresponding peak area of a 230 nm chromatogram as an extinction band satisfying the above conditions.

On the other hand, in the present invention, the extinction band of the impurity is an extinction band which does not satisfy at least one of the following conditions:

i) a peak having absorbance at 230 nm, 260 nm and 280 nm; or

ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; or

iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.

The absorbance band area (A _CONT ) of the impurity of the present invention can be obtained by calculating the corresponding peak area of the 230 nm chromatogram as an impurity absorption band satisfying the above conditions.

The purity analysis method of the extracellular endoplasmic reticulum of the present invention includes the step of calculating the purity of the extracellular endoplasmic reticulum from the value of the extinction band of the extracellular endoplasmic reticulum and the value of the extinction band of the impurity [step (e)].

The purity of the extracellular endoplasmic reticulum of the present invention includes those calculated using the following formula:

At this time, the A _EV is the extinction band area of the extracellular endoplasmic reticulum, and A _CONT is the extinction band area of the impurity.

As a result of analyzing the purity of the extracellular endoplasmic reticulum and the albumin mixture through one embodiment of the present invention, it was confirmed that the purity determined before the experiment and the purity calculated through the experiment were within the error range of ± 2%.

The present invention also provides a method of analyzing an extracellular endoplasmic reticulum.

The method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (a) of injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the same.

The detailed description thereof is as described above.

The method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of: (a) detecting an absorbance chromatogram for a first wavelength of the developed sample; (b) detecting a light intensity for a second wavelength different from the first wavelength of the developed sample And detecting the chromatogram (step (c)).

The first and second wavelengths of the present invention may be selected from the range of 200 nm to 800 nm, preferably 230 nm, 260 nm, 280 nm and 450 nm, but are not limited thereto Do not.

In one aspect of the present invention, the first wavelength and the second wavelength may be one or more values selected from the group consisting of 230 nm, 260 nm, 280 nm, and 330 to 450 nm.

In another embodiment of the present invention, the first wavelength is one of 330 nm to 450 nm, 230 nm, 260 nm and 280 nm, and the second wavelength is 200 nm to 800 nm May be one or more values to be selected.

As described above, a specific substance characteristically exhibits absorbance at a specific wavelength. The method for analyzing extracellular ER of the present invention is characterized in that the fractions eluted from the size exclusion chromatography are irradiated with different wavelengths to detect the absorbance for each wavelength and the difference of the absorbance by wavelength to determine the characteristics of the extracellular endoplasmic reticulum , It is possible to identify unknown impurities other than the extracellular endoplasmic reticulum and to compare the components of the sample.

It was confirmed that the extracellular endoplasmic reticulum and albumin exhibit completely different absorbance characteristics at wavelengths of 260 nm, 280 nm and 450 nm, respectively, according to one embodiment of the present invention.

The method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (d) of calculating an extinction band area of an extracellular vesicle or an extinction band area of an impurity from each of the above absorbance chromatograms.

The method of calculating the extracellular albumin extinction band area and the extinction extinction band area from the above-described absorbance chromatogram of the present invention is as described above.

The analyzing step of the present invention can analyze either the extracellular endoplasmic reticulum or the impurity singly, and the extracellular endoplasmic reticulum and the impurity at the same time.

The analyzing step of the present invention may be to calculate and analyze the ratio of the extracellular albumin absorbance band area measured at the second wavelength to the extracellular albumin absorbance band area measured at the first wavelength.

The analyzing step of the present invention may be performed by calculating the ratio of the impurity absorption band area measured at the second wavelength to the impurity absorption band area measured at the first wavelength.

In addition, the present invention provides a method for quantitative analysis of extracellular endoplasmic reticulum.

The method for quantitatively analyzing an extracellular vesicle of the present invention comprises the steps of injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column (step (a)), detecting the absorbance chromatogram for a specific wavelength of the developed sample (Step (b)), calculating the extinction band area of the extracellular endoplasmic reticulum from the absorbance chromatogram (step (c)), and calculating the total amount of extracellular endoplasmic reticulum from the extinction band area of the extracellular endoplasmic reticulum [(d) step].

The extracellular vesicle quantitative analysis method of the present invention utilizes the fact that the extracellular endoplasmic reticulum contained in the sample is linearly proportional to the absorption band area at a specific wavelength.

In the extracellular vesicle quantitative analysis method of the present invention, the wavelength range is as described above.

In the extracellular vesicle quantitative analysis method of the present invention, the method of calculating the extracellular extracorporeal absorbance band area from the above absorbance chromatogram is as described above.

The total amount of extracellular endoplasmic reticulum in the present invention can be calculated from the area of the extracellular extracellular extinction band derived from the above absorbance chromatogram using the following equation:

At this time, A _EV is the extinction band area of the extracellular endoplasmic reticulum, and a and b are constants.

The total amount of the specimen extracellular endoplasmic reticulum and the area of the extracellular endocardial extinction band identified in one embodiment of the present invention show the following linear relationship:

The method of analyzing the extracellular endoplasmic reticulum of the present invention can quickly and effectively analyze the total amount of the extracellular endoplasmic reticulum or impurities contained in the sample, and the purity of the extracellular endoplasmic reticulum without limiting the amount of the sample. In addition, it can be used to measure the yield and efficiency of the extracellular ER separation method by comparing and analyzing purity before and after extracellular ER separation and purification.

FIG. 1 is a schematic diagram of a method for analyzing purity, total amount, and yield of extracellular endoplasmic reticulum using size exclusion chromatography in various samples.

FIG. 2 is a schematic view (a) and a separation result (b, c) of a sample extracellular endoplasmic reticulum from a colorectal cancer cell line SW480 according to an embodiment of the present invention.

FIG. 3 shows the absorption chromatogram (a), nanoparticle analysis (b), and Westin blot analysis (c) results of a sample of extracellular endoplasmic reticulum using size exclusion chromatography.

FIG. 4 shows the results of analysis of extracellular endoplasmic reticulum and albumin using size exclusion chromatography by absorption spectrophotometry at various wavelengths.

FIG. 5 shows the results of analysis of the correlation between the 230 nm absorbance chromatogram of the extracellular endoplasmic reticulum (a) and albumin (b) and the total area and total amount of each peak using size exclusion chromatography.

FIG. 6 shows the results of analyzing the purity of the mixed solution in which extracellular ER and albumin were mixed at various ratios using size exclusion chromatography.

FIG. 7 shows the results of comparative analysis of the purity of extracellular endoplasmic reticulum isolated from colon cancer cell culture medium by different separation methods (ultracentrifugation, PEG precipitation).

FIG. 8 shows the results of comparative analysis of the purity of extracellular endoplasmic reticulum isolated from human body fluids (urine) by different separation methods (ultracentrifugation, PEG precipitation).

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to 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 examples.

Example 1 Isolation and Characterization of the Extracellular Endoplasmic Reticulum

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. A polyethylene glycol solution (final 8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) was added to the supernatant for the first purification of the extracellular endoplasmic reticulum and incubated at 4 ° C for 16 hours, xg for 30 minutes to dissolve the precipitated extracellular endoplasmic reticulum in HEPES buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4). The solution was applied to 30% -20% -5% Optiprep buoyant density gradient ultracentrifugation for 2 hours at 200,000 xg for secondary purification of the primary purified extracellular endoplasmic reticulum, After centrifugation, regions with an equal density (1.08-1. 12 g / ml) to the extracellular endoplasmic reticulum were harvested. For the third purification of the purified extracellular endoplasmic reticulum, the second purified sample was injected into a column packed with Sephacryl S500 connected to a high performance liquid chromatography (HPLC) system (10 x 100 mm) The high-purity extracellular endoplasmic reticulum was purified through fractionation by size, and the separation process of the endoplasmic reticulum endoplasmic reticulum was briefly shown in Fig. 2 (a).

In order to confirm the characteristics and purity of the extracellular endoplasmic reticulum, the distribution of extracellular endoplasmic reticulum markers (Alix, CD63, CD9) was confirmed by Western blotting and compared with the whole cell lysate of SW480 Purity can be confirmed (Fig. 2 (b)).

The shape and size (about 50 to 200 nm) of the extracellular endoplasmic reticulum were confirmed using a transmission electron microscope, and the result is shown in FIG. 2 (c).

Example 2: Absorption chromatogram of specimen extracellular endoplasmic reticulum using size exclusion chromatography and its analysis

In order to analyze the purity of the extracellular endoplasmic reticulum in the sample using size exclusion chromatography and spectroscopic analysis, the extracellular endoplasmic reticulum isolated in Example 1 was injected into a column (10 x 100 mm) filled with Sephacryl S500 And the absorbance was measured at 280 nm while developing with HEPES buffer at a flow rate of 1.0 ml / min. At the same time, the fraction eluted in minutes was collected to analyze the concentration of nanoparticles, and western blot was performed on each fraction to analyze the marker of the extracellular endoplasmic reticulum.

As a result, one absorbance band was observed at a elution time of 3.3 minutes in a 280 nm absorbance chromatogram as shown in Fig. 3 (Fig. 3 (a)). The nanoparticle analysis also showed a strong signal in the same fraction (FIG. 3 (b)) and a strong signal in the Western blot for the CD63 and CD9 markers (FIG. 3 (c)). From the results, it was found that the extracellular endoplasmic reticulum was eluted at 3.3 minutes in the Sephacryl 5600 size exclusion chromatography column, and the single peak was observed, indicating that the purity of the extracellular endoplasmic reticulum was very high.

Example 3. Analysis of multi-wavelength absorbance chromatograms of extracellular endoplasmic reticulum and albumin using size exclusion chromatography

In order to analyze the difference of absorbance by wavelength for each analyte, the absorbance chromatogram for each substance was recorded and analyzed at various wavelengths. Specifically, the same method as that used in Example 2 was used, and the sample was subjected to albumin, which is a main contaminant when the extracellular and extracellular endoplasmic reticulum were purified. Absorption chromatograms of 260 nm, 280 nm and 450 nm wavelengths were simultaneously recorded and analyzed.

As a result, as shown in Figs. 4 (a) and 4 (b), peaks were observed at 260 nm and 280 nm at about 3.3 minutes for the endoplasmic reticulum endoplasmic reticulum and at about 5.8 minutes for albumin. In addition, the absorbance at 280 nm of albumin was higher than that at 260 nm, whereas that of extracellular endoplasmic reticulum was inversely higher than that of 280 nm at 260 nm. In addition, it was confirmed that albumin had no absorbance at 450 nm, while a band corresponding to about 10% of absorbance at 260 nm was formed at 450 nm in the extracellular endoplasmic reticulum. This is related to the light scattering caused by the extracellular ERs being nanoparticles and is an important feature of nanoparticles.

The absorbance at various wavelengths (peak area) shown in Figs. 4 (a) and 4 (b) was calculated and the absorbance at various wavelengths was compared with the results shown in Fig. 4 (c). As a result, it was found that the extracellular endoplasmic reticulum had a characteristic that the ratio of absorbance per wavelength was distinct from that of albumin.

From this, it was found that by analyzing the spectroscopic characteristics of the sample developed by size exclusion chromatography, not only the total amount of extracellular fibrils or impurities in the sample can be analyzed, but also the physical and spectroscopic characteristics of the impurities can be analyzed .

Example 4. Absorption chromatogram analysis of extracellular ER and albumin using size exclusion chromatography at 230 nm and its application

In order to quantitatively analyze the material from the size exclusion chromatography and spectroscopic analysis results, the specimen extracellular endoplasmic reticulum and albumin were injected into the same Sephacryl 500 column as in Example 2. The specimen was then developed in a 230 nm absorbance chromatogram, The absorption band area of the sample was analyzed. The wavelength of 230 nm is known to be absorbed by relatively wide materials and high in sensitivity.

As a result, as shown in FIG. 5, it was found that the extinction band area of the extracellular endoplasmic reticulum eluting at 3.3 minutes and the extinction band area of albumin eluted at 7.7 minutes increased with increasing injection amount. Analysis of the relationship between the area of the extinction band and the injection amount showed that there is a high linear correlation as follows.

Extracellular endoplasmic reticulum (SW480 EVs):

Peak Area (mAU * min) at 230 nm = 3.760 * EV quantity (mg) - 0.380

Albumin (BSA):

Peak Area (mAU * min) at 230 nm = 4.765 * Albumin quantity (mg) - 1.491

In order to compare the difference of absorbance according to the mixing ratio of extracellular ER and albumin, various amounts of albumin were mixed with a certain amount of extracellular ER (20%, 33.33%, 50%, 66.67%, 80% , 88.89%) was injected into the size exclusion chromatography column, and the absorbance was measured at 230 nm.

As a result, as shown in FIG. 6, the extracellular albumin extinction band areas were almost the same in each of the mixed samples, while the albumin extinction band area increased in proportion to the injection amount (FIG. 6 (a), b)). The purity of the extracellular endoplasmic reticulum was calculated from the extracellular albumin extinction band area and albumin extinction band area, and it was confirmed that the purity calculated from the experiment was within the range of ± 2% with the expected purity (FIG. 6 c)). From this, it has been proved that the total amount and purity of the extracellular endoplasmic reticulum contained in the sample can be analyzed by measuring the absorbance at 230 nm of the eluate through size exclusion chromatography in the case of the sample sample.

Example 5. Comparative analysis of the yields of different extracellular elastomer separation methods in colon cancer cell culture

In order to compare the yields of the conventional extracellular fibrinolysis method by the method of the present invention, extracellular extracellular fluid was isolated from colon cancer cell culture medium using a conventional ultracentrifugal centrifugation method (100,000 xg, 2h) or a polyethylene glycol based precipitation method After the endoplasmic reticulum was separated, the purity of the extracellular endoplasmic reticulum was analyzed by the above purity analysis method. Extracellular ER samples isolated from colorectal cancer cell culture were injected into Cefacill S500 column, and size exclusion chromatography was developed. The absorbance at 230 nm of each eluted sample was measured and analyzed.

As a result, purity of the extracellular albumin extinction band area and other extinction band areas detected at a 230 nm absorbance chromatogram (FIG. 7 (a)) of each sample was calculated at 3.3 minutes. As a result, the polyethylene glycol- The extracellular vesicle purity of the method was about 69% and the purity of the ultra-high speed centrifugation method was about 43%. It was confirmed that the separation yield of the polyethylene glycol precipitation method was higher (Figs. 7 (b) and (c)).

In order to verify the purity analysis method of the present invention, analysis of protein sugar nanoparticles, which is a method of evaluating the purity of extracellular endoplasmic reticulum, was performed. As a result, extracellular endoplasmic reticulum of a sample isolated by a polyethylene glycol- (Fig. 7 (d)).

Example 6. Purity comparison analysis of different extracellular ERs in human urine

The purity of the extracellular endoplasmic reticulum isolated from a human urine sample was analyzed by the same method as in Example 5 using ultra-high-speed centrifugation (100,000 xg, 2h) or a polyethylene glycol-based precipitation method.

As a result, as shown in FIG. 8, the relative area of the extracellular albumin absorbance band detected at 3.3 min in a 230 nm absorbance chromatogram (FIG. 8 (a)) of each sample was calculated and the purity was analyzed. Showed a purity of less than 10% in all of the samples through the polyethylene glycol-based precipitation method (FIG. 8 (b), (c)).

From this, it can be understood that the method of the present invention can be utilized as an efficient yield analysis method of a method of separating extracellular endoplasmic reticulum from body fluids.

Claims (20)

1. (a) injecting a sample containing extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the sample;

   (b) detecting an absorbance chromatogram for a specific wavelength of the developed sample;

   (c) distinguishing the extinction band of the extracellular endoplasmic reticulum from the extinction band of the impurity from the extinction chromatogram of step (b);

   (d) calculating an absorption band area of the extracellular vesicle and an absorption band area of the impurity from the absorption chromatogram of the step (b); And

   (e) calculating the purity of the extracellular endoplasmic reticulum from the value of the extinction band of the extracellular endoplasmic reticulum and the value of the extinction band of the impurity

   A method for analyzing purity of extracellular endoplasmic reticulum .
2. The method according to claim 1,

   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, a plasma, a saliva, a tear, sweat, urine, feces, cerebrospinal fluid (CSF) , Amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermented food.

   A method for analyzing purity of extracellular endoplasmic reticulum.
3. The method according to claim 1,

   Wherein the specific wavelength is one or more values selected from the range of 200 nm to 800 nm.
4. The method according to claim 1,

   Wherein said specific wavelength is at least four wavelengths of at least 230 nm, 260 nm and 280 nm in the range of 330 to 450 nm.
5. The method according to claim 1,

   Wherein the extinction band of the extracellular endoplasmic reticulum satisfies the following conditions:

   i) a peak having absorbance at 230 nm, 260 nm and 280 nm;

   ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; And

   iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.
6. The method according to claim 1,

   Wherein the extinction band of the impurity does not satisfy one or more of the following conditions:

   i) a peak having absorbance at 230 nm, 260 nm and 280 nm;

   ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; or

   iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.
7. The method according to claim 1,

   The extinction band area (A _EV ) of the extracellular endoplasmic reticulum was calculated by calculating the area under the 230 nm peak satisfying the following conditions:

   i) a peak having absorbance at 230 nm, 260 nm and 280 nm;

   ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; And

   iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.
8. The method according to claim 1,

   The extinction band area (A _CONT ) of the impurity is calculated by calculating the area under 230 nm peak which does not satisfy at least one of the following conditions:

   i) a peak having absorbance at 230 nm, 260 nm and 280 nm;

   ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; or

   iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.
9. The method according to claim 1,

   The purity of the extracellular endoplasmic reticulum is calculated using the following formula

   Purity analysis of extracellular endoplasmic reticulum:

   

   A _EV is the extinction band area of the extracellular endoplasmic reticulum, and A _CONT is the extinction band area of the impurity.
10. (a) injecting a sample containing extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the sample;

    (b) detecting a light absorption chromatogram for the first wavelength of the developed sample;

    (c) detecting a light absorption chromatogram for a second wavelength different from the first wavelength of the developed sample; And

    (d) calculating an extinction band area of an extracellular vesicle or an extinction band area of an impurity from each of the above absorbance chromatograms.
11. 11. The method of claim 10,

    Wherein the first wavelength and the second wavelength are one or more values selected from the range of 200 nm to 800 nm.
12. 11. The method of claim 10,

    Wherein the first wavelength and the second wavelength are at least one value selected from the group consisting of 230 nm, 260 nm, 280 nm, and 330 to 450 nm.
13. 11. The method of claim 10,

    The first wavelength is one of 330 nm to 450 nm, 230 nm, 260 nm and 280 nm,

    Wherein the second wavelength is one or more values selected from the range of 200 nm to 800 nm.
14. 11. The method of claim 10,

    Wherein the analysis of the extracellular endoplasmic reticulum comprises analyzing the ratio of the extinction band area of the extracellular endoplasmic reticulum to each of the wavelengths.
15. 11. The method of claim 10,

    Wherein the analysis of the extracellular endoplasmic reticulum comprises analyzing the ratio of the extinction band area of the impurity to each wavelength.
16. (a) injecting a sample containing extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the sample;

    (b) detecting an absorbance chromatogram for a specific wavelength of the developed sample;

    (c) calculating an extinction band area of the extracellular endoplasmic reticulum from the absorbance chromatogram of step (b); And

    (d) calculating the total amount of extracellular endoplasmic reticulum from the extinction band area of said extracellular endoplasmic reticulum

    Quantitative analysis of extracellular endoplasmic reticulum.
17. 17. The method of claim 16,

    Wherein the specific wavelength is one or more values selected from the range of 200 nm to 800 nm.
18. 17. The method of claim 16,

    Wherein the specific wavelength is at least four wavelengths in a range of 330 to 450 nm, 230 nm, 260 nm and 280 nm.
19. 17. The method of claim 16,

    The extinction band area (A _EV ) of the extracellular endoplasmic reticulum was calculated by calculating the area under the 230 nm peak satisfying the following conditions: Quantitative analysis method of extracellular endoplasmic reticulum:

    i) a peak having absorbance at 230 nm, 260 nm and 280 nm;

    ii) a peak at a ratio of the absorbance at 260 nm to the absorbance at 280 nm (260 nm / 280 nm) of 1 or more; And

    iii) there is a peak for one or more wavelengths selected from the range of 330 nm to 450 nm.
20. 17. The method of claim 16,

    Wherein the total amount of the extracellular endoplasmic reticulum is calculated using the following equation

    Quantitative analysis of extracellular endoplasmic reticulum:

    

    A _EV is the extinction band area of the extracellular vesicle, and a and b are constants.

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