Method for concentrating extracellular vesicle and / or cell-free DNA

Abstract

To provide a method for concentrating extracellular vesicles and / or cell-free DNAs at high efficiency and low cost.SOLUTION: A method for concentrating extracellular vesicles and / or cell-free DNAs contained in a liquid sample includes: (i) a step of preparing a liquid sample containing extracellular vesicles and / or cell-free DNAs; (ii) a step of adding protamine sulfate or spermidine to the sample, by which an aggregate of the extracellular vesicles and / or cell-free DNAs, and protamine sulfate or spermidine is obtained; and (iii) a step of recovering the aggregate.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for concentrating extracellular vesicles and / or cell-free DNA. [Background technology]

[0002] Traditionally, in order to diagnose a disease, a biopsy was performed to take a part of the lesion from the living body. In recent years, liquid biopsy, which uses biological fluid samples, has become a less invasive testing method. Liquid biopsy is attracting attention for its ability to analyze circulating tumor cells (CTCs). ), cell-free DNA (cfDNA) such as circulating tumor DNA (ctDNA), and exon These include extracellular vesicles (EVs) such as endothelial cell membrane infiltrates.

[0003] cfDNA is extracted using conventional nucleic acid extraction methods using spin columns or magnetic beads. However, cfDNA is only available in nanograms per mL of biological fluid sample. It is present in very small amounts (at the gram level), so large volumes of biological fluid samples are required. In addition, EVs can be isolated by ultracentrifugation or by using antibodies that recognize antigens on the membrane surface. Although it can be extracted by immunoprecipitation using a conventional method (Non-Patent Document 2), the recovery efficiency and the operating procedure are still poor. There are many kits for extracting cfDNA and EVs. Although commercially available, they are relatively expensive. There is a need to establish a method for extracting this compound in high yields and at low cost. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Meddeb,R.et al, Clin.Chem., Vol.65, No.5, pp.623-633(2019) [Non-Patent Document 2] Yang,D.et al, Theranostics, Vol.10, No.8, pp.3684-3707(2020) Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention solves the problems of the prior art and provides a method for extracting extracellular vesicles from a biological fluid sample by a simple operation. and / or to provide a method for extracting cell-free DNA with high yield and low cost. It was done with a purpose. [Means for solving the problem]

[0006] As a result of intensive research, the inventors have discovered that by adding protamine sulfate or spermidine to a biological fluid sample, This allows for good enrichment of extracellular vesicles and cell-free DNA in biological fluid samples. And found that.

[0007] That is, according to one embodiment, the present invention relates to extracellular vesicles and / or extracellular vesicles contained in a liquid sample. or a method for concentrating cell-free DNA, comprising: (i) extracting extracellular vesicles and / or cells; (ii) providing a liquid sample containing free DNA; and (iii) treating the sample with protamine sulfate. adding spermidine or spermidine, thereby producing extracellular vesicles and / or serine. (iii) obtaining a condensate of free DNA with protamine sulfate or spermidine; and recovering the agglomerate.

[0008] The liquid sample is preferably a biological fluid sample.

[0009] The method further comprises: (iv) after step (iii), treating the aggregates with a protease. A further step of digestion may be included.

[0010] According to one embodiment, the present invention also provides a method for treating a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of protamine sulfate or spermidine. and a kit for concentrating extracellular vesicles and / or cell-free DNA. It is.

[0011] For the extraction of cell-free DNA, the kit may further comprise a protease. preferable. Effect of the Invention

[0012] According to the method of the present invention, protamine sulfate or spermidine is added to a biological fluid sample. This allows the detection of trace amounts of extracellular vesicles and / or cell-free DNA in biological fluid samples. Highly efficient and low-cost extraction of extracellular vesicles and / or cell-free DNA with enrichment This becomes possible. [Brief description of the drawings]

[0013] [Figure 1] Figure 1 shows an electrophoretic image of lipoproteins contained in the supernatant and pellet lysate (exosome extract) obtained from a plasma sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in this specification. It is not something that can be achieved.

[0015] According to a first embodiment, the present invention relates to an extracellular vesicle and / or a vesicle containing in a liquid sample. 1. A method for concentrating cell-free DNA, comprising: (i) extracting extracellular vesicles and / or cell-free DNA from the ... (ii) providing a liquid sample containing DNA; and (iii) adding to the sample protamine sulfate or The method further comprises the step of adding spermidine, thereby forming extracellular vesicles and / or self-ligating cells. (iii) obtaining a condensate of DNA with protamine sulfate or spermidine; and recovering the collection.

[0016] In the method of this embodiment, a liquid sample containing extracellular vesicles and / or cell-free DNA is Prepare and use.

[0017] "Extracellular vesicles" (hereinafter referred to as "EVs") are lipid bilayers secreted from cells. EVs are small membrane vesicles. They can be classified into exosomes based on their size and surface markers. They are broadly classified into EVs, microvesicles, and apoptotic bodies. s may be any of these or a mixture thereof. "cfDNA" (hereinafter referred to as "cellular DNA") is DNA that is lost due to cell death or damage. The term "free DNA fragments" refers to the free DNA fragments released from cells by digestion.

[0018] The extracellular vesicles and cell-free DNA in this embodiment are not secreted from any cells. It may be secreted from either normal or diseased cells. The type of cells from which EVs and cfDNA are derived is not particularly limited, and can be, for example, dendritic cells. , T cells, B cells, neural cells, stem cells, cancer cells, primary cultured cells derived from them, or The cfDNA in this embodiment may be a cell line or the like. The organism from which the cells are derived is not particularly limited, and any The vertebrate may be any of the following: mouse, rat, rabbit, pig, cow, goat, The subject is a mammal such as a monkey or a human, and particularly preferably a human.

[0019] The liquid sample in this embodiment contains extracellular vesicles and / or cell-free DNA. As long as it is, there is no particular limitation, and for example, a body fluid obtained from a living body or a cell cultured The body fluid may be, for example, blood, plasma, serum, saliva, urine, gastric juice, etc. , pancreatic juice, bile, sweat, tears, breast milk, nasal secretions, intestinal fluids, etc., but are not limited to these.

[0020] Protamine sulfate or spermidine is then added to the liquid sample. Aggregates of exovesicles and / or cell-free DNA with protamine sulfate or spermidine The final concentration of protamine sulfate added to the liquid sample is, for example, 100 μl. The range may be from 600 μg / ml to 6.5 mg / ml. The final concentration of spermidine added to the liquid sample may be in the range of mg / ml. The range may be from 0.8 μg / ml to 85 μg / ml, and preferably from 4 μg / ml to 4 The range may be 5 μg / ml. Add protamine sulfate or spermidine to the liquid sample. After that, it is preferable to incubate it at room temperature for a certain period of time (for example, 10 to 30 minutes). .

[0021] To stabilize EVs and cfDNA and further improve extraction efficiency, sulfate protease was used. Add other ingredients as appropriate before or at the same time as adding tamoxifen or spermidine. Such other ingredients may be added, for example, salts such as sodium chloride, EDT, Chelating agents such as A, buffer solutions such as PBS, tetraethylene glycol dimethyl ether Some examples include:

[0022] The extracellular vesicles and / or cell-free DNA were then co-cultured with protamine sulfate or spermidine. The aggregates are collected by, for example, centrifugation at 4°C for 10 to 30 minutes at 16,000 × g. The pellet can be recovered by centrifugation for 10 min.

[0023] The method of this embodiment then further comprises digesting the aggregates with a protease. Digestion of aggregates may improve the efficiency of extraction of cfDNA in particular. The protease in this embodiment is not particularly limited, and may be, for example, proteinase K. serine proteases such as trypsin, chymotrypsin; cathepsin B, cathepsin L , cathepsin K, papain, and other cysteine ​​proteases; pepsin, chymosin, cathepsin and aspartic acid proteases such as syn D. Alternatively, two or more types of proteases can be used in combination. The protease is preferably proteinase K. The concentration of the protease depends on the type of protease. For example, proteinase K is 10 mg / ml to 100 mg / ml. The concentration may be in the range of 15 mg / ml to 30 mg / ml. The digestion conditions may be appropriately changed depending on the type of protease. In the case of proteinase K, incubation may be carried out at room temperature to 70° C. for 10 to 30 minutes.

[0024] Interaction of extracellular vesicles and / or cell-free DNA with protamine sulfate or spermidine The aggregates or their protease digests can be dissolved in a buffer such as PBS and then The EVs / cfDNA can be extracted by a known method established in the past. For example, DNA can be extracted using spin columns or magnetic beads, and EVs can be separated by ultracentrifugation. These can be extracted by immunoprecipitation using antibodies against surface markers. There are also many commercially available kits for extracting EVs and / or cfDNA based on the method. In this embodiment, such a commercially available product can also be used.

[0025] The method of the present embodiment is capable of detecting the presence of protamine sulfate or spermidine in a sample by simply adding the protamine sulfate or spermidine. It is possible to enrich EVs and / or cfDNA in E. coli at high efficiency and low cost. Allows extraction of Vs and / or cfDNA.

[0026] According to a second embodiment, the present invention comprises protamine sulfate or spermidine. A kit for concentrating extracellular vesicles and / or cell-free DNA. In this embodiment, the term "extracellular vesicles" and "cell-free DNA" are defined as in the first embodiment. It is the same as:

[0027] The kit of this embodiment may further include a protease in addition to the above configuration. The "protease" in this embodiment is the same as that defined in the first embodiment. The kit of the embodiment includes a buffer for use in extracting EVs and / or cfDNA and a Further information may be included, such as other reagents, instructions for extracting EVs and / or cfDNA. That's fine. EXAMPLES

[0028] The present invention will be further described below with reference to examples. However, these examples are not intended to limit the scope of the present invention. This does not imply a determination.

[0029] <1. Extraction of cfDNA> (1) Preparation of liquid samples containing cfDNA As liquid samples containing cfDNA, plasma samples, urine samples, and culture supernatant samples were used as follows. It was prepared according to the procedure. Plasma samples: EDTA2K-containing blood collection tubes (BD Vacutainer blood collection tubes, BD Japan) Blood samples were collected from five healthy subjects and centrifuged at 2,000×g for 15 minutes. The supernatant was collected and The supernatant obtained by centrifugation at 13,000 rpm (16,060 × g) for 15 minutes was used as a plasma sample. The fee was set at Urine samples: Urine samples collected from five healthy subjects were centrifuged at 2,000 × g for 15 minutes. The urine was collected and centrifuged at 13,000 rpm for 15 minutes, and the resulting supernatant was used as a urine sample. Culture supernatant sample: Add 10% FCS (Fujifilm Wako Pure Chemical Industries, 556-33865) Human chronic bone marrow injury was treated with RPMI1640 (Fujifilm Wako Pure Chemical Industries, 189-02025) Myeloid leukemia cell line K562 (National Institute of Biomedical Innovation, Health and Nutrition JCRB cells) (obtained from the bank) was cultured in a 50 ml flask until it became confluent. The supernatant was collected and centrifuged at 2,000×g for 15 minutes. The supernatant obtained by centrifugation at 400 nm for 15 minutes was used as the culture supernatant sample.

[0030] (2) Extraction of cfDNA from plasma samples (2-1) Extraction of cfDNA using protamine sulfate 1 mL of plasma sample (n=10) was diluted with 1 / 20 volume of 5 M NaCl (FUJIFILM Wako Pure Chemical Industries, Ltd.). Add 50 μL of 1% sulfuric acid (1% sulphuric acid) and mix. Add protamine (FUJIFILM Wako Pure Chemical, 160-05193) (150 μL) and mix. After leaving it at room temperature for 10 minutes, it was centrifuged at 13,000 rpm for 10 minutes and the supernatant was removed. Add 200 μL of PBS to dissolve the pellet, then add 20 mg / mL of proteinase K. Add 40 μL of (ProK) (Roche Diagnostics, 3115836001) The mixture was left at room temperature for 30 minutes with or without the addition of 100% glycerol. High Pure PCR Template Preparation Kit cfDNA was extracted using a 30 mL aliquot of 100% PBS (Roche Diagnostics, 1796828). cfDNA was eluted using 50 μL of Elution Buffer.

[0031] (2-2) Extraction of cfDNA using spermidine To 1 mL of plasma sample (n=10), add 1 / 10 volume of 5M NaCl (100 μL). Then, 1 / 10 the amount of 0.01% spermidine (FUJIFILM Wako Pure Chemical Industries, Ltd., 19 1-13831) (100 μL) was added and mixed. After standing at room temperature for 10 minutes, Add 1 μL of tetraethylene glycol dimethyl ether (Tokyo Chemical Industry) and mix. The mixture was then left to stand at room temperature for 10 minutes, after which it was centrifuged at 13,000 rpm for 30 minutes and the supernatant was removed. 160 μL of PBS was added to dissolve the pellet, and 20 mg / mL ProK was added to After adding 0 μL or no 0 μL, the mixture was left at room temperature for 30 minutes. High Pure PCR Template Preparation Kit cfDNA was extracted using the Elution Kit. This was performed using n Buffer.

[0032] As a control, we used cobas cfDNA Sa, a dedicated kit for cfDNA extraction. Mple Preparation Kit (Roche Diagnostics, 072 cfDNA was extracted from the same volume (1 mL) of plasma samples (n=10) using the 47737190 Extracted.

[0033] The extracted cfDNA solution was analyzed using KAPA hg DNA quantification. 1. Included in the NIH and QC Kit (Roche Diagnostics, KK4966) Quantification was performed using 29 bp Primer Premix. ghtCycler® 96 System (Roche Diagnostics) ) was used to determine the concentration of the extracted cfDNA solution (mean ± SD).

[0034] [Reaction solution composition (10 μL)] ·cfDNA solution 1μL ·2×FastStart Essential DNA Green Master( Roche Diagnostics, 06402712001) 5μL Primer Premix 1μL Distilled water remaining

[0035] [Reaction conditions] Step 1 (1 cycle): 95℃ 10 minutes Steps 2-3 (40 cycles): 95℃ 10 seconds, 62℃ 30 seconds

[0036] The results are shown in Table 1. By agglutinating cfDNA with protamine sulfate, It was shown that cfDNA can be extracted with the same efficiency as that of a dedicated cfDNA extraction kit. Furthermore, commercially available cfDNA was purified by digesting protamine sulfate-cfDNA aggregates with ProK. It was possible to extract cfDNA more efficiently than with a dedicated DNA extraction kit. When spermidine was used, spermidine-cfDNA aggregates were digested with ProK. This allows for more efficient extraction of cfDNA than commercially available cfDNA extraction kits. These results suggest that the method using protamine sulfate or spermidine is a low-cost and It is possible to efficiently extract cfDNA from a sample. It was shown that cfDNA can be extracted with high efficiency by ProK treatment.

[0037] Table 1. Extraction of cfDNA from plasma samples [Table 1]

[0038] (3) Extraction of cfDNA from urine samples Instead of 1 mL of plasma sample, 2 mL of urine sample (n=5) was used, and the same procedure as in 1(2-1) above was repeated. The procedure described above allows for the use of protamine sulfate and ProK, as well as a commercially available cfDNA extraction kit. We compared the cfDNA extraction efficiency with a method using a fluorouracil-based antibody.

[0039] The results are shown in Table 2. The method using protamine sulfate and ProK was It was confirmed that cfDNA can be extracted more efficiently than the method using a dedicated NA extraction kit. It was.

[0040] Table 2. Extraction of cfDNA from urine samples [Table 2]

[0041] (4) Concentration of cfDNA in the sample Instead of 1 mL of plasma sample, 1 mL, 2.5 mL, 5 mL or 10 mL of urine sample or The culture supernatant samples (n=3 each) were treated with sulfuric acid in the same manner as in 1(2-1) above. Extract cfDNA using protamine and ProK and collect 50 µL of cfDNA eluate. Got it.

[0042] The results are shown in Tables 3 and 4. In both urine and culture supernatant samples, the starting amount was dependent on the amount of the sample. The results suggest that the use of protamine sulfate and ProK significantly increased the yield of cfDNA. The method was shown to be capable of concentrating cfDNA with high efficiency.

[0043] Table 3. Enrichment of cfDNA in urine samples [Table 3]

[0044] Table 4. Enrichment of cfDNA in culture supernatant samples [Table 4]

[0045] <2.Exosome extraction> (1) Extraction of exosomes from exosome-containing biofluid samples The suspension-adherent human colon cancer cell line COLO2 was cultured in RPMI1640 supplemented with 10% FCS. 01 (obtained from the JCRB Cell Bank of the National Institute of Biomedical Innovation, Health and Nutrition) The cells were cultured at 7°C until they became confluent. The cells were harvested and washed twice with PBS(-). The cells were cultured in RPMI 1640 without FCS for 48 hours. Centrifuge at 0 × g for 15 minutes and filter the collected supernatant into a sterile syringe with a pore size of 0.22 μm. The mixture was filtered using a filter (Hawach Scientific, SLPES2522S). The supernatant was diluted with Total Exosome Isolation Reagent (fr om cell culture media) (Thermo Fisher Scientific The mixture was incubated overnight at 4°C. The mixture was centrifuged at 400 rpm for 60 minutes, the supernatant was removed, and the pellet was suspended in PBS(-). Protein amount was measured using Qubit Protein Assay Kit (Thermo Fisher Scientific) Scientific, Q33211) and PBS(-) was used to measure protein. An exosome solution was obtained with an exosome concentration adjusted to 10 μg / mL.

[0046] For 475 μL of plasma and urine samples (n=3 each) prepared in 1(1) above After adding RNaseA (10 U) and incubating at 37°C for 30 minutes, the above exosomes were 25 μL of solution was added. The obtained exosome sample solution was diluted with 1 / 20 volume of 5M NaCl. 1 was added and mixed, and then 3 / 20 volume of 1% protamine sulfate was added and mixed; or The obtained exosome sample solution was mixed with 1 / 10 volume of 5M NaCl, and then Then, 1 / 10 volume of 0.01% spermidine was added and mixed. After leaving it at room temperature for 10 minutes, The mixture was centrifuged at 3,000 rpm for 30 minutes, and the supernatant was removed. 150 μL of PBS was added to the pellet. The exosomes were then dissolved in water to obtain an exosome extract. Total Exosome Isolation Kit (from plasma a) (Thermo Fisher Scientific, 4484450) or Total Exosome Isolation Kit (from urine) (ThermoFisher Exosomes were extracted from the same amount of the same sample using a 100% ethanolamine (Shar Scientific, 4484452). A mu extract was prepared.

[0047] Exosome extraction efficiency is assessed by quantifying miRNA purified from exosome extracts. Specifically, the High Pure miRNA Isolation The miR The NA was purified and eluted with 50 μL of Elution Buffer. anscriptor First Strand cDNA Synthesis K cDNA was synthesized using it (Roche Diagnostics, 0489703001). The primers were a stem-loop RT primer (5'-GTCAGAGGAGG TGCAGGGTCCGAGGTATTCGCACCTCCTCTGACTCAACA- 3': SEQ ID NO: 1) was used. The composition of the reaction solution was as described in the instruction manual attached to the kit (1 The reverse transcription reaction was carried out under the following conditions.

[0048] [Reaction conditions] Step 1 (1 cycle): 16℃ 30 minutes Steps 2-4 (60 cycles): 30℃ 30 seconds, 42℃ 30 seconds, 50℃ 1 second Step 5 (1 cycle): 85℃, 5 minutes

[0049] The resulting reaction solution was diluted 5-fold with TE buffer to obtain a cDNA solution. Real-time PCR for miR-21 was performed under the conditions listed below. The reaction was performed using the ler® 96 System (Roche Diagnostics). Reactions and analyses were performed, and Cp values ​​(mean ± SD) were calculated.

[0050] [Reaction solution composition (10 μL)] ·cDNA solution 2.5μL ·2×FastStart Essential DNA Probes Master (Roche Diagnostics, 6402682001) 5μL 10 μM forward primer (5'-GCCTGCTAGCTTATCAGACT GATG-3': SEQ ID NO:2) 0.2μL 10 μM reverse primer (5'-GTGCAGGGTCCGAGGT-3': sequence Row number 3) 0.2μL 10 μM Universal Probe Library probe #82 (Roche Diagnostics) Sticks, 04689054001) 0.2μL Distilled water remaining

[0051] [Reaction conditions] Step 1 (1 cycle): 95℃ 10 minutes Steps 2-3 (40 cycles): 95℃ 10 seconds, 60℃ 30 seconds

[0052] The results are shown in Tables 5 and 6. When protamine sulfate and spermidine were used, However, exosomes can be extracted with the same or higher efficiency as commercially available exosome extraction reagents. It was shown that.

[0053] Table 5. Cp values ​​of miR-21 extracted from plasma samples spiked with exosomes [Table 5]

[0054] Table 6. Cp values ​​of miR-21 extracted from urine samples spiked with exosomes [Table 6]

[0055] (2) Concentration of exosomes in samples Add the exosome solution prepared in 2(1) above to 1mL or 10mL of RPMI1640. The same procedure as above in 2(1) was repeated except that 20 μL or 200 μL of the liquid was added to the sample. The same procedure was used to isolate exosomes in the sample using protamine sulfate or spermidine. was extracted, and the Cp value for miR-21 (mean ± SD, n = 3) was calculated.

[0056] The results are shown in Tables 7 and 8. Methods using either protamine sulfate or spermidine However, it was confirmed that the yield of exosomes increased depending on the starting amount. Both the protamine sulfate and spermidine methods were highly efficient in enriching exosomes. It has been shown that it is possible to reduce

[0057] Table 7. Exosome enrichment in urine samples [Table 7]

[0058] Table 8. Exosome enrichment in culture supernatant samples [Table 8]

[0059] (3) Contamination of lipoproteins during exosome extraction Lipoproteins such as LDL and HDL exist in plasma, and lipoprotein particles Therefore, in order to perform accurate exosome analysis, It is necessary to eliminate the contamination of lipoproteins in the isosome extraction. The presence or absence of lipoprotein contamination in the method was confirmed.

[0060] Add 500 μL of the plasma sample (n=2) prepared in 1(1) above to 1 / 20 volume of 5M NaCl. l (FUJIFILM Wako Pure Chemicals, 191-01665) was added and mixed, and then 3 / 20 volume was added. 100 ml of 1% protamine sulfate was added and mixed; or 1 / 10 volume of 5M NaCl was added and mixed. The mixture was mixed, and 1 / 10 volume of 0.01% spermidine was added and mixed. After standing for 1 h, the mixture was centrifuged at 13,000 rpm for 30 min, and the supernatant was collected. 0 μL of PBS was added to obtain pellet dissolution solution (exosome extract). Total Exosome Isolation K, a commercially available exosome extraction reagent Using it (from plasma), the supernatant and pellet lysate were obtained from the same amount of the same sample. The supernatant and pellet solution were then purified with QuickGel LIPO (Helena Laboratory, J715 ) and electrophoresis was performed using Eparizer 2 (Helena Laboratory). The results of electrophoresis are shown as images. The band intensities were quantified using ImageJ (analysis was performed in triplicate, and the mean values ​​were expressed as ± SD was calculated).

[0061] The results are shown in Figure 1. Lane 1: Supernatant obtained from plasma sample 1 by protamine treatment; Lane 2: Pellet solution obtained by protamine treatment from plasma sample 1; Lane 3: Plasma Lane 3: Supernatant obtained by protamine treatment from sample 2; lane 4: Supernatant obtained by protamine treatment from plasma sample 2 Lane 5: pellet lysate obtained by spermidine treatment from plasma sample 1 Lane 5: supernatant obtained by spermidine treatment from plasma sample 1; lane 6: pellet lysate obtained by spermidine treatment from plasma sample 1 Lane 7: Supernatant obtained by spermidine treatment from plasma sample 2; Lane 8: Plasma sample Lane 9: pellet lysate obtained by spermidine treatment from plasma sample 1; lane 10: commercial plasma sample 1 Lane 10: Supernatant obtained by treatment with a commercial reagent from plasma sample 1 Lane 10: Pellet lysate, lane 11: Supernatant obtained from plasma sample 2 by treatment with a commercial reagent, lane Lane 12: Pellet solution obtained from plasma sample 2 by treatment with a commercially available reagent; lane 13: Plasma sample Lane 13: Plasma sample 1, and lane 14: Plasma sample 2. The band intensities in FIG. 1 are shown in Table 9. In pellet lysates prepared with rotamine or spermidine, lipoproteins were The amount of exosomes was very small, whereas the amount of exosomes prepared using a commercially available exosome extraction reagent was very small. The pellet solution was found to be significantly contaminated with lipoproteins. The method of extracting exosomes using protamine or spermidine has been It is possible to minimize contamination by foreign substances and prepare highly pure exosomes. It was shown that:

[0062] Table 9. Lipoprotein contamination of exosome extracts [Table 9]

Claims

1. A method for concentrating extracellular vesicles and / or cell-free DNA contained in a liquid sample. So, (i) preparing a liquid sample containing extracellular vesicles and / or cell-free DNA; and, (ii) adding protamine sulfate or spermidine to the sample; The extracellular vesicles and / or cell-free DNA are then combined with protamine sulfate or spermidine. Agglomerates with dimethylformamide were obtained, (iii) recovering the agglomerate; The method includes:

2. The method of claim 1 , wherein the liquid sample is a biological fluid sample.

3. (iv) after said step (iii), a step of digesting said aggregates with a protease; The method of claim 1 or 2, further comprising:

4. Extracellular vesicles and / or self-binding proteins comprising protamine sulfate or spermidine. - A kit for concentrating DNA.

5. Cell-free DNA comprising protamine sulfate or spermidine sulfate and a protease A kit for concentrating A.

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