CN111505264A - Exosome separation method, immunomagnetic beads and kit - Google Patents
Exosome separation method, immunomagnetic beads and kit Download PDFInfo
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- CN111505264A CN111505264A CN201910090464.9A CN201910090464A CN111505264A CN 111505264 A CN111505264 A CN 111505264A CN 201910090464 A CN201910090464 A CN 201910090464A CN 111505264 A CN111505264 A CN 111505264A
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- magnetic beads
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- exosome
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention relates to an exosome separation method, immunomagnetic beads and a kit. The invention provides a method for separating exosomes, which comprises the step of contacting a sample containing exosomes from a subject with nanobeads to separate the exosomes from the sample, wherein the size of the nanobeads ranges from 30nm to 600nm, and the number of the nanobeads is 1x109‑2x1012Each per milliliter of sample. The invention also provides a nano magnetic bead, a kit and a method for preparing the nano magnetic bead. The method and the product have the advantages of convenient synthesis of magnetic beads and low cost; the method is suitable for various systems including blood plasma and cell supernatant; the dosage of the antibody is less; high exosome capture efficiency and the like.
Description
Technical Field
The present invention relates to the fields of chemistry and medicine. Specifically, the invention relates to an exosome separation method, immunomagnetic beads and a kit. The invention also relates to a nano magnetic bead, a preparation method of the nano magnetic bead, application of the nano magnetic bead in the medical field such as immunoassay, for example, application in exosome separation, and a kit containing the nano magnetic bead.
Background
Exosomes are membrane vesicles that are secreted by most cells and are secreted to the outside of the cell. In addition to playing an important role in cellular communication, there is increasing evidence that some cellular contents of exosomes, including proteins, micrornas, mrnas, fusion genes, etc., are closely related to the onset of human malignant tumors; in addition, exosomes play an important role in diagnosis, treatment, prognosis and the like of diseases. For this reason, it is desirable to find a simple, efficient, high-purity and automatable method for exosome isolation. Among them, the magnetic bead-based affinity capture exosome technology is particularly emphasized by its simple operation process, easy washing, separation and automation. See, e.g., Oksvold, m.p., a.neuroauter, and k.w.pedersen, Magnetic bead-based isolation of exosomes.methods Mol Biol, 2015.1218: p.465-81, Pedersen, K.W., B.Kierulf, andA.Neurauter, Specific and general Isolation of excellar visicles with magnetic beads.methods Mol Biol, 2017.1660: p.65-87.
At present, magnetic beads used for affinity capture of exosomes are mostly used
See, e.g., publication nos. CN106289926A, CN107893051A, CN108085338A, CN106967747A, US20130273544a1, EP3289361a4, WO2015112382a1, WO2018033929a1, etc. These magnetic beads are all in
Having modified specific antibodies, and methods of using the modified specific antibodies
To specifically capture exosomes.
See patent or patent application publication nos. US8658733B2, US20080139399a1, US20070299249a1, EP1693387B1, WO2010125170a1, US6984702B2 and US6787233B1, all of Thermo Fisher Scientific et al.
The existing method for capturing exosome by magnetic bead affinity needs to consume a large amount of antibody, and the cost of the magnetic bead method is very high due to the expensive price of the antibody. With DynabeadsTMMyOneTMCarboxylic Acid, cat 65012 for example, manufacturer's instructions indicate the antibodies and binding
The mass ratio of (A) to (B) is about 0.1: 1. Other magnetic beads coated with antibody are known from documents for performing liquid biopsies, such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A, etc., and the mass ratio of antibody to magnetic beads is also disclosed to be between (0.01-1): 1. The relatively high ratio of antibody to magnetic beads causes these methods to consume large amounts of antibody, increasing the cost of preparing exosome-capture magnetic beads.
Patent documents such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A and CN106381286A disclose the following preparation methods:
s1 preparation of magnetic nanoclusters;
s1.1 ferrous salt is reduced to Fe under the action of ammonia water3O4Nanoparticles;
s1.2 addition of oleic acid to Fe3O4Nano particles and generating magnetic nano clusters through high-temperature and high-pressure reaction;
s2 preparation of amino modified magnetic microspheres;
s2.1, adding ammonia water, a silanization reagent and an aminosilane coupling agent into the magnetic nano-cluster, and reacting for 1-3 days;
preparation of S3 hydrazine modified part a;
s3.1, carrying out hydrazine group modification on the amino-modified magnetic microsphere or antibody by using SANH (p-propylhydrazone pyridine carboxylic acid N-hydroxysuccinimide ester, CAS: 362522-50-7) with the molar equivalent of 10-50 times;
preparing an aldehyde modified B part of S4;
s4.1, performing aldehyde group modification on the amino-modified magnetic microsphere or antibody by using SFB (4-formylbenzoic acid N-succinimidyl ester, CAS: 60444-78-2) with the molar equivalent of 5-20 times.
S5 tumor cell capture.
The above method has troublesome immunomagnetic bead synthesis.
Still other documents such as CN106289926A, CN107893051A, etc. disclose the following methods:
s1 is based on
Preparation of exosome-capturing magnetic beads
S1.1 modification with streptavidin
S1.2 in the above
Adding a certain volume of antibody for incubation;
affinity capture of S2 exosomes
S3 serum was diluted for exosome capture.
The magnetic beads used in the method are imported
Therefore, there is a need in the art to develop a simpler immunomagnetic bead preparation method, a new exosome capture method and a high-efficiency, low-cost exosome capture kit with proprietary intellectual property rights.
Disclosure of Invention
The inventors have discovered, through research, a new and improved exosome capture method, a new and improved immunomagnetic bead, and related uses and kits. In some embodiments, it has been found that the amount of antibody consumed by the methods and/or products of the invention is significantly reduced. In some embodiments, it has been found that the capture efficiency of the methods and/or product exosomes of the present invention is significantly improved. In some embodiments, the methods and/or products of the invention have been found to have a broader range of applicability, for example, to capture exosomes in an unconcentrated/pre-enriched cell supernatant.
In some embodiments, the present invention provides a method of isolating exosomes, the method comprising contacting a sample containing exosomes from a subject with nanobeads, thereby isolating exosomes from the sample, wherein the nanobeads have a size in the range of 30-600nm, such as 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310hm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 570nm, 560nm, 580nm, 590nm, 600nm, or any range therebetween, such as a range of 30-150 nm. In some embodiments, it has been found that the capture efficiency of exosomes can be significantly improved and/or a widely applicable exosome capture method can be provided by adjusting the size of the magnetic beads for exosome separation, given the same total mass of the magnetic beads. In some embodiments, the size of the magnetic beads employed in the methods of the invention is preferably similar or identical to the size of the exosomes. In some embodiments, the size of the magnetic beads employed in the methods of the invention is preferably in the range of 30-600nm, such as 32nm, 45nm, 58nm, 62nm, 76nm, 88nm, 95nm, 106nm, 112nm, 125nm, 136nm, 143nm, 155nm, 166nm, 178nm, 184nm, 195nm, 205nm, 216nm, 228nm, 235nm, 246nm, 258nm, 262nm, 275nm, 286nm, 295nm, 306nm, 313nm, 322nm, 332nm, 345nm, 358nm, 365nm, 376nm, 382nm, 393nm, 404nm, 416nm, 428nm, 432nm, 445nm, 456nm, 466nm, 478nm, 483nm, 495nm, 502nm, 512nm, 524nm, 535nm, 546nm, 558nm, 569nm, 592nm, 600nm, or any range therebetween, such as 30-150 nm.
In some embodiments, the nanobead used in the separation method has one or more of the following properties: 1) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers; 2) has superparamagnetism; 3) from 8 to 12nm of Fe3O4The nano particles are agglomerated; 4) having a conjugate, e.g. an antibody to an exosome surface protein, e.g. exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, aNXA5, F L OT1, ICAM1, A L IX, GM130, ICAM-1, SNAP, MHC I/II, H L A-G, Integrins, Claudins, Tim4, 5) an antibody having a conjugate, e.g. a linker protein, e.g. an avidin, e.g. a linker selectively binding to exosomes, e.g. a linker selectively binding to exosome surface proteins or modified exosome surface proteins, e.g. CD9, CD63, CD81, CD44, CD31, Rab5b, EpTSTSG 101, HSP90, HSP70, ANXA5, F L OT1, ICAM1, A L IX, GM130, ICAM-1, SNAP, MHC I/II, H L A-G, Integrins, Claudin 4, a modified magnetic bead, or a modified magnetic bead of said protein, e.g. by addition of a plurality of said linker proteins, e.g. a ligand, e.g. a monoclonal antibody, optionally binding to exosome 1, such as a monoclonal antibody by addition of a linker protein, e.g. a monoclonal antibody to exosome, a monoclonal antibody, e.g. a monoclonal antibody, binding to an antibody9-2 x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5 x 1012To (c) to (d); and/or 7) the mass ratio of antibody or linker to magnetic bead is between (0.00005-0.005) to 1, such as 0.00005: 1, 0.0005: 1, 0.005: 1, or any range therebetween. In some embodiments, it has been found that by using magnetic beads having surface-modified thiols, e.g., surface-modified thiol-groups introduced by thiol-functionalized cross-linkers, the amount of magnetic beads is 1x10 per ml of sample9-2 x 1012At the same time, the amount of antibody used can be significantly reduced, and/or the capture efficiency of exosomes can be significantly improved. In some embodiments, the magnetic beads employed in the methods of the present invention preferably have superparamagnetism. In some embodiments, the magnetic beads employed in the methods of the inventionCan be made of 8-12nm Fe3O4The invention also relates to a method for the isolation of exosomes, which comprises the step of binding to exosomes, which is performed by a ligand binding to exosomes, which is performed by a bead-ligand binding to exosomes, which is a ligand, which is capable of binding to exosomes, which is selectively bound by a ligand, which is capable of binding to exosomes, which is performed by a ligand, which is capable of binding to exosomes, which is selectively binding to exosomes, which is performed by a ligand, which is capable of binding to exosomes, which is performed by a ligand, which is performed by binding to exosomes, which is an antibody, which is capable of binding to exosomes, which is performed by binding to a ligand, which is performed by a ligand, which is an antibody, which is performed by a ligand, which is performed by binding to exosomes, which is selectively binding to a ligand, which is specific for example a ligand, which is performed in an antibody, which is specific for example a ligand, which is performed in embodiments of binding to a ligand, which is specific for example a ligand, which is performed in embodiments of binding to exosomes, which is an antibody, which is performed by a ligand which is an antibody, which is specific for example a ligand which is an antibody, which is specific for example a ligand which is used in embodiments of antibody, which is used in embodiments of an antibody, which is a ligand which is an antibody, which is specific for example in embodiments of antibody, which is capable of binding to a ligand of binding to exosomeCoupling imido butyric acid-N-succinimidyl ester with magnetic beads. In some embodiments, the modification of the antibody can be achieved by coupling a linker, such as avidin, to the magnetic beads with a crosslinking agent, such as 4-maleimidobutyrate-N-succinimidyl ester. In some embodiments, the exosome surface protein may be linked to an avidin reaction on magnetic beads via a biotin avidin reaction.
In some embodiments, the amount of magnetic beads added employed in the methods of the present invention is not particularly limited. In some embodiments, the amount of magnetic beads employed in the methods of the present invention can be 1x10 per milliliter of sample9-2 x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5 x 1012In the meantime. In some embodiments, it has been found that the preferred addition amount of the magnetic beads can achieve sufficient separation of exosomes, improving the separation efficiency of exosomes. In some embodiments, the mass ratio of the antibody or linker to the magnetic bead in the methods of the invention is not particularly limited, but preferably the methods of the invention employ a reduced mass ratio of the antibody or linker to the magnetic bead using conventional methods. In some embodiments, the mass ratio of antibody or linker to magnetic bead in the methods of the invention is between (0.00005-0.005): 1, e.g., 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween.
In some embodiments, the source of the sample from which exosomes are isolated in the methods of the invention is not particularly limited. In some embodiments, the sample is from a bodily fluid, e.g. from a human subject, e.g. from a patient, e.g. a tumor patient, e.g. from blood, serum, serosal fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, mucosal secretions of secretory tissues and organs, vaginal secretions, milk, tears, ascites, e.g. from pleura, pericardium, peritoneum, abdomen or other body cavities, e.g. from a cell culture, e.g. from a cell supernatant of a concentrated/pre-enriched exosome, e.g. a cell supernatant which has not been concentrated. In some embodiments, the methods and/or products of the invention have been found to have a broader range of applicability, for example, to capture exosomes in an unconcentrated/pre-enriched cell supernatant.
In some embodiments, the methods of the invention further comprise one or more of the following features: adding blocking agent such as bovine serum albumin, human serum albumin, and amino ligand such as glycine into the sample; incubate to capture exosomes, e.g., for 1-4 hours at 37 ℃, or for 2-6 hours at room temperature, or overnight at 4 ℃.
In some embodiments, the present invention provides a nanobead having one or more of the following properties: 1) a size range in the range of 30-600nm, such as 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, or any range therebetween, such as 30-150 nm; 2) has superparamagnetism; 3) from 8 to 12nm of Fe3O4Nanoparticles agglomerated, 4) antibodies with a conjugate, e.g., an antibody to an exosome surface protein, e.g., exosome surface protein, CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT1, ICAM1, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim4, 5) antibodies with a conjugate, e.g., a linker protein, e.g., avidin,e.g.a linker which selectively binds to exosomes, e.g.a linker which selectively binds to exosome surface protein or modified exosome surface protein, e.g.to exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT1, ICAM1, A L IX, GM130, ICAM-1, SNAP, MHC I/II, H L A-G, Integrins, udeClains, Tim4 or a modified one or more of the above mentioned proteins, which modification may be e.g.an exosome surface protein modified by a ligand of said linker, e.g.an exosome surface protein modified by biotin, 6) an amount of magnetic beads per ml of sample to be detected9-2 x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5 x 1012To (c) to (d); 7) the mass ratio of the antibody magnetic beads is between (0.00005-0.005): 1, such as 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween; and/or 8) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers. In some embodiments, the nanobead provided by the present invention is preferably particularly suitable for use in the method for isolating exosomes of the present invention. In some embodiments, it has been found that the amount of antibody consumed by the nanobead of the present invention is significantly reduced. In some embodiments, it has been found that the capture efficiency of the nanobead exosomes of the present invention is significantly improved. In some embodiments, it has been found that the nanobead of the present invention has a broader range of applicability, e.g. it can be used to capture exosomes in unconcentrated/pre-enriched cell supernatant.
In some embodiments, the present invention provides a kit comprising a nanobead described herein. In some embodiments, the kit is particularly suitable for use in the method of isolating exosomes of the present invention. In some embodiments, the invention provides a kit for the isolation of exosomes comprising nanobeads as described herein. In some embodiments, the kit may further comprise other suitable components, such as components for exosome isolation. In some embodiments, the kits of the invention may further comprise a blocking agent, such as bovine serum albumin, human serum albumin, an amino ligand, such as glycine and the like. In some embodiments, it has been found that the amount of antibody consumed by the kits of the invention is significantly reduced. In some embodiments, it has been found that the capture efficiency of the kit exosomes of the present invention is significantly improved. In some embodiments, the kits of the invention have been found to have a broader range of applicability, e.g. to capture exosomes in unconcentrated/pre-enriched cell supernatants.
In some embodiments, the present invention provides a method for preparing a nanobead, the method comprising reacting a trivalent iron salt, a thiol-functionalized cross-linking agent, a stabilizing agent in coordination with a reduction reaction, and a reducing agent in a vessel at high temperature and high pressure to obtain the nanobead. In some embodiments, the nanobead of the present invention is a surface-modified thiol nanobead. In some embodiments, the methods of the invention provide surface-modified thiol nanobeads with functionalized cross-linkers. In some embodiments, the method of the present invention is a one-step method, i.e., the synthesis and surface modification of magnetic beads can be accomplished by a single step. In some embodiments, the invention provides an efficient and simple method for synthesizing immunomagnetic beads by one-step synthesis and surface modification of the magnetic beads. In some embodiments, magnetic beads prepared by the methods of the invention are particularly suitable for use in the exosome separation methods and kits of the invention. In some embodiments, it has been found that the amount of antibody consumed by the nanobead of the present invention is significantly reduced. In some embodiments, it has been found that the capture efficiency of the nanobead exosomes of the present invention is significantly improved. In some embodiments, it has been found that the nanobead of the present invention has a broader range of applicability, e.g. it can be used to capture exosomes in unconcentrated/pre-enriched cell supernatant.
In some embodiments, the present invention provides a one-step method for preparing a surface-modified thiol nanoparticle. In some embodiments, the iron salts that can be used in the methods of the present invention are not particularly limited. In some embodiments, the ferric salts can include ferric chloride, ferric sulfate, ferric acetate, ferric nitrate, ferric phosphate, ferric citrate, ferric pyrophosphate, and the like. In some embodiments, magnetic beads of the present invention can be prepared using a suitable cross-linking agent. In some embodiments, the thiol-functionalized cross-linking agent may include methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester and/or poly (ethylene glycol) 2-mercaptoethyl ether acetic acid, and the like. In some embodiments, the stabilizers that may be employed in the method of the present invention are not particularly limited, and for example, the stabilizers that may be employed in the complex reduction reaction include sodium acetate, trisodium citrate, urea, and the like. In some embodiments, the reducing agent that may be employed in the method of the present invention is not particularly limited, and for example, the reducing agent that may be employed includes ethylene glycol and the like. In some embodiments, the surfactant that may be employed in the method of the present invention is not particularly limited, and for example, the surfactant that may be employed includes polyethylene glycol, polyvinylpyrrolidone, and the like.
In some embodiments, the process of the invention may be carried out at a suitable reaction temperature, for example the reaction temperature may be in the range of 150 to 240 ℃. In some embodiments, the process of the invention may be carried out for a suitable time, for example the time of reaction may be in the range of 7 to 72 hours.
In some embodiments, the reaction components in the methods of the present invention may be appropriately selected. For example, in some embodiments, the ferric salt, the mercapto-functionalized cross-linking agent, the stabilizer and the surfactant are present in a co-reduction reaction in a mass ratio of 1 to (0-0.1) to (1.4-3.5) to (0.4-1.3), such as 1 to (0, 0.02, 0.04, 0.06, 0.08, 0.1 or any range therebetween) to (1.4, 1.5, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5 or any range therebetween) to (0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or any range therebetween).
In some embodiments, the method of the invention further comprises the step of coupling the nanomagnetic beads to a conjugate, in some embodiments, the conjugate of the magnetic beads employed in the method of the invention is not particularly limited in some embodiments, the conjugate or conjugate may be, for example, an antibody to an exosome surface protein, for example, exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L, optionally one or more antibodies that bind to an exosome surface protein in some embodiments, the invention may be a magnetic bead selectively bound to an exosome surface protein in a sample, thereby achieving isolation of the exosome in some embodiments, the modified exosome may be a bead-ligand that binds to an exosome surface protein, for example, a ligand, such as a ligand, or a ligand that may be selectively bound to an exosome in some embodiments, e.g, a ligand, or a ligand that may be used in embodiments of an exosome surface protein, in the invention, e.g. a antibody, a antibody that is a ligand, a ligand that binds to a ligand, in embodiments, a ligand, or a ligand that is specifically binds to a ligand, in an exosome surface protein, in embodiments, a antibody that is specifically in an exosome surface protein, in an antibody, in embodiments, a antibody that is an exosome 72, a cell L, a cell, or a cell, or a cell, or a cell, or cellA protein. In some embodiments, the amount of magnetic beads added employed in the methods of the present invention is not particularly limited. In some embodiments, the amount of magnetic beads employed in the methods of the present invention can be 1x10 per milliliter of sample9-2 x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5 x 1012In the meantime. In some embodiments, it has been found that the preferred addition amount of the magnetic beads can achieve sufficient separation of exosomes, improving the separation efficiency of exosomes. In some embodiments, the mass ratio of the antibody or linker to the magnetic bead in the methods of the invention is not particularly limited, but preferably the methods of the invention employ a reduced mass ratio of the antibody or linker to the magnetic bead using conventional methods. In some embodiments, the mass ratio of antibody or linker to magnetic bead in the methods of the invention is between (0.00005-0.005): 1, e.g., 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween.
In some embodiments, the magnetic beads prepared by the methods of the present invention have one or more of the following properties: 1) a size range in the range of 30-600nm, such as 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, or any range therebetween, such as 30-150 nm; 2) has superparamagnetism; 3) by8-12nm Fe3O4Nanoparticles agglomerated, 4) an antibody with a conjugate, such as an antibody, e.g. an antibody to an exosome surface protein, e.g. exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L or an antibody to one or more of these, 5) an antibody with a conjugate, e.g. a linker protein, e.g. an avidin, e.g. a linker that selectively binds to an exosome, e.g. an exosome surface protein or modified exosome surface protein, e.g. a linker that selectively binds to an exosome surface protein CD L, Rab5, a magnetic bead, tsxa 101, tsg. a magnetic bead, a ligand modified by a ligand, a9-2 x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic bead, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5 x 1012To (c) to (d); 7) the mass ratio of the antibody magnetic beads is between (0.00005-0.005): 1, such as 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween; and/or 8) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers.
The inventors have found that exosome capture methods and products can be significantly improved, in particular existing exosome capture methods and products can be significantly improvedConventional methods and magnetic bead affinity capture kits, e.g. based on
The exosome affinity capturing method of (1). It has been found that the efficiency of exosome capture can be significantly improved. In some embodiments, the methods and products of the present invention have been found to be based on quantitation of exosome capture by ddPCR and the housekeeping gene GAPDH
Compared with the exosome capturing method, the capturing efficiency of the exosome consuming 0.5 mu g of the antibody in the cell supernatant is 27 times higher than that of the antibody consuming
The capture efficiency (consumption of 13.5. mu.g antibody) was much higher; for plasma, the capture efficiency of the invention for exosomes consuming 0.5. mu.g of antibody is 9 times higher than the consumption of antibody
The capture efficiency (consumption of 4.5. mu.g antibody) was still higher. Compared with the conventional exosome capture kit, the invention has higher capture efficiency for exosomes in cell supernatant as well as plasma.
The inventors found that the magnetic bead synthesis process described in the prior art methods such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A is rather complicated and needs to be performed by Fe3O4Reducing the nano particles, generating magnetic nano clusters at high temperature and high pressure, and generating magnetic microspheres coated by aminated silicon dioxide under the action of ammonia water, a silanization reagent, an aminosilane coupling agent and the like. The inventor researches and discovers that the synthesis and modification of the magnetic beads can be realized by a one-step method. For example, in some embodiments, the present invention achieves that magnetic microspheres can be synthesized by a one-step process. In some embodiments, the method may employ ferric chloride hexahydrate, sodium acetate, polyethylene glycol, methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl esterThe ester and the glycol react under high temperature and high pressure. Surface modification and antibody coating in the prior art have also been found to be cumbersome. For example, after the magnetic bead is aminated, hydrazine and aldehyde-based antibodies need to be further modified to complete the modification. In contrast, the present invention only needs to react the prepared magnetic beads with a reactive reagent such as hydroxysuccinimide acid for 15min and then add the antibody or avidin to incubate.
Thus, in some embodiments, the invention provides a low antibody to magnetic bead ratio coated, high magnetic bead content exosome capture magnetic bead.
In some embodiments, preferably, the magnetic beads are prepared in one step by a forced lysis method.
In some embodiments, it is preferred that the size of the magnetic beads is the same as the size of the exosomes, being 30-150 nm.
In some embodiments, preferably, the magnetic beads are superparamagnetic.
In some embodiments, it is preferred that the magnetic beads consist of 8-12nm Fe3O4The nano particles are agglomerated.
In some embodiments, preferably, the antibody is an antibody to a protein commonly used in exosomes, including but not limited to antibodies to one or more of CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT1, ICAM1, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim4, and the like.
In some embodiments, preferably, the low antibody to magnetic bead ratio refers to a mass ratio of antibody to magnetic bead of between (0.00005-0.005): 1;
in some embodiments, preferably, the high content of magnetic beads refers to the number of magnetic beads in 1 × 10 per ml of cell supernatant/plasma9-1 x 1012Between
In some embodiments, the present invention provides a method for preparing an exosome-capture magnetic bead, which may comprise the following steps: s1: preparing magnetic beads; s2: modification of antibodies.
In some embodiments, the magnetic beads in step S1 are prepared by a one-step reaction via a forced lysis method.
In some embodiments, the magnetic beads in step S1 are preferably prepared by the following method:
1) adding a trivalent ferric salt, a cross-linking agent such as methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, a stabilizing agent such as sodium acetate, a surfactant such as polyethylene glycol, a reducing agent such as ethylene glycol into a high-temperature reaction kettle, and heating and reacting at 150-240 ℃ for 7-72 hours to obtain the magnetic beads.
In some embodiments, the modification of the antibody in step S2 is performed by coupling the antibody to magnetic beads under the action of 4-maleimidobutyrate-N-succinimidyl ester.
In some embodiments, the modification of the antibody in step S2 can be further obtained by coupling avidin to magnetic beads under the action of 4-maleimidobutyrate-N-succinimidyl ester and linking the avidin on the magnetic beads through a biotin avidin reaction.
In some embodiments, the invention provides a kit for isolating exosomes in a cell supernatant.
In some embodiments, preferably, the cell supernatant comprises a cell supernatant that has been concentrated/pre-enriched for exosomes and a cell supernatant that has not been concentrated.
In some embodiments, it is preferable that the kit for separating exosome in cell supernatant comprises the above-mentioned exosome capture magnetic beads, BSA (bovine serum albumin) or HSA (human serum albumin), and an amino ligand such as glycine and the like.
In some embodiments, exosome isolation in the concentrate/pre-enrichment treated cell supernatant may comprise the steps of:
s1 adding 1-10 times volume of 0.11% -0.2% BSA solution into the concentrated/pre-enriched cell supernatant;
s2 adding 0.001% -0.1% of mouse IgG to the system of the step S1;
s3 is added into the system of the step S21X10 cell supernatants per ml9-1x 1012Magnetic beads;
s4 mass ratio of the antibody to the magnetic bead in the step S2 is between 0.00005-0.005: 1;
s5 incubating the system of step S4 at 37 ℃ for 1-4 hours, or at room temperature for 2-6 hours, or at 4 ℃ overnight; and finishing the capture of the exosomes.
In some embodiments, exosome isolation in cell supernatant that has not been treated for exosome concentration/pre-enrichment may comprise the steps of:
s1, adding BSA with the mass fraction of 0.1-2% relative to the cell supernatant into the cell supernatant which is not subjected to concentration treatment;
s2 adding 0.001% -0.1% of mouse IgG to the system of the step S1;
s3 adding 1x10 per ml of cell supernatant to the system of the step S29-1 x 1012Magnetic beads;
s4 mass ratio of the antibody to the magnetic bead in the step S2 is between 0.00005-0.005: 1;
s5 incubating the system of step S4 at 37 ℃ for 1-4 hours, or at room temperature for 2-6 hours, or at 4 ℃ overnight; and finishing the capture of the exosomes.
In some embodiments, the invention provides a kit for isolating exosomes in plasma.
In some embodiments, it is preferable that the kit for separating exosomes in plasma comprises the above-described exosome capture magnetic beads, BSA or HSA, and mouse IgG.
In some embodiments, exosome separation in plasma may comprise the steps of:
s1 adding 1-5 times volume of 0.12% -0.2% BSA or HSA solution into plasma;
s2 adding 0.001% -0.1% of mouse IgG to the system of the step S1;
s3 adding 1x10 per ml of plasma into the system of the step S29-1x 1012Magnetic beads;
s4 mass ratio of the antibody to the magnetic bead in the step S2 is between 0.00005-0.005: 1;
s5 incubating the system of step S4 at 37 ℃ for 1-4 hours, or at room temperature for 2-6 hours, or at 4 ℃ overnight; and finishing the capture of the exosomes.
In some embodiments, the antibodies conjugated to the present invention are antibodies to proteins commonly used in exosomes, including, but not limited to, one or more of CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT1, ICAM1, A L IX, GM130, ICAM-1, SNAP, MHC I/II, H L A-G, Integrins, Claudins, Tim4, and the like.
In some embodiments, the conjugated antibodies in the methods and products of the present invention further comprise a method of first modifying the avidin outside the magnetic beads and then linking the above antibodies by a biotin avidin reaction.
In some embodiments, the BSA in the methods and products of the invention may be replaced with HSA.
In some embodiments, the cell supernatant in the methods and products of the invention may alternatively be described as a cell culture medium.
In some embodiments, the methods and products of the invention have applications ranging from, but not limited to, cell supernatants, plasma, serum, urine, milk, cerebrospinal fluid, tumor ascites, saliva samples.
In some embodiments, the methods and products of the present invention have one or more of the following advantages:
1) the synthesis and surface modification of the magnetic beads can be completed by a one-step method, and the synthesis of the immunomagnetic beads is convenient;
2) the magnetic beads have the same size of 30-150nm as the exosomes;
3) the coated magnetic beads need less antibodies, and the mass ratio of the antibodies to the magnetic beads is (0.00005-0.005) to 1; the magnetic beads of the present invention need only be consumed in cell supernatant and plasma, respectively
The 1/27 and 1/9 antibodies in the antibody pair were compared
Higher exosome capture efficiency; magnetic beads of the present invention are used in cell supernatants and plasma
The capture efficiency for exosomes in cell supernatant and plasma can be 28-fold and 2.4-fold, respectively, at the same antibody consumption. Therefore, the method and the magnetic beads can effectively reduce the consumption of antibodies and improve the capture efficiency of exosomes, and the method and the product have low cost and high efficiency;
4) the cost is reduced by matching the magnetic beads synthesized by the one-step method with extremely low antibody dosage;
5) the exosome capture kit can be applied to concentrated and pre-enriched cell supernatants and blood plasma, and is also suitable for cell supernatants which are not concentrated and pre-enriched. The methods and products of the invention have higher exosome capture efficiency than conventional exosome capture kits, and a greater range of uses (capture of exosomes in unconcentrated/pre-enriched cell supernatants).
Drawings
Figure 1 shows SEM characterization of synthetic magnetic beads.
Fig. 2 shows the hysteresis loop of the synthetic beads.
FIG. 3 shows the effect of varying the ratio of reaction components on the preparation of magnetic beads and coupled proteins; wherein FIG. 3A shows the ratios of thiol cross-linking agent, NaAc, PEG, and ferric chloride hexahydrate, where + represents satisfactory magnetic beads; -indicating unsatisfactory magnetic beads, all numbers appearing in the table being the mass ratio of the substance to ferric chloride hexahydrate; fig. 3B shows the effect of the ratio of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester addition on the effect of magnetic beads on coupling proteins.
FIG. 4 shows the coupling efficiency of magnetic beads synthesized by different methods to proteins (expressed by relative fluorescence intensity with respect to the present magnetic beads).
FIG. 5 shows the magnetic beads of the present invention in the supernatant of H3122 cells
Comparison of exosome capture efficiency (ddPCR positive droplet number quantification).
FIG. 6 shows the consumption of the same 0.5. mu.g of antibody, magnetic beads of the invention and
comparison of exosome capture efficiency in cell supernatants (ddPCR GAPDH copy number quantification).
FIG. 7 shows the magnetic beads of the invention in plasma with different amounts of antibody consumed
Comparison of exosome capture efficiency (ddPCR positive droplet number quantification).
FIG. 8 shows the consumption of the same 0.5. mu.g of antibody, magnetic beads of the invention and
comparison of exosome capture efficiency in plasma (ddPCR GAPDH copy number quantification).
FIG. 9 shows the ratio of coated beads with different antibodies to coated beads in the present invention
Comparison of magnetic beads for exosome capture efficiency in cell supernatants (ddPCR positive droplet number quantification).
FIG. 10 shows a comparison of the efficiency of exosome capture in plasma for magnetic beads of different particle size, same antibody content, total mass of magnetic beads (ddPCR GAPDH copy number quantification).
FIG. 11 shows the comparison of the capture efficiency of exosomes in cell supernatants (ddPCR positive droplet number quantification) for the same antibody content, same magnetic bead size, different number of magnetic beads in the present invention.
FIG. 12 shows the comparison of the number of different magnetic beads of the present invention with respect to the efficiency of exosome capture in plasma for the same antibody content (ddPCR positive droplet number quantification).
FIG. 13 shows the use of magnetic beads of the present invention in cell supernatants and plasma along with a control cell supernatant Exosome capture kit (Exosome-Human CD 63) Isolation/Detection Reagent，InvitrogenTM) And control plasma exosome capture kit (ExoCap)TMJSR L ife Sciences) capture exosomes, and WB characterization.
FIG. 14 shows the magnetic beads of the present invention in cell supernatant with a control Exosome capture kit (Exosome-HumanCD63Isolation/Detection Reagent, Invitrogen)TM) Comparison of exosome capture efficiency (ddPCRGAPDH copy number quantification).
FIG. 15 shows the magnetic beads of the present invention and a control exosome capture kit (ExoCap) in plasmaTMJSR L ifeSectens) comparison of exosome capture efficiency (ddPCR GAPDH copy number quantification).
FIG. 16 shows the capture of exosomes (ddPCRGAPDH copy number quantification) by inventive exosome magnetic beads in pre-concentrated/non-pre-concentrated cell supernatants.
FIG. 17 shows a control exosome capture kit (ExoCap) with magnetic beads of the present invention coupled to antibodies in plasma using an avidin antibody systemTMJSR L ife Sciences) comparison of exosome capture efficiency (ddPCR GAPDH copy number quantification).
Detailed Description
Compared with the prior art, the invention has one or more of the following advantages and outstanding effects: the invention can complete the synthesis and surface modification of the magnetic beads by a one-step method, and the synthesis of the immunomagnetic beads is convenient.
The magnetic beads of the present invention need only be consumed in cell supernatant and plasma, respectively
The 1/27 and 1/9 antibodies in the antibody pair were compared
Higher exosome capture efficiency; magnetic beads of the present invention are used in cell supernatants and plasma
The capture efficiency of the exosome in cell supernatant and plasma can reach 28 times respectively by consuming the same amount of the antibodyAnd 2.4 times. Therefore, the magnetic bead can effectively reduce the consumption of the antibody and simultaneously improve the capture efficiency of the exosome. Is a low-cost and high-efficiency exosome capturing kit.
The exosome capture kit of the present invention has higher exosome capture efficiency than the control exosome capture kit, and a larger range of use (capturing exosomes in the unconcentrated/pre-enriched cell supernatant).
The method for synthesizing the magnetic beads is simple and is obviously different from the complicated methods in the prior art (such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A and the like). The magnetic beads in the invention are simpler to synthesize and can be obtained through one-step reaction. One non-limiting example of a synthetic magnetic bead is as follows:
example 1 Synthesis of magnetic beads, antibody coating and blocking (FIGS. 1 and 2)
Adding 3.12g of ferric trichloride hexahydrate, 0.23g of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate and 2.4g of polyethylene glycol into 100m L of ethylene glycol, uniformly stirring, and reacting in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic beads.
After the magnetic beads are washed by ethanol, 0.01 mu mol/m L4-maleimide butyric acid-N-succinimide ester is added for reaction for 15min, after washing, the antibody is added according to the proportion that the ratio of the magnetic beads to the CD63 antibody (similar products are obtained by the same process by using other exosome surface specific antibodies) is 1: 0.0005, and the magnetic bead antibody is incubated overnight to complete the coating of the magnetic bead antibody.
The coated beads were blocked with 5% (w/v) serum albumin overnight and the exosome-capture beads were prepared.
Scanning electron microscopy characterization of the synthesized beads is shown in fig. 1, and the hysteresis loop of the synthesized beads is shown in fig. 2:
it can be seen from fig. 1 that the size of the magnetic beads in the present invention is between 30-150nm (exactly the same size as the exosomes). From the hysteresis loop in fig. 2, it can be seen that the saturation magnetic strength of the magnetic bead in the present invention is 60emu/g, and it is superparamagnetic. Therefore, the exosome capture magnetic bead is convenient and simple to synthesize.
Example 2 determination of extent of addition of reagents in Synthesis of magnetic beads (FIG. 3A)
Adding 3.12g of ferric trichloride hexahydrate into 100m L g of ethylene glycol, adding methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, sodium acetate and polyethylene glycol according to a certain proportion, stirring uniformly, reacting in a high-pressure reaction kettle at 220 ℃ for 8h, and performing characterization judgment on the obtained substance, wherein the magnetic beads are seriously agglomerated or do not form magnetic beads or the size of the magnetic beads is not within the range of 30-600nm and is judged to be unqualified (-), and the formed monodisperse magnetic beads within the range of 30-600nm are judged to be qualified (+), and the result is shown in fig. 3A.
As can be seen from FIG. 3A, when the mass ratio of the ferric salt, the thiol-functionalized cross-linking agent, the stabilizer and the surfactant which are carried out by the reduction reaction is 1: 0-0.1: 1.4-3.5: 0.4-1.3, the magnetic beads meeting the requirements can be synthesized.
Example 3 determination of the optimum amount of thiol-functionalized crosslinker to be added (FIG. 3B)
Adding 3.12g of ferric trichloride hexahydrate, a certain mass of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate and 2.4g of polyethylene glycol into 100m L of ethylene glycol, uniformly stirring, and reacting in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic beads.
After the magnetic beads are washed by ethanol, 0.01 mu mol/M L4-maleimide butyric acid-N-succinimide ester is added to react for 15min, after washing, the magnetic beads are placed into BSA marked by 10 mu M fluorescent dye Cy5, the magnetic beads are rotated and incubated for 2h at room temperature, PBST is washed twice and once by PBS, fluorescence is detected by a fluorescence microscope, and the coating efficiency of the protein is judged according to the fluorescence intensity.
It can be seen from FIG. 3B that the best capture efficiency is achieved when the mass ratio of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester to ferric chloride hexahydrate is 0.7: 1.
Example 4 other alternatives to synthetic magnetic beads (FIG. 4)
3.12g of ferric sulfate, 0.23g of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate and 2.4g of polyethylene glycol are added into 100m L of ethylene glycol, and after being uniformly stirred, the mixture reacts in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic beads ①.
Ferric acetate, ferric nitrate, ferric phosphate, ferroferric citrate, ferric pyrophosphate and the like are used for replacing ferric sulfate to prepare similar products through the same process.
3.12g of ferric chloride hexahydrate, 0.23g of poly (ethylene glycol) 2-mercaptoethyl ether acetic acid, 4.2g of sodium acetate and 2.4g of polyethylene glycol were added to 100m L of ethylene glycol, and after stirring uniformly, the mixture was reacted in a muffle furnace at 220 ℃ for 8 hours to obtain a magnetic bead ②.
3.12g of ferric sulfate, 0.23g of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate and 2.4g of polyvinylpyrrolidone are added into 100m L of ethylene glycol, and after being uniformly stirred, the mixture reacts in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic bead ③.
3.12g of ferric chloride hexahydrate, a certain mass of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate, 0.72g of trisodium citrate and 2.4g of polyethylene glycol are added into 100m L of ethylene glycol, the mixture is uniformly stirred and then reacts in a muffle furnace at 200 ℃ for 10 hours to obtain magnetic beads ④, and urea is used for replacing trisodium citrate to prepare similar products through the same process.
After judging whether the magnetic beads meet the requirements, after the magnetic beads are washed by ethanol, 0.01 mu mol/M L4-maleimide butyric acid-N-succinimide ester is added for reaction for 15min, after washing, the magnetic beads are put into BSA marked by 10 mu M fluorescent dye Cy5, and are incubated for 2h at room temperature.
The results show that the above alternatives can prepare satisfactory magnetic beads and can coat antibodies, but according to the scheme, ferric trichloride hexahydrate, methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, sodium acetate and polyethylene glycol are used as reactants, and the effect is best (shown as original magnetic beads in the figure). In conclusion, the invention can complete the synthesis and surface modification of the magnetic beads by a one-step method, and the synthesis of the immunomagnetic beads is rapid and convenient.
(2) The existing method for capturing exosome by magnetic bead affinity needs to consume a large amount of antibody, and the antibody is expensiveResulting in a very high cost of the magnetic bead method. With DynabeadsTMMyOneTMCarboxylic Acid, cat 65012, as an example, and the official gazette of antibodies and antibodies thereto
The mass ratio of (A) to (B) is about 1: 10. And for some self-synthesized magnetic beads coated with antibodies for liquid biopsy (CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A and the like), the mass ratio of the antibodies to the magnetic beads is also (0.01-1) to 1, but for the invention, the mass ratio of the antibodies to the magnetic beads is (0.00005-0.005) to 1, thereby greatly saving the use amount of the antibodies, greatly reducing the preparation cost of the exosome affinity magnetic beads, but not reducing the exosome capture efficiency.
The operation method comprises the following steps:
example 5 exosomes (magnetic beads of the invention) were captured in concentrated/pre-enriched cell supernatant and used for exosome quantitative detection. (FIG. 5, FIG. 6)
Adding a culture medium without exosomes into a culture dish with the density of H3122 cells (a lung cancer cell line) of about 80%, culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium of 20m L to 130 mu L through an ultrafiltration tube of 30KDa to obtain the culture medium pre-enriched with exosomes.
6 times of volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG are added into the supernatant of 130 mu L cell subjected to exosome concentration/pre-enrichment treatment, and after uniform mixing, exosome capture magnetic beads (the mass ratio of CD63 to magnetic beads is 0.0005: 1) in the invention with the content of 0.5 mu gCD63 (an exosome surface-specific antibody) are added, and the mixture is subjected to rotary incubation at 4 ℃ overnight to obtain exosome capture magnetic beads.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and10μL ddPCRTMsupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in the exosomes was quantified, and the content of exosomes was reflected by the content of housekeeping gene GAPDH.
Example 6 Capture of exosomes in concentrated/pre-enriched cell supernatant
(FIG. 5, FIG. 6)
Adding a culture medium without exosomes into a culture dish with the density of H3122 cells (a lung cancer cell line) of about 80%, culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium of 20m L to 130 mu L through an ultrafiltration tube of 30KDa to obtain the culture medium pre-enriched with exosomes.
Adding 6 times volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into two equal amounts of exosome concentration/pre-enrichment treated 130 mu L cell supernatants, respectively, mixing well, adding 4.5 mu g and 13.5 mu g of CD63 antibody (similar product is obtained by the same process using other exosome surface-specific antibodies)
The magnetic beads with the exosomes captured were obtained by rotary incubation at 4 ℃ overnight.
Will trap exosomes
Using miRNeasy SerumRNA was extracted from the plasmid Advanced Kit (RNA extraction Kit from QIAGEN), the RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (NodUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in the exosomes was quantified, and the exosome content was reflected by the number of positive drops of housekeeping gene GAPDH.
Combining example 5 with example 6, and referring to FIGS. 5 and 6, it can be seen that the exosome capture kit of the present invention has a ratio in cell supernatant
Much higher exosome capture efficiency: consuming the same antibody, the exosome capturing kit of the invention can achieve the capturing efficiency of exosome in cell supernatant
About 28 times of the total weight of the composition;
after 27 times of the antibody of the exosome capture kit is consumed, the capture efficiency of the exosome capture kit on cell supernatant is still lower than that of the exosome capture kit.
Example 7 capture of exosomes (magnetic beads of the invention) in plasma (FIG. 7, FIG. 8)
12000g of plasma was centrifuged for 10min to collect the supernatant, 1000. mu. L of a 0.14% BSA solution and 0.002% (w/v) of mouse IgG were added to 400. mu. L of plasma, and after mixing well, 0.5. mu.g of the exosome-capturing magnetic beads of the present invention (mass ratio of CD63 to magnetic beads 0.0005: 1) containing CD63 was added and incubated overnight at 4 ℃ with rotation.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMIn the exosomeHousekeeping gene GAPDH was quantified and exosome content was reflected in positive drops of housekeeping gene GAPDH.
Example 8 capturing exosomes in plasma
(FIG. 7, FIG. 8)
Centrifuging 12000g plasma for 10min to obtain supernatant, adding 1000 μ L% BSA solution and 0.002% (w/v) mouse IgG into 400 μ L plasma, mixing, adding 0.5 μ g, 1.5 μ g, and 4.5 μ g CD63 antibody (similar product obtained by the same process using other exosome surface-specific antibody)
The cells were incubated at 4 ℃ overnight with rotation.
Will trap exosomes
RNA was extracted using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), the RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (N0 dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in the exosomes was quantified, and the exosome content was reflected by the number of positive drops of housekeeping gene GAPDH.
Combining example 7 with example 8, and referring to FIGS. 7 and 8, it can be seen that the exosome capture kit of the present invention has a ratio in plasma
Much higher exosome capture efficiency: consuming the same antibody, the exosome capturing kit of the invention can achieve the capturing efficiency of exosome in plasma
About 240% of;
the capture efficiency of the exosome capture kit on plasma exosomes is still lower than that of the exosome capture kit after 9 times of the antibody is consumed.
Example 9 exploration of the coating ratio of the magnetic beads according to the present invention for the antibody and magnetic beads while ensuring the efficiency of exosome capture (FIG. 9)
Adding a culture medium without exosomes into a culture dish with the density of H3122 cells (a lung cancer cell line) of about 80%, culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium of 20m L to 130 mu L through an ultrafiltration tube of 30KDa to obtain the culture medium pre-enriched with exosomes.
Adding 6 times of volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into supernatant of 130 mu L cells subjected to exosome concentration/pre-enrichment treatment, uniformly mixing, adding 0.01-1 mu g of exosome capture magnetic beads (the mass of the magnetic beads is 0.2mg, and the mass ratio of CD63 to the magnetic beads is (0.00005-0.005): 1) with the content of CD63 (an exosome surface specific antibody), and performing rotary incubation at 4 ℃ overnight to obtain the exosome capture magnetic beads.
Adding 6 times volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into two equal amounts of exosome concentration/pre-enrichment treated 130 mu L cell supernatants, respectively, mixing well, and adding 20 mu g CD63 antibody (similar product is obtained by the same process using other exosome surface-specific antibody)
(
0.2mg by mass, CD63 and
the mass ratio is 0.1: 1), and the magnetic beads with the exosomes are obtained after rotary incubation at 4 ℃ overnight.
Magnetic beads with exosome captured were treated with miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit from QIAGEN) extracts RNA, reverse transcribes the RNA to cDNA, and adds 8. mu. L reverse-transcribed cDNA, 1. mu. L GAPDH primer, 1. mu. L enzyme-free water, and 10. mu. L ddPCR to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the number of positive drops of housekeeping gene GAPDH, and the results are shown in FIG. 9.
The result shows that the good exosome capturing effect can be obtained when the mass ratio of the antibody to the magnetic bead in the magnetic bead is (0.00005-0.005) to 1. The capture effect of the exosome is better than that of the antibody and the magnetic bead with the mass ratio of 0.1: 1
Greatly saves the dosage of the antibody and improves the capture efficiency of the exosome.
Example 10 exploring the effect of different sized magnetic beads on exosome capture efficiency (FIG. 10)
3.12g of ferric chloride hexahydrate, 0.23g of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 4.2g of sodium acetate and 2.4g of polyethylene glycol are added into 100m L of ethylene glycol, and after being uniformly stirred, the mixture reacts in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic beads with the particle size of 30-150 nm.
The synthesis method of the magnetic beads with different sizes comprises the steps of adding 3.38g of ferric trichloride hexahydrate, 0.23g of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, 9g of sodium acetate and 2.5g of polyethylene glycol into 100m L g of ethylene glycol, uniformly stirring, and then reacting in a muffle furnace at 200 ℃ for 8-72h to obtain the magnetic beads with the size of 200-800nm, wherein the size of the magnetic beads can be adjusted by controlling the reaction time, for example, the magnetic beads with the size of 200nm are obtained in 8h, and the magnetic beads with the size of 800nm are obtained in 72 h.
After the magnetic beads are washed by ethanol, 0.01 mu mol/m L4-maleimide butyric acid-N-succinimide ester is added for reaction for 15min, after washing, the antibody is added according to the proportion that the ratio of the magnetic beads to the CD63 antibody (similar products are obtained by the same process by using other exosome surface specific antibodies) is 1: 0.0005, and the magnetic bead antibody is incubated overnight to complete the coating of the magnetic bead antibody.
The coated beads were blocked with 5% (w/v) serum albumin overnight and the exosome-capture beads were prepared.
12000g of plasma was centrifuged for 10min to obtain the supernatant, 1000. mu. L of 0.14% BSA solution and 0.002% (w/v) of mouse IgG were added to 400. mu. L of plasma, and after mixing, 0.5. mu.g of the exosome-capturing magnetic beads of the present invention (each having a CD63 content) (the mass of each magnetic bead was 0.2mg) were added thereto, followed by overnight incubation at 4 ℃ with rotation.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the concentration of housekeeping gene GAPDH, and the results are shown in FIG. 10.
The results show that the particle size decreases with the mass of the beads in the range of 800-30nm for the same amount of antibody added and the same mass of beads. The capture efficiency of the immunomagnetic beads to the plasma exosomes is gradually improved, which is probably due to the number N ═ m/(4/3 ^ R) of the magnetic beads3Rho), wherein m is the total mass of the magnetic beads, R is the radius of the magnetic beads, and rho is the density of the magnetic beads. It can be seen that the number of magnetic beads and R are equal to each other under the condition of a certain m3In inverse proportion. Therefore, the number of 100nm magnetic beads under the same mass can reach more than 500 times of 800nm, and the magnetic beads (smaller magnetic beads) of the invention improve the capture efficiency of exosomes through more magnetic beads under the condition of the same total mass of the magnetic beads.
Example 11 exploring the effect of different bead additions on exosome capture efficiency (FIG. 11, FIG. 12)
Adding a culture medium without exosomes into a culture dish with the density of H3122 cells (a lung cancer cell line) of about 80%, culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium of 20m L to 130 mu L through an ultrafiltration tube of 30KDa to obtain the culture medium pre-enriched with exosomes.
Adding 6 times of volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into the supernatant of 130 mu L cells subjected to exosome concentration/pre-enrichment treatment, uniformly mixing, and adding 0.5 mu g of exosome capturing magnetic beads (the number of the added magnetic beads is 1x 10) with the content of CD63 (an exosome surface-specific antibody) in the invention6-1 x 1012And (b) were subjected to rotary incubation at 4 ℃ overnight to obtain magnetic beads with the exosomes captured.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the number of droplets positive for the housekeeping gene GAPDH, and the results are shown in FIG. 11.
12000g of plasma was centrifuged for 10min to collect the supernatant, 1000. mu. L of 0.14% BSA solution and 0.002% (w/v) of mouse IgG were added to 400. mu. L of plasma, and after mixing well, 0.5. mu.g of the exosome-capturing magnetic beads of the present invention (mass ratio of CD63 to magnetic beads 0.0005: 1) containing CD63 was added and incubated overnight at 4 ℃ with rotation.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in the exosome was quantified, and the content of exosome was reflected by the number of positive drops of housekeeping gene GAPDH, and the structure is shown in FIG. 12.
From FIGS. 11 and 12, we can see that the number of captured magnetic beads is larger than 1 × 10 for cell supernatant9When the method is used, the capture of the cell supernatant exosomes by the capture magnetic beads can reach the maximum value; for plasma, the number of captured magnetic beads is more than 1x1011The capture of plasma exosomes by the capture magnetic beads can be maximized. The number of exosomes reported in this result and data (https:// www.thermofisher.com/cn/zh/home/life-science/cell-analysis/exosomes/exosomes-binder-q-and-a. html) -the number of exosomes in the cell was 109About 10 per m L in plasma11The number/m L is about the same.
The recommended number of magnetic beads per ml of sample is 1x10, considering cost efficiency issues9-1 x 1012In the meantime.
In summary, the magnetic beads of the present invention need only be consumed in cell supernatants and plasma, respectively
The 1/27 and 1/9 antibodies in the antibody pair were compared
Higher exosome capture efficiency; magnetic beads of the present invention are used in cell supernatants and plasma
The capture efficiency for exosomes in cell supernatant and plasma can be 28-fold and 2.4-fold, respectively, at the same antibody consumption. Therefore, the magnetic bead can effectively reduce the consumption of the antibody and simultaneously improve the capture efficiency of the exosome.
(3) Existing magnetic bead affinity capture exosome kit and based on same
The exosome affinity capturing method of (2) is not efficient for capturing exosomes. The invention has a control exosome capture kit and is based on
The Exosome affinity capture method of (1) has higher capture efficiency (in the examples described herein, the control Exosome capture kit in cell supernatant is Exosome-Human CD63Isolation/Detection Reagent, InvitrogenTM(ii) a In the plasmaThe control exosome capture kit of (1) is ExoCapTM，JSR Life Sciences)。
The operation method comprises the following steps:
example 12 exosomes (magnetic beads of the invention and control exosome capture kit) were captured in concentrated/pre-enriched cell supernatant and characterized with Western Blot. (FIG. 13)
Adding a culture medium without exosomes into a culture dish with the density of about 80% of H2228 cells (a non-small cell lung cancer cell line), culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane with the diameter of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium with the diameter of 20m L to 130 mu L through an ultrafiltration tube with the diameter of 30KDa to obtain the culture medium with the preconcentration of exosomes.
The kit treatment method comprises the steps of adding 6 times of volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into concentrated/pre-enriched 130 mu L cell supernatant, uniformly mixing, adding 0.5 mu g of the exosome capture magnetic beads (the mass ratio of CD63 to the magnetic beads is 0.0005: 1) with the content of CD63, and performing rotary incubation at 4 ℃ overnight to obtain the exosome capture magnetic beads.
The control exosome capture kit treatment method comprises the steps of taking exosome capture magnetic beads in a 100 mu L kit, washing the exosome capture magnetic beads with 0.1% BSA solution for 3 times, adding 70 mu L of 0.1% BSA solution into supernatant of 130 mu L cells subjected to equivalent exosome concentration/pre-enrichment treatment respectively, and performing rotary incubation at 4 ℃ overnight to obtain the exosome capture magnetic beads.
Washing the magnetic beads with the captured exosomes twice with PBST and once with PBS, adding 40 mu L lysis solution for lysis, adding 10 mu L L applying Buffer after 30min of lysis, boiling for 5min at 95 ℃, sampling the 14 mu L supernatant after magnetic separation, carrying out 80V electrophoresis for 30min, and carrying out 120V electrophoresis for 1 h.
After electrophoresis is finished, membrane is switched, and then 5% (w/v) of PBST solution of milk powder is used for sealing, and sealing is carried out for 1h at room temperature;
rabbit anti-human EpCAM (epithelial cell adhesion molecule, a protein rich in tumor patient exosomes) was added at a ratio of 1: 1000 and incubated overnight at 4 ℃;
washing with WB (Western blot) for 3 times, and incubating with HRP-labeled goat anti-rabbit IgG at 1: 10000 for 1h at room temperature;
after washing with WB (Western blot) washing solution 3 times, the mixture was developed by adding a developing solution. The results are shown in FIG. 13.
The result shows that in cell supernatant, exosomes extracted by the exosome capture kit can show obvious bands when being detected by WB, and the obvious bands of exosomes extracted by the control exosome kit are very light when being detected by WB under the same condition. This demonstrates from a protein perspective that the exosome capture kit of the present invention has a higher exosome capture efficiency than the control exosome kit.
Example 13 exosomes (magnetic beads of the invention and control exosome capture kit) were captured in plasma and characterized with WesternBlot. (FIG. 13)
The treatment method of the kit comprises the steps of centrifuging 12000g of plasma for 10min to obtain a supernatant, adding 1000 mu L of 0.14% BSA solution into 300 mu L of the plasma, adding 0.002% (w/v) of mouse IgG, uniformly mixing, adding 0.5 mu g of the exosome capture magnetic beads with the content of CD63 (the mass ratio of CD63 to the magnetic beads is 0.0005: 1), and performing rotary incubation at 4 ℃ overnight.
The control exosome capture kit treatment method comprises the steps of taking 500 mu L avidin-coupled magnetic beads, carrying out magnetic separation, removing supernatant, carrying out resuspension by using 1m L Washing Buffer (provided by the kit), adding 5 mu L0 (1 mu g/mu L1) biotin-CD63, carrying out room-temperature rotary incubation for 1h, carrying out magnetic separation, removing supernatant, Washing 3 times by using 0.5m L Washing Buffer, carrying out resuspension by using 0.5m L Washing Buffer for later use, taking 100 mu L magnetic beads, carrying out magnetic separation, removing supernatant, adding 300 mu L Treame Buffer (provided by the kit), carrying out resuspension, adding 300 mu L plasma, carrying out overnight rotary incubation at room temperature, carrying out magnetic separation, removing supernatant after finishing incubation, adding 1m L Washing Buffer, carrying out resuspension and transferring to a new tube, carrying out magnetic separation, removing supernatant, carrying out rewashing by using 1m L Washing Buffer, carrying out resuspension once again by using 100 mu L Washing Buffer for later use.
12000g of plasma was centrifuged for 10min to obtain supernatant, and 100. mu. L of plasma was added to the magnetic beads, and the mixture was incubated overnight at 4 ℃ with rotation to obtain magnetic beads with exosomes captured.
Washing the magnetic beads with the captured exosomes twice with PBST and once with PBS, adding 40 mu L lysis solution for lysis, adding 10 mu L L applying Buffer after 30min of lysis, boiling for 5min at 95 ℃, sampling the 14 mu L supernatant after magnetic separation, carrying out 80V electrophoresis for 30min, and carrying out 120V electrophoresis for 1 h.
After electrophoresis is finished, membrane is switched, and then 5% (w/v) of PBST solution of milk powder is used for sealing, and sealing is carried out for 1h at room temperature;
rabbit anti-human EpCAM (epithelial cell adhesion molecule, a protein rich in tumor patient exosomes) was added at a ratio of 1: 1000 and incubated overnight at 4 ℃;
washing with WB (Western blot) for 3 times, and incubating with HRP-labeled goat anti-rabbit IgG at 1: 10000 for 1h at room temperature;
after washing with WB (Western blot) washing solution 3 times, the mixture was developed by adding a developing solution. The results are shown in FIG. 13.
The result shows that in plasma, the exosome extracted by the exosome capture kit can show obvious bands when being detected by WB, and the exosome extracted by the control exosome kit under the same condition can not detect the bands when being detected by WB. This demonstrates from a protein perspective that the exosome capture kit of the present invention has a higher exosome capture efficiency than the control exosome kit.
Example 14 exosomes (magnetic beads of the invention versus control exosome capture kit) were captured in concentrated/pre-enriched cell supernatant and a comparison of capture efficiency was made with ddPCR. (FIG. 14)
Adding a culture medium without exosomes into a culture dish with the density of about 80% of H2228 cells (a non-small cell lung cancer cell line), culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, centrifuging for 30min at 2000g, taking the supernatant, filtering the supernatant through a filter membrane with the diameter of 0.22 mu m to obtain a cell supernatant without cell and apoptotic bodies, and ultrafiltering and concentrating the culture medium with the diameter of 20m L to 130 mu L through an ultrafiltration tube with the diameter of 30KDa to obtain the culture medium with the preconcentration of exosomes.
The kit treatment method comprises the steps of adding 6 times of volume of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG into concentrated/pre-enriched 130 mu L cell supernatant, uniformly mixing, adding 0.5 mu g of the exosome capture magnetic beads (the mass ratio of CD63 to the magnetic beads is 0.0005: 1) with the content of CD63, and performing rotary incubation at 4 ℃ overnight to obtain the exosome capture magnetic beads.
The control exosome capture kit treatment method comprises the steps of taking exosome capture magnetic beads in a 100 mu L kit, washing the exosome capture magnetic beads with 0.1% BSA solution for 3 times, adding 70 mu L of 0.1% BSA solution into supernatant of 130 mu L cells subjected to equivalent exosome concentration/pre-enrichment treatment respectively, and performing rotary incubation at 4 ℃ overnight to obtain the exosome capture magnetic beads.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the concentration of housekeeping gene GAPDH, and the results are shown in FIG. 14.
The result shows that the capture effect of the exosome capture kit on exosomes in cell supernatant is far better than that of a control exosome capture kit. This example demonstrates from a nucleic acid perspective that the exosome capture kit of the present invention has higher exosome capture efficiency in cell supernatant than the control exosome capture kit.
Example 15 capture of exosomes in plasma (magnetic beads of the invention versus a control exosome capture kit) and comparison of capture efficiency with ddPCR. (FIG. 15)
The treatment method of the kit comprises the steps of centrifuging 12000g of plasma for 10min to obtain a supernatant, adding 1000 mu L of 0.14% BSA solution into 300 mu L of the plasma, adding 0.002% (w/v) of mouse IgG, uniformly mixing, adding 0.5 mu g of the exosome capture magnetic beads with the content of CD63 (the mass ratio of CD63 to the magnetic beads is 0.0005: 1), and performing rotary incubation at 4 ℃ overnight.
The control exosome capture kit treatment method comprises the steps of taking 500 mu L avidin-coupled magnetic beads, carrying out magnetic separation, removing supernatant, carrying out resuspension by using 1m L Washing Buffer (provided by the kit), adding 5 mu L0 (1 mu g/mu L1) biotin-CD63, carrying out room-temperature rotary incubation for 1h, carrying out magnetic separation, removing supernatant, Washing 3 times by using 0.5m L Washing Buffer, carrying out resuspension by using 0.5m L Washing Buffer for later use, taking 250 mu L magnetic beads, carrying out magnetic separation, removing supernatant, adding 300 mu L Treame Buffer (provided by the kit), carrying out resuspension, adding 300 mu L plasma, carrying out overnight rotary incubation at room temperature, carrying out magnetic separation, removing supernatant after finishing incubation, adding 1m L Washing Buffer, carrying out resuspension and transferring to a new tube, carrying out magnetic separation, removing supernatant, carrying out rewashing by using 1m L Washing Buffer, carrying out resuspension once again by using 250 mu L Washing Buffer for later use.
12000g of plasma was centrifuged for 10min to obtain supernatant, and 100. mu. L of plasma was added to the magnetic beads, and the mixture was incubated overnight at 4 ℃ with rotation to obtain magnetic beads with exosomes captured.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the concentration of housekeeping gene GAPDH, and the results are shown in FIG. 15.
The result shows that the capture effect of the exosome capture kit on exosomes in plasma is far better than that of a control plasma exosome capture kit.
Example 15 exosomes (magnetic beads of the invention) were captured in unconcentrated/pre-enriched cell supernatant and used for exosome quantification. (FIG. 16)
Adding an exosome-free culture medium into a culture dish with the density of H2228 cells (a non-small cell lung cancer cell strain) of about 80%, culturing for 3 days, collecting the culture medium, centrifuging for 10min at 300g, taking the supernatant, and centrifuging for 30min at 2000g to take the supernatant; the supernatant was filtered through a 0.22 μm filter to obtain a cell supernatant free of apoptotic bodies.
0.4g BSA solution and 0.4ng mouse IgG were added to 20m L unconcentrated/pre-enriched supernatant of H2228 cells (a non-small cell lung cancer cell line), mixed well, added with the exosome-capturing magnetic beads of the present invention (mass ratio of CD63 to magnetic beads is 0.0005: 1) at 0.5. mu. gCD63, and incubated overnight at 4 ℃ with rotation to obtain exosome-capturing magnetic beads.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (No dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in the exosomes was quantified, and the content of exosomes was reflected by the content of housekeeping gene GAPDH.
The results show that the magnetic beads of the present invention are not only suitable for capturing exosomes in the pre-enriched cell supernatant, but also suitable for capturing exosomes in the cell supernatant which is not pre-enriched.
Example 16 exosomes were captured in plasma using magnetic beads of the present invention avidin antibody magnetic beads and compared to a commercially available kit. (FIG. 17)
Washing the magnetic beads with ethanol, adding L4-maleimide butyric acid-N-succinimide ester of 0.01 mu mol/m to react for 15min, adding avidin according to the ratio of 1: 0.005 of the magnetic beads to the avidin after washing, and incubating overnight to complete the coating of the magnetic bead antibody.
The coated beads were blocked with 5% (w/v) serum albumin overnight, washed with PBS, then biotinylated CD63 antibody (similar to that obtained with other exosome surface-specific antibodies in the same procedure) was added at a ratio of 1: 0.0005, incubated for 1h, washed with PBS, and the exosome-capturing beads were prepared.
The treatment method of the kit comprises the steps of centrifuging 12000g of plasma for 10min to obtain a supernatant, adding 1000 mu L of 0.14% BSA solution into 300 mu L of the plasma, adding 0.002% (w/v) of mouse IgG, uniformly mixing, adding 0.5 mu g of the exosome capture magnetic beads with the content of CD63 (the mass ratio of CD63 to the magnetic beads is 0.0005: 1), and performing rotary incubation at 4 ℃ overnight.
The control exosome capture kit treatment method comprises the steps of taking 500 mu L avidin-coupled magnetic beads, carrying out magnetic separation, removing supernatant, carrying out resuspension by using 1m L Washing Buffer (provided by the kit), adding 5 mu L0 (1 mu g/mu L1) biotin-CD63, carrying out room-temperature rotary incubation for 1h, carrying out magnetic separation, removing supernatant, Washing 3 times by using 0.5m L Washing Buffer, carrying out resuspension by using 0.5m L Washing Buffer for later use, taking 250 mu L magnetic beads, carrying out magnetic separation, removing supernatant, adding 300 mu L Treame Buffer (provided by the kit), carrying out resuspension, adding 300 mu L plasma, carrying out overnight rotary incubation at room temperature, carrying out magnetic separation, removing supernatant after finishing incubation, adding 1m L Washing Buffer, carrying out resuspension and transferring to a new tube, carrying out magnetic separation, removing supernatant, carrying out rewashing by using 1m L Washing Buffer, carrying out resuspension once again by using 250 mu L Washing Buffer for later use.
12000g of plasma was centrifuged for 10min to obtain supernatant, and 100. mu. L of plasma was added to the magnetic beads, and the mixture was incubated overnight at 4 ℃ with rotation to obtain magnetic beads with exosomes captured.
The exosome-captured magnetic beads were subjected to RNA extraction using the miRNeasy Serum/Plasma Advanced Kit (RNA extraction Kit of QIAGEN), RNA was reverse-transcribed into cDNA, and 8. mu. L of the reverse-transcribed cDNA, 1. mu. L of GAPDH primer, 1. mu. L of enzyme-free water, and 10. mu. L of ddPCR were added to each ddPCR wellTMSupermix for Probes (N0 dUTP). After PCR amplification, the DNA was amplified using Bio-Rad QX200TMThe housekeeping gene GAPDH in exosomes was quantified, and the content of exosomes was reflected by the concentration of housekeeping gene GAPDH, and the results are shown in FIG. 17.
The result shows that the capture effect of the exosome capture kit of the invention on exosomes in plasma by using the avidin antibody is far better than that of the control plasma exosome capture kit.
To summarize the examples, both protein and nucleic acid perspectives demonstrate that the exosome capture kit of the present invention has higher exosome capture efficiency in both cell supernatant as well as serum than the control exosome capture kit, and a greater range of uses (capture of exosomes in unconcentrated/pre-enriched cell supernatant). In summary, the synthesis and surface modification of the magnetic beads can be completed by a one-step method, and the synthesis of the immunomagnetic beads is convenient.
Without being bound by a particular theory, in the present invention, it is possible to use the magnetic beads on more thiol small beads (e.g., 1X10 per ml sample)9-2 x 1012Magnetic beads) to capture exosomes, since the number of exosomes in a sample is limited, more magnetic beads means fewer exosomes are captured per magnetic bead, thereby avoiding exosomesThe steric hindrance of the exosome on the magnetic bead improves the capture efficiency of the exosome; and simultaneously, because each magnetic bead only needs to capture a small amount of exosomes, the coating concentration of the antibodies on the magnetic beads can be reduced, and the antibodies are saved. Therefore, the invention provides a technical scheme with low antibody consumption and high exosome capture efficiency.
The magnetic beads of the present invention need only be consumed in cell supernatant and plasma, respectively
The 1/27 and 1/9 antibodies in the antibody pair were compared
Higher exosome capture efficiency; magnetic beads of the present invention are used in cell supernatants and plasma
The capture efficiency for exosomes in cell supernatant and plasma can be 28-fold and 2.4-fold, respectively, at the same antibody consumption. Therefore, the magnetic bead can effectively reduce the consumption of the antibody and simultaneously improve the capture efficiency of the exosome.
Both protein and nucleic acid perspectives demonstrate that the exosome capture kit of the present invention has higher exosome capture efficiency in both cell supernatant as well as serum than the control exosome capture kit, and a greater range of uses (capture of exosomes in unconcentrated/pre-enriched cell supernatant).
Claims (13)
1. A method of isolating exosomes, the method comprising contacting a sample containing exosomes from a subject with nanobeads, thereby isolating exosomes from the sample, wherein the nanobeads have a size in the range of 30-600nm, such as any of the ranges 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 540nm, 530nm, 560nm, 570nm, 580nm, 590nm, or any range therebetween, for example in the range of 30-150 nm.
2. The method of claim 1, wherein the nanobead has one or more of the following properties: 1) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers; 2) has superparamagnetism; 3) from 8 to 12nm of Fe3O4Nanoparticles agglomerated, 4) an antibody with a conjugate, e.g. an antibody to an exosome surface protein, e.g. CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L, an antibody with a conjugate, e.g. a linker protein, e.g. a linker that binds selectively to an exosome, e.g. an exosome surface protein or modified exosome surface protein, e.g. a linker that binds selectively to an exosome surface protein, e.g. CD L, Rab5, a magnetic bead, tsxa, tsg. a magnetic bead, a ligand, HSP 72, a ligand modified by an antibody to an exosome surface protein, HSP 130, HSP 72, a ligand modified by an antibody, a monoclonal antibody, a ligand, a monoclonal antibody, a ligand, a monoclonal antibody for a monoclonal9-2x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5x 1012To (c) to (d); and/or 7) the mass ratio of antibody to magnetic bead or linker to magnetic bead is between (0.00005-0.005) to 1, such as 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween.
3. The method of claim 1 or 2, wherein the sample is from a body fluid, e.g. from a human subject, e.g. from a patient, such as a tumor patient, e.g. from blood, serum, serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, mucosal secretions of secretory tissues and organs, vaginal secretions, milk, tears, ascites, e.g. from fluid from the pleura, pericardium, peritoneum, abdomen or other body cavities, e.g. from a cell culture, e.g. from a cell supernatant of a concentrated/pre-enriched exosomes, e.g. a cell supernatant which has not been concentrated.
4. The method of any one of claims 1-3, wherein the method comprises one or more of the following features: adding blocking agents such as bovine serum albumin, human serum albumin, glycine to the sample; incubate to capture exosomes, e.g., for 1-4 hours at 37 ℃, or for 2-6 hours at room temperature, or overnight at 4 ℃.
5. A nanobead, such as for use in the method of any one of claims 1 to 4, having one or more of the following properties: 1) a size range in the range of 30-600nm, such as 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, or any range therebetween, such as in the range of 30-150nm(ii) a 2) Has superparamagnetism; 3) from 8 to 12nm of Fe3O4Nanoparticles agglomerated, 4) an antibody with a conjugate, such as an antibody, e.g. an antibody to an exosome surface protein, e.g. exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L or an antibody to one or more of these, 5) an antibody with a conjugate, e.g. a linker protein, e.g. an avidin, e.g. a linker that selectively binds to an exosome, e.g. an exosome surface protein or modified exosome surface protein, e.g. a linker that selectively binds to an exosome surface protein CD L, Rab5, a magnetic bead, tsxa 101, tsg. a magnetic bead, a ligand modified by a ligand, a9-2x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic beads, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5x1012To (c) to (d); 7) the mass ratio of the antibody or linker to the magnetic bead is between (0.00005-0.005): 1, e.g., 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween; and/or 8) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers.
6. A kit, e.g. for isolating exosomes, comprising nanobeads of claim 5.
7. The kit of claim 6, wherein the kit further comprises a blocking agent, such as bovine serum albumin, human serum albumin, an amino ligand, such as glycine and the like.
8. A method for preparing the surface-modified thiol nano magnetic bead in one step, for example, the method for preparing the nano magnetic bead in one step as claimed in claim 5, the method comprising reacting a trivalent iron salt, a thiol-functionalized cross-linking agent, a stabilizer for reduction reaction, and a reducing agent in a container at high temperature and high pressure to obtain the nano magnetic bead.
9. The method of claim 8, wherein the ferric salts include ferric chloride, ferric sulfate, ferric acetate, ferric nitrate, ferric phosphate, ferric citrate, ferric pyrophosphate, and the like; the thiol-functionalized cross-linking agent comprises methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, poly (ethylene glycol) 2-mercaptoethyl ether acetic acid; the stabilizer for the complex reduction reaction comprises sodium acetate, trisodium citrate, urea and the like; the reducing agent includes ethylene glycol and the like; the surfactant includes polyethylene glycol, polyvinylpyrrolidone, and the like.
10. The process of claim 8 or 9, wherein the temperature of the reaction is between 150 and 240 ℃; the reaction time is 7-72 hours.
11. The method of any one of claims 8 to 10, wherein the ferric salt, thiol-functionalized cross-linking agent, stabilizer and surfactant are present in a co-reduction reaction in a mass ratio of 1 to (0 to 0.1) to (1.4 to 3.5) to (0.4 to 1.3), such as 1 to (0, 0.02, 0.04, 0.06, 0.08, 0.1 or any range therebetween) to (1.4, 1.5, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.5 or any range therebetween) to (0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or any range therebetween).
12. The method according to any of claims 8-11, wherein the method further comprises the step of coupling the nanobead to a conjugate, such as an antibody, e.g. an antibody of an exosome surface protein, such as exosome surface proteins CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L, the conjugate, e.g. a linker, e.g. an adaptor protein, e.g. an avidin, e.g. a linker which binds selectively to an exosome surface protein or a modified exosome surface protein, e.g. a linker which binds selectively to an exosome surface protein, e.g. CD L, a linker, a L, a ligand, a L, a ligand, a L, a ligand, or a ligand, a antibody for example a ligand for a antibody for.
13. The method of any one of claims 8-12, wherein the nanobead has one or more of the following properties: 1) a size range in the range of 30-600nm, such as 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm, 590nm, 600nm, or any range therebetween, such as 30-150nm, e.g. 30-150nmA range; 2) has superparamagnetism; 3) from 8 to 12nm of Fe3O4Nanoparticles agglomerated, 4) an antibody with a conjugate, such as an antibody, e.g. an antibody to an exosome surface protein, e.g. exosome surface protein CD9, CD63, CD81, CD44, CD31, Rab5b, EpCAM, TSG101, HSP90, HSP70, ANXA5, F L OT L, ICAM L, a L IX, GM130, ICAM-1, SNAP, MHC I/II, H L a-G, Integrins, Claudins, Tim L or an antibody to one or more of these, 5) an antibody with a conjugate, e.g. a linker protein, e.g. an avidin, e.g. a linker that selectively binds to an exosome, e.g. an exosome surface protein or modified exosome surface protein, e.g. a linker that selectively binds to an exosome surface protein CD L, Rab5, a magnetic bead, tsxa 101, tsg. a magnetic bead, a ligand modified by a ligand, a9-2x 1012Magnetic beads, e.g. 1x109Magnetic beads, 1.5X 109Magnetic bead, 5X109Magnetic bead, 8X109Magnetic beads, 1X1010Magnetic beads, 1.5X 1010Magnetic bead, 5X1010Magnetic bead, 8X1010Magnetic bead, 1X1011Magnetic beads, 1.5X 1011Magnetic bead, 5X1011Magnetic bead, 8X1011Magnetic beads, 1X1012Magnetic beads, 1.5X 1012Magnetic beads, 2X 1012Magnetic beads or any range therebetween, e.g. 1.5x 1011-1.5x 1012To (c) to (d); 7) the mass ratio of the antibody or linker to the magnetic bead is between (0.00005-0.005): 1, e.g., 0.00005: 1, 0.0005: 1, 0.005: 1 or any range therebetween; and/or 8) surface-modified thiols, for example, introduced via thiol-functionalized crosslinkers.
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