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WO2018030511A1 - Procédé de détection de protéine présente dans une membrane d'exosomes - Google Patents

Procédé de détection de protéine présente dans une membrane d'exosomes Download PDF

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Publication number
WO2018030511A1
WO2018030511A1 PCT/JP2017/029064 JP2017029064W WO2018030511A1 WO 2018030511 A1 WO2018030511 A1 WO 2018030511A1 JP 2017029064 W JP2017029064 W JP 2017029064W WO 2018030511 A1 WO2018030511 A1 WO 2018030511A1
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cancer
exosome
biological sample
cea
protein
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Japanese (ja)
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省三 横山
裕機 山上
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Wakayama Medical University
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Wakayama Medical University
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

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  • the present invention relates to a method for detecting a protein present in an exosome membrane.
  • Exosomes are vesicles with a diameter of about 40 nm to about 150 nm that are secreted from cells, and contain proteins, mRNA, miRNA and other molecules. Since exosomes secreted from tumor cells contain different molecules depending on tumor cell types, exosome-derived molecules are attracting attention as biomarkers. In recent years, cancer diagnosis using exosomes derived from cancer cells in serum as tumor markers has been reported (Non-patent Documents 1 and 2). However, the measurement of proteins that are supposed to be present on the exosome membrane is limited to molecules such as CD9, CD63, and CD81, and there are many membrane proteins such as CEA family and integrins that are related to cell function. Nevertheless, there are no reports measuring these proteins on the exosome membrane.
  • CEA carcinoembryonic antigen
  • CEACAM CEA-related cell adhesion molecule
  • PSG pregnancy-specific glycoprotein
  • CEA belongs to the CEACAM group together with CEACAM 1, CEACAM 3, CEACAM 4, CEACAM 6, CEACAM 7 and CEACAM 8.
  • CEA is used as a diagnostic marker for cancer recurrence, there is no report that it is used as a marker for early diagnosis of cancer.
  • CEA family members other than CEA are used as tumor markers.
  • An object of the present invention is to provide a highly accurate detection method for a protein present in an exosome membrane. Another object of the present invention is to provide a method for detecting cancer, particularly a method for detecting distant metastasis of cancer.
  • the present inventors have intensively studied to solve the above-mentioned problems. As a result, it is not possible to detect CEA present in the exosome membrane simply by detecting exosomes obtained from serum, but bovine serum albumin (bovine It was found by chance that CEA present in the exosome membrane can be detected in the presence of serum (albumin®; BSA). In addition, when a remote metastasis diagnosis of colorectal cancer was performed using the measured value of CEA present in the exosome membrane, it was also found that a highly accurate diagnosis was possible.
  • the present invention provides the following.
  • a method for detecting a protein present in an exosome membrane in a biological sample A method comprising: contacting an exosome isolated from the biological sample dispersed in a bovine serum albumin solution with an antibody against the protein, and detecting a complex of the protein and the antibody.
  • the concentration of the bovine serum albumin solution is 1% (w / v).
  • the concentration of exosomes isolated from the biological sample dispersed in the bovine serum albumin solution is 1.5 to 10 times the concentration of exosomes in the biological sample. 3].
  • the concentration of exosomes isolated from the biological sample dispersed in the bovine serum albumin solution is 2.5 to 5 times the concentration of exosomes in the biological sample. the method of. [6] The method according to any one of [1] to [5], further comprising a step of thawing the sample at 4 ° C. to 60 ° C. when the biological sample is a frozen sample. [7] The method according to [6], wherein the melting step is performed at 4 ° C to 37 ° C.
  • the detection method of the present invention it is possible to detect a protein present in an exosome membrane with high accuracy.
  • the biological sample is a frozen sample, it is possible to detect the protein present in the exosome membrane, and more accurate detection is possible by setting the melting temperature to an appropriate temperature.
  • the above-described highly accurate detection method it is possible to detect cancer, particularly detection of distant metastasis of cancer.
  • FIG. 2A is a diagram showing changes in the amount of exosomes in serum due to differences in the melting temperature of frozen serum
  • FIG. 2B is a diagram showing changes in measured values of CEA present in the exosome membrane.
  • Each graph connects the measured values of CEA derived from the 8 patients with colorectal cancer.
  • FIG. 4 is a diagram showing an ROC curve of exosomes in serum when the melting temperature of frozen serum is 4 ° C., 25 ° C., or 37 ° C., and a diagram showing an ROC curve of CEA in serum (B). . It is a figure which shows the ROC curve of CEA (eCEA) of exosome and CEA in serum.
  • eCEA CEA
  • the present invention provides a method for detecting a protein present in an exosome membrane in a biological sample (hereinafter abbreviated as “exosome membrane protein”) (hereinafter sometimes abbreviated as “the detection method of the present invention”). To do.
  • the method is (1) contacting an exosome isolated from the biological sample dispersed in a bovine serum albumin (BSA) solution with an antibody against the protein; (2) including detecting a complex of the protein and the antibody.
  • BSA bovine serum albumin
  • the present invention also provides a test method for diagnosing distant metastasis of cancer based on the above detection method (hereinafter sometimes abbreviated as “test method of the present invention”).
  • the method is (1) The concentration of exosome membrane protein in the biological sample derived from the subject is measured by the detection method of the present invention, and (2) the protein concentration obtained in (1) is compared with the reference value. Including doing. As a result of the comparison, when the protein concentration in the subject is higher than the reference value, it can be diagnosed that there is distant metastasis of cancer.
  • the biological sample used in the detection method of the present invention is not particularly limited as long as it is a sample obtained from a living body.
  • the sample obtained from a living body include body fluids and cell culture supernatants, preferably body fluids.
  • body fluid include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal discharge, semen, pleural effusion, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, and lymph fluid, preferably Serum or plasma.
  • the culture supernatant of the cell include a culture supernatant containing no exosome other than the exosome derived from the cell.
  • a culture supernatant for example, after culturing the target cell under suitable conditions, the medium is replaced with a serum-free medium in order to remove exosomes other than the cell-derived exosome, and the culture solution is obtained.
  • the supernatant obtained by centrifuging is mentioned.
  • the biological sample may not be frozen or may be frozen.
  • the method for isolating exosomes from a biological sample is not particularly limited. Examples of such methods include ultracentrifugation, sucrose cushioning, density gradient centrifugation, immunoprecipitation, polymer precipitation, and size exclusion.
  • exosomes can be isolated from a biological sample using a commercially available kit. Examples of such kits include ExoQuick TM (System Biosciences), miRCURY TM Exosome Isolation Kit (Takara Bio), Total Exosome Isolation (Life Technologies), ExoTrap TM Exosome Isolation Spin Column Kit for Protein Research (Cosmo Bio), Examples include exoEasy Maxi Kit (Qiagen).
  • a method for dispersing exosomes isolated from a biological sample in a bovine serum albumin (BSA) solution is not particularly limited.
  • a method of dispersing an exosome pellet isolated from a biological sample with a BSA solution a method of adding BSA after dispersing the pellet with MilliQ water or the like can be mentioned.
  • the concentration of BSA in the BSA solution is not particularly limited, but the lower limit is preferably 0.5% (w / v) or more, more preferably 0.8% (w / v) or more, and further preferably 1% (w / v) or more,
  • the upper limit is preferably 5% (w / v) or less, more preferably 4% (w / v) or less, and further preferably 3% (w / v) or less.
  • the concentration of BSA is preferably 0.5% (w / v) to 5% (w / v), more preferably 0.8% (w / v) to 4% (w / v), and 1% (w / v) to 3 % (w / v) is more preferable, and 1% (w / v) is particularly preferable.
  • the exosome membrane protein to be detected in the detection method of the present invention is a protein that is present on the exosome membrane and that is partly exposed to the extent that it has antigenicity and can react with an antibody.
  • tetraspanin cell adhesion molecule
  • antigen presentation related protein cytokine receptor and the like
  • membrane of the exosome in the body fluid of a cancer patient are mentioned.
  • tetraspanin include CD9, CD37, CD63, and CD81.
  • the cell adhesion molecule include integrin, CEA family member, ICAM-1 (intercellular adhesion molecule-1), CD31 and the like.
  • antigen presentation related protein examples include MHC I and MHC II.
  • cytokine receptors examples include EGFRvIII.
  • CD9, CD24, CD91, CD147, caveolin-1, glypican-1, and EpCAM Epidermal growth factor receptors
  • glycoproteins CD9, CD37, CD63, integrin, CEA family members ICAM-1, CD31, MHC I, MHC II, EGFRvIII, CD24, CD147, glypican-1, EpCAM are preferred, CEA family Members are more preferred.
  • Examples of CEA family members include CEA, CEACAM 1, CEACAM 3, CEACAM 4, CEACAM 6, CEACAM 7, CEACAM 8, and preferably CEA, CEACAM 1, and CEACAM 6.
  • the antibody used in the detection method of the present invention may be either a polyclonal antibody or a monoclonal antibody, and can be prepared by a well-known immunological technique. Commercially available antibodies can also be used.
  • the antibody may be an antibody-binding fragment (eg, Fab, F (ab ′) 2 ) or a recombinant antibody (eg, a single chain antibody).
  • the method for detecting an exosome membrane protein in a biological sample is not particularly limited as long as it uses an antigen-antibody reaction.
  • examples of such methods include ELISA (Enzyme-Linked ImmunoSorbent Assay), chemiluminescent enzyme immunoassay (Chemiluminescent Enzyme Imuno Asssay; CLEIA method), chemiluminescence immunoassay (Chemiluminescent Immunomm Asssay; CLIA method), electrolysis Examples include chemiluminescence immunoassay (Electro-chemiluminescence immunoassay; CLI ECLIA method), radioimmunoassay (RIA method), fluorescent antibody method, latex agglutination method, Western blotting method and the like.
  • the exosome membrane protein can be detected using a commercially available kit containing an antibody specific for the exosome membrane protein.
  • kits include, for example, CEA (human) ELISA kit (abnova, Taipei city, Taiwan), Human CEACAM-1 DuoSet (R & D Systems, Minneapolis, MN), Human CEACAM-6 DuoSet (R & D Systems, Minneapolis, MN ), Carcinoembryonic antigen kit CEA Abbott, Architect i2000 (Abbott Japan, Chiba, Japan) and the like.
  • the mode, order, and specific method are not particularly limited as long as these antibodies can interact with the protein in the solution.
  • the contact can be performed, for example, by adding the solution to a plate on which the antibody is immobilized.
  • the exosome membrane protein in the solution can be separated by means such as SDS-PAGE, transferred to a membrane and fixed, and then contacted with an antibody.
  • the time for maintaining such contact is not particularly limited as long as it is sufficient for the antibody and exosome membrane protein to bind to form a complex, but it is usually several seconds to several tens of hours.
  • the temperature condition for the contact is usually 4 ° C. to 50 ° C., preferably 4 ° C. to 37 ° C., and most preferably 15 ° C. to 37 ° C.
  • pH conditions for conducting the reaction are preferably pH 5.0 to pH 9.0, and particularly preferably pH 6.0 to pH 8.0.
  • exosome membrane protein is detected by sandwich ELISA, but is not limited to this method as long as it uses an antigen-antibody reaction.
  • the sandwich ELISA method is performed as follows. First, the primary antibody is immobilized on the well surface of an ELISA plate. Next, blocking is performed to prevent nonspecific adsorption to the well surface, and then an exosome isolated from a biological sample is added and brought into contact with the antibody to form a complex. After the exosomes that have not bound to the antibody are removed by washing, a labeled secondary antibody is added to the well to form a complex, and detection and quantification are performed using the label. Quantification can be performed by a method known per se, such as quantifying the signal detected based on the label used using a calibration curve created using a standard sample, or measuring the protein concentration. .
  • the concentration of exosomes isolated from the biological sample dispersed in the BSA solution is higher than the concentration of exosomes in the biological sample, Furthermore, highly accurate detection is possible. Examples of such a case include a case where an exosome pellet isolated from a biological sample is dispersed in a smaller amount of BSA solution than the biological sample.
  • the concentration of exosomes isolated from the biological sample dispersed in the BSA solution is preferably 1.5 to 10 times, more preferably 2 to 7.5 times the concentration of exosomes in the biological sample. More preferably, it is 2.5 to 5 times.
  • the detection method of the present invention further includes a step of thawing the sample at a specific temperature when the biological sample is a frozen sample.
  • the lower limit of the melting temperature is preferably 4 ° C or higher, more preferably 10 ° C or higher, further preferably 20 ° C or higher, and the upper limit is preferably 60 ° C or lower, more preferably 50 ° C or lower, further preferably 40 ° C or lower, 37 ° C.
  • the melting temperature is preferably 4 ° C. to 60 ° C., more preferably 4 ° C. to 37 ° C., further preferably 10 ° C. to 33 ° C., still more preferably 20 ° C. to 30 ° C., and particularly preferably 25 ° C.
  • the mechanism by which the measured value changes due to the difference in melting temperature is that when the exosome membrane protein is a glycoprotein, the glycan hydration change due to freezing and thawing Is presumed to change the reactivity of the antibody. Therefore, when using frozen serum, the exosome membrane protein to be detected is preferably a glycoprotein.
  • the remote metastasis of cancer can be detected by the detection method of the present invention.
  • cancer for example, when the exosome membrane protein in the biological sample to be detected is a CEA family member, colon cancer, small intestine cancer, biliary tract cancer, stomach cancer, esophageal cancer, liver cancer, pancreatic cancer, breast cancer , Lung cancer, medullary thyroid cancer, uterine cancer, ovarian cancer, and the like, preferably colon cancer, stomach cancer, and esophageal cancer, more preferably colon cancer.
  • exosome membrane protein in the biological sample to be detected is CD24, EpCAM, ovarian cancer, etc.
  • CD91 is lung cancer, etc.
  • CD9 is colon cancer, etc.
  • CD63 caveolin-1
  • pancreatic cancer breast cancer and the like, but it is not limited to these combinations.
  • the concentration of exosome membrane protein in a biological sample derived from a subject is measured, and the protein concentration and the reference value are calculated.
  • the reference value includes a cut-off point set based on an ROC curve prepared for a patient population with known cancer (particularly, distant metastasis of cancer), and an exosome membrane protein in a biological sample derived from a healthy person Can be mentioned.
  • the concentration of exosome membrane protein in a biological sample derived from a subject is compared with a reference value, and that the protein concentration in the subject is higher than the reference value is an indicator of distant metastasis of cancer Become.
  • the detection method of the present invention collects exosome membrane protein detection data and measurement data of the protein concentration, and from these data, the probability that cancer exists or cancer has distantly metastasized. Analyzes and evaluates possibilities.
  • Cutoff point is a value that can satisfy both high sensitivity and high specificity when a disease is determined based on the value. For example, among subjects with cancer, a value that shows a high positive rate in subjects with the presence of distant metastasis of cancer and a high negative rate in subjects without the presence of distant metastasis of cancer is used as a cut-off point Can be set. In the inspection method of the present invention, when using a cut-off point, it is desirable to set the cut-off point in advance.
  • the method for calculating the cut-off point is well known in this field. For example, when ROC curves are created using analysis software based on measured values of exosomal membrane proteins in biological samples in a group of cancer patients with known presence or absence of metastasis, when sensitivity and specificity are as close to 100% as possible Find the value (ie, the cut-off value corresponding to the point on the ROC curve that is closest to the coordinates (0,1)) and use that value as the cut-off point for determining the cancer metastasis in the subject be able to. For example, when investigating whether a subject has distant metastasis of colorectal cancer using CEA present in the exosome membrane in serum, thaw frozen serum at 25 ° C.
  • BSA 1% (w / v) BSA Is used to disperse the exosome pellet in the serum (the concentration of exosome in the dispersed liquid is 5 times the concentration of exosome in the serum), and a suitable cutoff point is 2.29 ng / mL
  • this value can vary depending on the number of populations and other conditions, and is not limited to this value.
  • Example 1 Change in CEA measurement value of exosome in serum The exosome pellet obtained from the same patient was dispersed using different solvents, and the change in the measurement value of CEA present in the exosome membrane was examined. This study included 8 patients with colorectal cancer who underwent surgery.
  • Exosomes were obtained from serum using ExoQuick TM Exosome Precipitation Solution (System Bioscience, Palo Alto, CA, USA). Specifically, 63 ⁇ l of ExoQuick TM Exosome Precipitation Solution was added to 250 ⁇ l of thawed serum, incubated at room temperature for 30 minutes, then centrifuged (1500 ⁇ g, 30 minutes, 4 ° C.), and the supernatant was removed by aspiration. Exosome pellets were obtained.
  • exosome CEA MilliQ water (250 ⁇ l or 50 ⁇ l) or 1% (w / v) BSA (250 ⁇ l or 50 ⁇ l) was added to the exosome pellet obtained from 250 ⁇ l of serum to disperse the pellet. .
  • these four solutions were prepared and exosomal CEA was measured. The measurement was performed using CEA (human) ELISA kit (abnova, Taipei city, Taiwan).
  • Example 2 Change in measured value of CEA of exosome in serum by melting temperature of frozen serum Thaw frozen serum at different temperatures, and change the amount of exosome in serum and the measured value of CEA present in exosome membrane. Examined. This study included 8 patients with colorectal cancer who underwent surgery.
  • Exosome quantification and exosome CEA measurement Exosome preparation is the same as in Example 1 except that frozen serum collected from the same patient was thawed at 4 ° C, 25 ° C, or 37 ° C for 30 minutes. The same method was used. Exosome quantification was performed using EXOCET Exosome Quantitation Kit (System Bioscience, Palo Alto, CA, USA). Exosome CEA was measured in the same manner as in Example 1 except that 50 ⁇ l of 1% (w / v) BSA was used to disperse the exosome pellet.
  • Example 3 Change in the diagnosis rate of distant metastasis using the CEA value of exosomes in serum with the melting temperature of frozen serum Thaw frozen serum at different temperatures and measure CEA present in the exosome membrane in the serum. An ROC curve was created to compare the presence or absence of distant metastasis in colorectal cancer patients. This study included 32 patients with colorectal cancer who underwent surgery.
  • Exosome CEA was prepared in the same manner as in Example 1 except that frozen serum collected from the same patient was thawed at 4 ° C, 25 ° C, or 37 ° C for 30 minutes. went. Exosome CEA was measured in the same manner as in Example 1 except that 50 ⁇ l of 1% (w / v) BSA was used to disperse the exosome pellet. For comparison, CEA in serum was measured using serum separated immediately after blood collection. Serum was separated by the same method as in Example 1. CEA in serum was measured using an oncofetal antigen kit CEA Abbott (Abbott Japan) and architect i2000 (Abbott Japan).
  • ROC curves were prepared for exosome CEA and serum CEA. The accuracy was examined based on the area under the ROC curve. Appropriate cut-off points were selected from the ROC curve and showed sensitivity, specificity, and correct classification. For statistical processing, the STATA software program ver. 13 (StataCorp, College Station, TX, USA) was used.
  • the CEA value of exosomes obtained from serum thawed at 25 ° C may be superior to the CEA value of exosomes obtained from serum thawed at other temperatures and the CEA value in serum. It was suggested.
  • the table which selected the appropriate cutoff point (Cut point), and compared sensitivity (Sensitivity), specificity (Specificity), and correct classification (Correctly Classified) is shown below (Table 1).
  • the cut-off point of CEA of exosome obtained from serum thawed at 25 ° C was 2.29 ng / mL
  • the sensitivity was 83.33%
  • the specificity was 96.15%
  • the correct classification was 93.75%. This result suggests that the diagnosis of distant metastasis of colorectal cancer using the exosome CEA value may have high diagnostic accuracy.
  • Example 4 Comparison of Remote Metastasis Diagnosis Rates Using Serum Exosome CEA and Serum CEA -Validation study- Frozen serum was thawed at 25 ° C., and CEA present in the exosome membrane in the serum was measured to create an ROC curve to compare the presence or absence of distant metastasis in colorectal cancer patients. This study included 100 patients with colorectal cancer who underwent surgery.
  • Example 1 Measurement of CEA of Exosome Exosome was prepared in the same manner as in Example 1. That is, 250 ⁇ l of frozen serum was thawed at 25 ° C. for 30 minutes, and an exosome pellet was obtained from the serum using ExoQuick TM Exosome Precipitation Solution (System Bioscience, Palo Alto, Calif., USA). Exosome CEA was measured in the same manner as in Example 1 except that 50 ⁇ l of 1% (w / v) BSA was used to disperse the exosome pellet. For comparison, serum CEA was measured by the same method as in Example 3.
  • Example 5 Possibility of remote metastasis diagnosis using exosomal CEACAMs (CEACAM 1 and CEACAM 6) Can CEA family members CEACAM 1 and CEACAM 6 also be used for remote metastasis diagnosis of colorectal cancer? I investigated. This study included 16 patients with colorectal cancer who underwent surgery.
  • Exosome CEACAM 6 was measured in the same manner as exosome CEACAM 1 except that Human CEACAM-6 DuoSet (R & D Systems, Minneapolis, Minn.) Was used. For comparison, CEACAM 1 and CEACAM 6 in serum were measured. Serum that had been stored frozen at ⁇ 20 ° C. was used for the measurement. Serum CEACAM 1 and CEACAM 6 were measured using Human CEACAM-1 DuoSet and Human CEACAM-6 DuoSet.
  • results Table 3 shows the results.
  • the measured values of exosome CEACAM 1 (eCEACAM1) and exosome CEACAM 6 (eCEACAM6) were high in stage 4, a stage with distant metastasis. From these results, it was suggested that the measured values of CEACAM 1 and CEACAM 6 of exosome may be useful for cancer diagnosis, particularly for remote metastasis diagnosis.
  • the detection method of the present invention it is possible to detect exosome membrane protein with high accuracy. Further, even when the biological sample is a frozen sample, it is possible to detect exosome membrane protein with high accuracy. Furthermore, it is possible to detect distant metastasis of cancer using the above detection method.

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Abstract

La présente invention concerne un procédé de détection de protéines présentes dans des membranes d'exosomes dans un échantillon biologique, le procédé comprenant une étape de mise en contact des exosomes isolés dudit échantillon biologique avec des anticorps desdites protéines, dans un état dispersé dans une solution d'albumine de sérum bovin, ce qui permet de détecter des complexes formés desdites protéines et desdits anticorps.
PCT/JP2017/029064 2016-08-12 2017-08-10 Procédé de détection de protéine présente dans une membrane d'exosomes Ceased WO2018030511A1 (fr)

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JP7687963B2 (ja) * 2019-12-23 2025-06-03 富士レビオ株式会社 細胞外小胞の回収方法、および採血管
KR20230044429A (ko) * 2020-07-08 2023-04-04 메르시 바이오애널리틱스, 인코포레이티드 폐암의 검출을 위한 조성물 및 방법
WO2025004683A1 (fr) * 2023-06-30 2025-01-02 国立大学法人大阪大学 Procédé d'examen cancéreux et agent de traitement cancéreux

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