CN111505264B

CN111505264B - Exosome separation method, immunomagnetic beads and kit

Info

Publication number: CN111505264B
Application number: CN201910090464.9A
Authority: CN
Inventors: 盛滔
Original assignee: Shanghai 3D Medicines Co Ltd
Current assignee: Shanghai 3D Medicines Co Ltd
Priority date: 2019-01-30
Filing date: 2019-01-30
Publication date: 2024-05-17
Anticipated expiration: 2039-01-30
Also published as: CN111505264A

Abstract

The invention relates to an exosome separation method, immunomagnetic beads and a kit. The present invention provides a method of isolating exosomes comprising contacting a sample containing exosomes from a subject with nanomagnetic beads, thereby isolating exosomes from the sample, wherein the nanomagnetic beads range in size from 30-600nm, the number of nanomagnetic beads being 1x10 ⁹-2x10¹² per milliliter of sample. The invention also provides a nano magnetic bead, a kit and a method for preparing the nano magnetic bead. Advantages of the method and the product of the invention include convenient synthesis of magnetic beads and low cost; is suitable for various systems including plasma and cell supernatant; the dosage of the antibody is small; high exosome capturing efficiency, etc.

Description

Exosome separation method, immunomagnetic beads and kit

Technical Field

The present invention relates to the fields of chemistry and medicine. In particular, 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 medical fields such as immunodetection, for example, application in exosome separation, and a kit comprising the nano magnetic bead.

Background

Exosomes are membrane vesicles that are secreted by most cells and to the outside of the cell. In addition to playing an important role in cellular communications, there is growing evidence that some cellular contents of exosomes, including proteins, microRNA, mRNA, fusion genes, etc., have a close relationship with the onset of human malignancies; in addition, exosomes play an important role in diagnosis, treatment, prognosis, etc. of diseases. For this reason, it is desirable to find a simple, efficient, high purity and automatable method for isolation of exosomes. Among them, the affinity capture exosome technology based on magnetic beads is particularly attractive due to the simple operation process, easy cleaning, separation and automation. See, for example Oksvold,M.P.,A.Neurauter,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 Generic Isolation of Extracellular Vesicles with Magnetic Beads.Methods Mol Biol,2017.1660：p.65-87.

At present, most of magnetic beads used for affinity capture of exosomes areSee, for example, publication number CN106289926A、CN107893051A、CN108085338A、CN106967747A、US20130273544A1、EP3289361A4、WO2015112382A1、WO2018033929A1, etc. These beads are all atThe specific antibodies are modified and then the/>, the specific antibodies are modifiedExosomes are specifically captured.See patent or patent application publications US8658733B2, US20080139399A1, US20070299249A1, EP1693387B1, WO2010125170A1, US6984702B2 and US6787233B1 by Thermo FISHER SCIENTIFIC et al.

The existing method for affinity capture of exosomes by using magnetic beads requires a large amount of antibodies, and the high price of the antibodies leads to the very high cost of the magnetic bead method. Taking Dynabeads ^TM MyOne^TM carboxilic Acid, cat No. 65012 as an example, the manufacturer's instructions point out antibodies toThe mass ratio of (2) is about 0.1:1. Other references, such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A, which disclose antibodies coated on magnetic beads for liquid biopsies, also disclose that the mass ratio of the antibodies to the magnetic beads is between (0.01-1) to 1. The relatively high ratio of antibody to magnetic beads makes these methods require the consumption of a large amount of antibody, which increases the cost of preparation of exosome-capturing magnetic beads.

Patent literature such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286a discloses the following preparation method:

s1, preparing magnetic nanoclusters;

S1.1 ferrous salt is reduced into Fe ₃O₄ nano particles under the action of ammonia water;

s1.2, adding oleic acid into Fe ₃O₄ nano particles, and generating magnetic nanoclusters through high-temperature high-pressure reaction;

s2, preparing amino modified magnetic microspheres;

S2.1, adding ammonia water, a silanization reagent and an aminosilane coupling agent into the magnetic nanoclusters, and reacting for 1-3 days;

S3, preparing a hydrazino-modified part A;

S3.1, performing hydrazino modification on the amino modified magnetic microsphere or antibody by using SANH (p-propylhydrazone picolinic acid N-hydroxysuccinimide ester, CAS: 362522-50-7) with the molar equivalent of 10-50 times;

s4, preparing an aldehyde group modified part B;

S4.1 the amino-modified magnetic microspheres or antibodies were aldehyde-modified with 5-20 molar equivalents of SFB (N-succinimidyl 4-formylbenzoate, CAS: 60444-78-2).

S5 capturing tumor cells.

The above method is troublesome for synthesizing immunomagnetic beads.

Still other documents such as CN106289926A, CN107893051a et al disclose the following methods:

S1 is based on Preparation of exosome-trapping magnetic beads

S1.1 modification with streptavidin

S1.2 aboveAdding a certain volume of antibody to perform incubation;

Affinity capture of S2 exosomes

S3, the serum is diluted and then exosome capture is carried out.

The method uses magnetic beads as inlet

Therefore, there is a need in the art to develop simpler methods of immunomagnetic bead preparation, new exosome capture methods and high-efficiency, low-cost exosome capture kits with proprietary intellectual property.

Disclosure of Invention

The inventors have found, 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 antibodies consumed by the methods and/or products of the invention is significantly reduced. In some embodiments, the methods of the invention and/or the capture efficiency of the product exosomes have been found to be 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 be useful for capturing exosomes in unconcentrated/pre-enriched cell supernatants.

In some embodiments, the invention provides a method of isolating exosomes, the method comprising contacting a sample containing exosomes from a subject with nanomagnetic beads, thereby isolating exosomes from the sample, wherein the nanomagnetic beads range in size from 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,550nm,560nm,570nm,580nm,590nm,600nm, or any range therebetween, such as the range of 30-150 nm. In some embodiments, it has been found that by sizing the magnetic beads for exosome separation with the same total mass of the beads, the efficiency of exosome capture can be significantly improved and/or a widely applicable exosome capture method can be provided. In some embodiments, the size of the magnetic beads employed in the methods of the invention is preferably approximately or the same as the exosome size. 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,572nm,581nm,592nm,600nm, or any range therebetween, such as the range of 30-150 nm.

In some embodiments, the nanomagnetic beads employed in the separation method possess one or more of the following properties: 1) Surface-modified mercapto groups, for example, introduced by a mercapto-functionalized crosslinking agent; 2) Has superparamagnetism; 3) Is formed by agglomerating Fe ₃O₄ nano particles with the particle size of 8-12 nm; 4) An antibody having a conjugate, e.g., an antibody, e.g., an exosome surface protein, e.g., an antibody to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4, 5) a conjugate, e.g., a linker protein, e.g., an avidin, e.g., a linker that selectively binds to an exosome surface protein or a modified exosome surface protein, e.g., a linker that selectively binds to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or modified proteins described above, e.g., ligand-modified exosome surface proteins via the linker, for example by biotin-modified exosurface proteins, 6) the beads are added in an amount of 1 x 10 ⁹-2 x 10¹² beads, for example 1 x 10 ⁹ beads, 1.5 x 10 ⁹ beads, 5 x 10 ⁹ beads, 8 x 10 ⁹ beads, 1 x 10 ¹⁰ beads, 1.5 x 10 ¹⁰ beads, 5 x 10 ¹⁰ beads, 8 x 10 ¹⁰ beads, 1 x 10 ¹¹ beads, 1.5 x 10 ¹¹ beads, 5 x 10 ¹¹ beads, 8 x 10 ¹¹ beads, 1 x 10 ¹² beads, 1.5 x 10 ¹² beads, 2 x 10 ¹² beads or any range therebetween, for example between 1.5 x 10 ¹¹-1.5 x 10¹² per milliliter of sample; and/or 7) the mass ratio of antibody or linker to magnetic beads 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 surface modified thiol-modified magnetic beads, e.g., introduced by thiol-functionalized cross-linking agents, the amount of antibody used can be significantly reduced and/or the efficiency of exosome capture significantly increased at a magnetic bead level of 1 x 10 ⁹-2 x 10¹² per milliliter of sample. In some embodiments, the magnetic beads employed in the methods of the invention preferably have superparamagnetism. In some embodiments, the magnetic beads employed in the methods of the present invention may be agglomerated from 8-12nm Fe ₃O₄ nanoparticles. In some embodiments, the magnetic beads employed in the methods of the invention may have conjugates. In some embodiments, the conjugates of magnetic beads employed in the methods of the invention are not particularly limited. In some embodiments, the conjugate or conjugate may be, for example, an antibody, such as an antibody to an exosome surface protein, such as an antibody to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4. In some embodiments, the magnetic beads of the invention selectively bind to exosomes in the sample by means of conjugated antibodies, thereby effecting separation of exosomes. In some embodiments, the conjugate may be, for example, a linker. The linker that can be used in the method of the present invention is not particularly limited as long as it can specifically link the exosomes to the magnetic beads of the present invention. In some embodiments, the linker may be, for example, a linker protein, such as avidin. In some embodiments, exosomes are specifically bound to the magnetic beads of the invention by a linker protein. In some embodiments, the linker may be, for example, a linker that selectively binds to an exosome, such as a linker that selectively binds to an exosome surface protein or a modified exosome surface protein, such as a linker that selectively binds to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or modified proteins described above. In some embodiments, the modification may be, for example, an exocrine surface protein modified by a ligand of the linker. In some embodiments, if the magnetic beads of the present invention are modified with a linker, such as avidin, the exocrine proteins may be modified with a ligand of the linker, i.e., biotin. Thus, in some embodiments, for example, exosome surface proteins may be modified by biotin. In some embodiments, the magnetic bead modification may be obtained by coupling an antibody to a crosslinking agent such as 4-maleimidobutyric acid-N-succinimidyl ester to the magnetic bead. In some embodiments, modification of the antibody may be obtained by coupling a linker, such as avidin, to a magnetic bead under the influence of a cross-linking agent, such as 4-maleimidobutyric acid-N-succinimidyl ester. In some embodiments, the exosome surface protein may be linked to the avidin reaction on the magnetic beads via a biotin avidin reaction.

In some embodiments, the amount of magnetic beads employed in the methods of the present invention is not particularly limited. In some embodiments, the amount of beads employed in the methods of the invention may be 1 x 10 ⁹-2 x 10¹² beads, e.g., 1 x 10 ⁹ beads, 1.5 x 10 ⁹ beads, 5 x 10 ⁹ beads, 8 x 10 ⁹ beads, 1 x 10 ¹⁰ beads, 1.5 x 10 ¹⁰ beads, 5 x 10 ¹⁰ beads, 8 x 10 ¹⁰ beads, 1 x 10 ¹¹ beads, 1.5 x 10 ¹¹ beads, 5 x 10 ¹¹ beads, 8 x 10 ¹¹ beads, 1 x 10 ¹² beads, 1.5 x 10 ¹² beads, 2 x 10 ¹² beads, or any range therebetween, e.g., between 1.5 x 10 ¹¹-1.5 x 10¹². In some embodiments, it has been found that the preferred addition of the above-described magnetic beads enables sufficient separation of exosomes, improving the separation efficiency of exosomes. In some embodiments, the mass ratio of antibody or linker to magnetic beads in the methods of the invention is not particularly limited, but preferably the methods of the invention employ conventional methods to reduce the mass ratio of antibody or linker to magnetic beads. 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) to 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 the exosomes are isolated in the methods of the invention is not particularly limited. In some embodiments, the sample is derived 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, mucous secretions of secretory tissues and organs, vaginal secretions, milk, tears, ascites, e.g., from the pleura, pericardium, peritoneum, abdomen, or other body cavity, e.g., from a cell culture, e.g., from a cell supernatant, e.g., a concentrated/pre-enriched exosome cell supernatant, e.g., a cell supernatant that 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 be useful for capturing exosomes in unconcentrated/pre-enriched cell supernatants.

In some embodiments, the methods of the invention further comprise one or more of the following features: adding a blocking agent, such as bovine serum albumin, human serum albumin, amino ligands, such as glycine, etc., to the sample; incubation to capture exosomes, for example, incubation at 37 ℃ for 1-4 hours, or at room temperature for 2-6 hours, or at 4 ℃ overnight.

In some embodiments, the present invention provides a nanomagnetic bead that possesses one or more of the following properties: 1) The size range is 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 the range of 30-150 nm; 2) Has superparamagnetism; 3) Is formed by agglomerating Fe ₃O₄ nano particles with the particle size of 8-12 nm; 4) An antibody having a conjugate, such as an antibody, e.g., an antibody to one or more of the exosome proteins, e.g., exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4, 5) a conjugate, e.g., a linker protein, e.g., an avidin, e.g., a linker that selectively binds to an exosome surface protein or a modified exosome surface protein, e.g., one or more of the proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or a modified exosome surface protein, e.g., a ligand-modified exosome surface protein, e.g., an exosome surface protein modified by biotin, 6) a bead amount of 1 x 10 ⁹-2 x 10¹² beads, e.g., 1 x 10 ⁹ beads, 1.5 x 10 ⁹ beads, 5 x 10 ⁹ beads, 8 x 10 ⁹ beads, 1 x 10 ¹⁰ beads, 1.5 x 10 ¹⁰ beads, 5 x 10 beads, 8 x 10 beads, ¹¹-1.5 x 10¹² x 10 beads, 6) or any bead range of between 1 x 10, e.g., 1 x 35, 6) beads, e.g., 1 x 10 x 5 x 35 beads, 6) for a sample to be detected; 7) The mass ratio of the antibody magnetic beads is between (0.00005-0.005) to 1, such as 0.00005: 1,0.0005: 1,0.005:1 or any range therebetween; and/or 8) surface-modified sulfhydryl groups, for example, surface-modified sulfhydryl groups introduced by a sulfhydryl-functional crosslinking agent. In some embodiments, the nanomagnetic beads provided herein are preferably particularly suitable for use in the methods of the invention for separating exosomes. In some embodiments, it has been found that the amount of antibodies consumed by the nanomagnetic beads of the present invention is significantly reduced. In some embodiments, it has been found that the efficiency of capturing the exosomes of the nanomagnetic beads of the present invention is significantly improved. In some embodiments, the nanomagnetic beads of the present invention have been found to have a broader range of applicability, for example, to be able to be used to capture exosomes in unconcentrated/pre-enriched cell supernatants.

In some embodiments, the invention provides a kit comprising the nanomagnetic beads described herein. In some embodiments, the kit is particularly suitable for use in the methods of the invention for isolating exosomes. In some embodiments, the invention provides a kit for isolation of exosomes comprising the nanomagnetic beads described herein. In some embodiments, the kit may further comprise other suitable components, for example 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, amino ligands, such as glycine, and the like. In some embodiments, the amount of antibody consumed by the kits of the invention has been found to be significantly reduced. In some embodiments, the capture efficiency of the kit exosomes of the invention has been found to be significantly improved. In some embodiments, the kits of the invention have been found to have a broader range of applicability, for example, to be useful for capturing exosomes in unconcentrated/pre-enriched cell supernatants.

In some embodiments, the present invention provides a method of preparing a nanomagnetic bead, the method comprising reacting a ferric salt, a thiol-functionalized cross-linking agent, a stabilizer that cooperates with a reduction reaction, and a reducing agent at an elevated temperature and pressure in a vessel, thereby obtaining a nanomagnetic bead. In some embodiments, the nanomagnetic beads of the present invention are surface-modified sulfhydryl-containing nanomagnetic beads. In some embodiments, the methods of the present invention provide surface-modified sulfhydryl-containing nanomagnetic beads via a functionalized crosslinking agent. In some embodiments, the methods of the invention are one-step methods, i.e., the synthesis and surface modification of magnetic beads can be accomplished by a one-step method. In some embodiments, the invention provides an efficient and convenient method for synthesizing immunomagnetic beads by completing synthesis and surface modification of the magnetic beads in one step. In some embodiments, the 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 antibodies consumed by the nanomagnetic beads of the present invention is significantly reduced. In some embodiments, it has been found that the efficiency of capturing the exosomes of the nanomagnetic beads of the present invention is significantly improved. In some embodiments, the nanomagnetic beads of the present invention have been found to have a broader range of applicability, for example, to be able to be used to capture exosomes in unconcentrated/pre-enriched cell supernatants.

In some embodiments, the present invention provides a method of preparing surface-modified sulfhydryl-containing nanomagnetic beads in a one-step process. 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 salt may include ferric chloride, ferric sulfate, ferric acetate, ferric nitrate, ferric phosphate, ferric citrate, ferric pyrophosphate, and the like. In some embodiments, the magnetic beads of the present invention may be prepared using a suitable crosslinking 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, stabilizers that may be employed in the methods of the present invention are not particularly limited, e.g., stabilizers that may be employed in conjunction with the reduction reaction include sodium acetate, trisodium citrate, urea, and the like. In some embodiments, the reducing agent that may be employed in the methods of the present invention is not particularly limited, e.g., the reducing agent that may be employed includes ethylene glycol and the like. In some embodiments, the surfactant that may be employed in the methods of the present invention is not particularly limited, e.g., surfactants that may be employed include polyethylene glycol, polyvinylpyrrolidone, and the like.

In some embodiments, the process of the present 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 methods of the invention may be carried out for a suitable period of time, for example, the reaction time may be from 7 to 72 hours.

In some embodiments, the reaction components in the methods of the invention may be appropriately selected. For example, in some embodiments, the ferric salt, the mercapto-functional crosslinker, the stabilizer, and the surfactant in combination with the reduction reaction are present in a mass ratio of 1: (0-0.1): (1.4-3.5): (0.4-1.3), such as 1: (0,0.02,0.04,0.06,0.08,0.1 or any range therebetween): (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): (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 methods of the invention further comprise the step of coupling the nanomagnetic beads to a conjugate. In some embodiments, the conjugates of magnetic beads employed in the methods of the invention are not particularly limited. In some embodiments, the conjugate or conjugate may be, for example, an antibody, such as an antibody to an exosome surface protein, such as an antibody to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4. In some embodiments, the magnetic beads of the invention selectively bind to exosomes in the sample by means of conjugated antibodies, thereby effecting separation of exosomes. In some embodiments, the conjugate may be, for example, a linker. The linker that can be used in the method of the present invention is not particularly limited as long as it can specifically link the exosomes to the magnetic beads of the present invention. In some embodiments, the linker may be, for example, a linker protein, such as avidin. In some embodiments, exosomes are specifically bound to the magnetic beads of the invention by a linker protein. In some embodiments, the linker may be, for example, a linker that selectively binds to an exosome, such as a linker that selectively binds to an exosome surface protein or a modified exosome surface protein, such as a linker that selectively binds to one or more of exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or modified proteins described above. In some embodiments, the modification may be, for example, an exocrine surface protein modified by a ligand of the linker. In some embodiments, if the magnetic beads of the present invention are modified with a linker, such as avidin, the exocrine proteins may be modified with a ligand of the linker, i.e., biotin. Thus, in some embodiments, for example, exosome surface proteins may be modified by biotin. In some embodiments, the amount of magnetic beads employed in the methods of the present invention is not particularly limited. In some embodiments, the amount of beads employed in the methods of the invention may be 1 x10 ⁹-2 x 10¹² beads, e.g., 1 x10 ⁹ beads, 1.5 x10 ⁹ beads, 5x 10 ⁹ beads, 8 x10 ⁹ beads, 1 x10 ¹⁰ beads, 1.5 x10 ¹⁰ beads, 5x 10 ¹⁰ beads, 8 x10 ¹⁰ beads, 1 x10 ¹¹ beads, 1.5 x10 ¹¹ beads, 5x 10 ¹¹ beads, 8 x10 ¹¹ beads, 1 x10 ¹² beads, 1.5 x10 ¹² beads, 2x 10 ¹² beads, or any range therebetween, e.g., between 1.5 x10 ¹¹-1.5 x 10¹². In some embodiments, it has been found that the preferred addition of the above-described magnetic beads enables sufficient separation of exosomes, improving the separation efficiency of exosomes. In some embodiments, the mass ratio of antibody or linker to magnetic beads in the methods of the invention is not particularly limited, but preferably the methods of the invention employ conventional methods to reduce the mass ratio of antibody or linker to magnetic beads. 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) to 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 invention possess one or more of the following properties: 1) The size range is 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 the range of 30-150 nm; 2) Has superparamagnetism; 3) Is formed by agglomerating Fe ₃O₄ nano particles with the particle size of 8-12 nm; 4) An antibody having a conjugate, such as an antibody, e.g., an antibody to one or more of the exosome proteins, e.g., exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4, 5) a conjugate, e.g., a linker protein, e.g., an avidin, e.g., a linker that selectively binds to an exosome surface protein or a modified exosome surface protein, e.g., one or more of the proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or a modified exosome surface protein, e.g., a ligand-modified exosome surface protein, e.g., an exosome surface protein modified by biotin, 6) a bead amount of any of 1 x 10 ⁹-2 x 10¹² beads, e.g., 1 x 10 ⁹ beads, 1.5 x 10 ⁹ beads, 5 x 10 ⁹ beads, 8 x 10 ⁹ beads, 1 x 0 ¹⁰ beads, 1.5 x 10 ¹⁰ beads, 5 x 10 beads, 8 x 10 beads, ¹¹-1.5 x 10¹² x 10 beads, 6) or any of beads in the range of 1.5 x 35, e.g., 1 x 35 beads, 6) between 1 x 10 x 35 beads, e.g., 1 x 35, 6) per milliliter of sample to be detected; 7) The mass ratio of the antibody magnetic beads is between (0.00005-0.005) to 1, such as 0.00005: 1,0.0005: 1,0.005:1 or any range therebetween; and/or 8) surface-modified sulfhydryl groups, for example, surface-modified sulfhydryl groups introduced by a sulfhydryl-functional crosslinking agent.

The inventors have found that exosome capture methods and products can be significantly improved, in particular existing conventional methods and magnetic bead affinity capture kits, e.g. based onExosome affinity capture method of (2). It has been found that the efficiency of exosome capture can be significantly improved. In some embodiments, quantification of exosome capture numbers by ddPCR and housekeeping gene GAPDH has been found to be based onIn comparison with the exosome capturing method of the present invention, the capturing efficiency of the present invention for exosome, which consumes 0.5. Mu.g of antibody in the cell supernatant, is 27 times the consumption of antibodyThe capture efficiency (consuming 13.5 μg antibody) was much higher; for plasma, the invention consumed 0.5 μg antibody to exosome capture efficiency/>, 9 times the antibody consumptionThe capture efficiency (4.5. Mu.g of antibody consumed) was also high. Compared with the conventional exosome capturing kit, the invention has higher capturing efficiency on exosomes in cell supernatant and plasma.

The inventor finds that the magnetic bead synthesis process described in the prior method such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A is quite complicated, and the magnetic microsphere coated with aminated silicon dioxide is generated by the processes of reduction of Fe ₃O₄ nano particles, high-temperature and high-pressure generation of magnetic nanoclusters, generation of aminated silicon dioxide under the action of ammonia water, a silylation agent, an aminosilane coupling agent and the like. The inventors have found that the synthesis and modification of magnetic beads can be achieved by a one-step process. For example, in some embodiments, the invention contemplates that the magnetic microspheres can be synthesized by a one-step process. In some embodiments, the method may be accomplished using ferric trichloride hexahydrate, sodium acetate, polyethylene glycol, methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, ethylene glycol at elevated temperature and pressure. It has been found that surface modifications and antibody coatings of the prior art are equally cumbersome. For example, antibodies that further modify hydrazine groups and aldehyde groups are required to accomplish the modification after the amination modification of the magnetic beads. In contrast, the prepared magnetic beads are only required to be reacted with a reactant such as hydroxysuccinimide acid for 15min and then added with an antibody or avidin for incubation.

Thus, in some embodiments, the invention provides a low antibody bead ratio coated, high bead content exosome trapping bead.

In some embodiments, preferably, the magnetic beads are prepared in one step by forced cleavage.

In some embodiments, it is preferred that the size of the magnetic beads is the same as the exosome size, ranging from 30 to 150nm.

In some embodiments, preferably, the magnetic beads are superparamagnetic.

In some embodiments, it is preferred that the magnetic beads are agglomerated from 8-12nm Fe ₃O₄ nanoparticles.

In some embodiments, it is preferred that the antibody is an antibody to a protein commonly used by exosomes, including but not limited to antibodies to one or more of the proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 and the like.

In some embodiments, preferably, the low antibody magnetic bead ratio refers to a mass ratio of antibody to magnetic bead of between (0.00005-0.005) to 1;

In some embodiments, preferably, the high magnetic bead content refers to a number of magnetic beads per milliliter of cell supernatant/plasma of between 1 x 10 ⁹-1 x 10¹²

In some embodiments, the invention provides a method of preparing exosome-capturing magnetic beads, which may include the steps of: s1: preparing magnetic beads; s2: modification of antibodies.

In some embodiments, the magnetic beads in step S1 are prepared by a forced cleavage method through a one-step reaction.

In some embodiments, the magnetic beads in step S1 are preferably prepared by the following method:

1) Adding 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 and a reducing agent such as ethylene glycol into a high-temperature reaction kettle, and heating and reacting for 7-72 hours at 150-240 ℃ to obtain the magnetic beads.

In some embodiments, the modification of the antibody in step S2 is obtained by coupling the antibody to magnetic beads under the action of 4-maleimidobutyric acid-N-succinimidyl ester.

In some embodiments, the modification of the antibody in step S2 may also be obtained by coupling avidin to the magnetic beads under the action of 4-maleimidobutyric acid-N-succinimidyl ester and reacting the avidin on the magnetic beads via 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 concentrated/pre-enriched exosome cell supernatant and a cell supernatant that has not been subjected to a concentration treatment.

In some embodiments, it is preferred that the kit for separating exosomes in the cell supernatant comprises the exosome-capturing magnetic beads described above, BSA (bovine serum albumin) or HSA (human serum albumin), and an amino ligand such as glycine, etc.

In some embodiments, the exosome separation in the concentrated/pre-enriched treated cell supernatant may comprise the steps of:

s1, adding 0.11% -0.2% BSA solution with the volume of 1-10 times to the concentrated/pre-enriched cell supernatant;

s2, adding 0.001% -0.1% of mouse IgG into the system of the step S1;

s3, adding 1X 10 ⁹-1x 10¹² magnetic beads per milliliter of cell supernatant into the system of the step S2;

S4, the mass ratio of the antibody to the magnetic beads in the step S2 is (0.00005-0.005) to 1;

s5, incubating the system of the step S4 at 37 ℃ for 1-4 hours, or at room temperature for 2-6 hours, or at 4 ℃ overnight; and (5) finishing the capturing of the exosomes.

In some embodiments, performing exosome isolation in the cell supernatant that has not been subjected to 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 into the system of the step S1;

S3, adding 1X 10 ⁹-1 x 10¹² magnetic beads per milliliter of cell supernatant into the system of the step S2;

In some embodiments, the invention provides a kit for separating exosomes in plasma.

In some embodiments, preferably, the kit for separating exosomes in plasma comprises the exosome-capturing magnetic beads, BSA or HSA described above, and murine IgG.

In some embodiments, the exosome separation in plasma may comprise the steps of:

S1, adding 0.12% -0.2% BSA or HSA solution with the volume of 1-5 times to the blood plasma;

s2, adding 0.001% -0.1% of mouse IgG into the system of the step S1;

s3, adding 1X 10 ⁹-1x 10¹² magnetic beads per milliliter of plasma into the system of the step S2;

In some embodiments, the conjugated antibodies of 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,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4 or the like.

In some embodiments, the conjugated antibodies in the methods and products of the invention also include methods of modifying avidin outside the magnetic beads prior to attachment of the antibodies via biotin-avidin reactions.

In some embodiments, 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 find application in a range of applications including, 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 as the exosomes and are 30-150nm;

3) The amount of the antibody required for coating the magnetic beads is less, and the mass ratio of the antibody to the magnetic beads is (0.00005-0.005) to 1; in the cell supernatant and plasma, the magnetic beads of the invention are consumed only The 1/27 and 1/9 antibodies of (C1/C9)Higher exosome capture efficiency; in cell supernatant and plasma, the magnetic beads of the invention andThe same amount of antibody was consumed, and the capturing efficiency for exosomes in cell supernatant and plasma could reach 28-fold and 2.4-fold, respectively. Therefore, the method and the magnetic beads can effectively reduce the consumption of the antibody, and simultaneously improve the exosome capturing efficiency, and are low-cost and high-efficiency exosome capturing methods and products;

4) The magnetic beads synthesized by the one-step method are matched with extremely low antibody consumption to reduce the cost;

5) The exosome capture kit can be applied to concentrated and pre-enriched cell supernatants and plasma, and is also applicable to 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 use (capturing 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 magnetic beads.

FIG. 3 shows the effect of varying the ratio of the reaction components on the preparation of magnetic beads and coupled proteins; wherein FIG. 3A shows the ratio of thiol crosslinker, naAc, PEG, and ferric chloride hexahydrate, wherein +represents satisfactory magnetic beads; -representing 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 addition ratio of methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester on the effect of magnetic bead coupled protein.

FIG. 4 shows the coupling efficiency of magnetic beads synthesized by different methods to proteins (expressed as relative fluorescence intensity relative to the present magnetic beads).

FIG. 5 shows the beads of the present invention in the supernatant of H3122 cellsComparison of exosome capture efficiency (ddPCR positive droplet count quantification).

FIG. 6 shows the same consumption of 0.5. Mu.g antibody, magnetic beads according to the invention andComparison of exosome capture efficiency in cell supernatants (DDPCR GAPDH copy number quantification).

FIG. 7 shows the magnetic beads of the present invention and the consumption of different amounts of antibodies in plasmaComparison of exosome capture efficiency (ddPCR positive droplet count quantification).

FIG. 8 shows the same 0.5. Mu.g of antibody consumption, magnetic beads according to the invention andComparison of exosome capture efficiency in plasma (DDPCR GAPDH copy number quantification).

FIG. 9 shows the magnetic beads and the magnetic beads with different coating ratios of the antibodies in the present inventionComparison of the efficiency of exosome capture in the cell supernatant by magnetic beads (ddPCR positive droplet count quantification).

Figure 10 shows a comparison (DDPCR GAPDH copy number quantification) of the exosome capture efficiency in plasma for magnetic beads of different particle sizes, the same antibody content, and the total mass of the beads.

FIG. 11 shows a comparison of the number of different beads in the present invention versus the efficiency of exosome capture in the cell supernatant (ddPCR positive droplet count quantification) for the same antibody content and the same bead size.

FIG. 12 shows a comparison of the number of different magnetic beads in the present invention with respect to the efficiency of exosome capture in plasma (ddPCR positive droplet count quantification) for the same antibody content.

FIG. 13 shows WB characterization performed after exosomes were captured in cell supernatants and plasma using the magnetic beads of the invention and control cell supernatant Exosome capture kit (Exosome-Human CD63 Isolation/Detection Reagent, invitrogen ^TM) and control plasma Exosome capture kit (ExoCap ^TM, JSR LIFE SCIENCES).

FIG. 14 shows a comparison of the Exosome capture efficiency (DDPCR GAPDH copy number quantification) of the magnetic beads of the invention versus a control Exosome capture kit (Exosome-Human CD63Isolation/Detection Reagent, invitrogen ^TM) in cell supernatants.

Figure 15 shows a comparison (DDPCR GAPDH copy number quantification) of the exosome capture efficiency of the inventive magnetic beads versus a control exosome capture kit (ExoCap ^TM, JSR LIFE SCIENCES) in plasma.

FIG. 16 shows the capture of exosomes (DDPCR GAPDH copy number quantification) in pre-concentrated/non-pre-concentrated cell supernatants by the exosome magnetic beads of the invention.

Figure 17 shows a comparison (DDPCR GAPDH copy number quantification) of exosome capture efficiency in plasma after coupling antibodies with the magnetic beads of the invention using an avidin antibody system, with a control exosome capture kit (ExoCap ^TM, JSR LIFE SCIENCES).

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.

In the cell supernatant and plasma, the magnetic beads of the invention are consumed onlyThe 1/27 and 1/9 antibodies of (C1/C2)Higher exosome capture efficiency; in cell supernatant and plasma, the magnetic beads of the invention are used in combination withThe same amount of antibody was consumed, and the capturing efficiency for exosomes in cell supernatant and plasma could reach 28-fold and 2.4-fold, respectively. Therefore, the magnetic beads can effectively reduce antibody consumption and improve the exosome capturing efficiency. 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 greater range of use (capturing exosomes in unconcentrated/pre-enriched cell supernatants).

The method for synthesizing the magnetic beads is simple and is obviously different from the complicated method in the prior art (such as CN106279421A, CN106432504A, CN106366197A, CN106279422A, CN106366196A, CN106366195A, CN106381286A and the like). The magnetic bead synthesis is simpler, and the magnetic bead 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 (FIG. 1, FIG. 2)

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 are added into 100mL of ethylene glycol, and the mixture is stirred uniformly and then reacted 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/mL of 4-maleimidobutyric acid-N-succinimidyl ester is added for reaction for 15min, after washing, the antibodies are added according to the ratio of the magnetic beads to the CD63 antibodies (similar products are obtained by using other exosome specific antibodies through the same process) of 1:0.0005, and the magnetic bead antibodies are coated by incubation overnight.

The coated magnetic beads were blocked with 5% (w/v) serum albumin overnight and the exosome trapping magnetic beads were prepared.

The scanning electron microscope characterization of the synthesized beads is shown in fig. 1, and the hysteresis loop of the synthesized beads is shown in fig. 2:

From FIG. 1, it can be seen that the magnetic beads of the present invention are between 30-150nm in size (exactly the same as the exosomes). From the hysteresis loop in FIG. 2, it can be seen that the saturation magnetic strength of the magnetic beads in the present invention is 60emu/g and is superparamagnetic. Therefore, the exosome trapping magnetic beads are synthesized conveniently and simply.

Example 2 determination of the extent of addition of reagents during the Synthesis of magnetic beads (FIG. 3A)

Adding 3.12g of ferric trichloride hexahydrate into 100mL of ethylene glycol, adding methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, sodium acetate and polyethylene glycol according to a certain proportion, uniformly stirring, reacting for 8 hours at 220 ℃ in a high-pressure reaction kettle, and judging the characterization of the obtained substance, wherein magnetic beads are seriously agglomerated or are not formed into magnetic beads or the size is not in the range of 30-600nm, and judging as unqualified (-); the formation of monodisperse beads in the 30-600nm range was judged to be acceptable (+), and the results are shown in FIG. 3A.

From FIG. 3A, it can be seen that when the mass ratio of the ferric salt, the mercapto-functionalized cross-linking agent, the stabilizer for the reduction reaction and the surfactant 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 optimal amount of thiol-functional crosslinker (FIG. 3B)

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 are added into 100mL of ethylene glycol, and the mixture is stirred uniformly and then reacted in a muffle furnace at 220 ℃ for 8 hours to obtain the magnetic beads.

After washing the magnetic beads with ethanol, 0.01. Mu. Mol/mL of 4-maleimidobutyric acid-N-succinimidyl ester was added to react for 15min, and after washing, the magnetic beads were put into 10. Mu.M fluorescent dye Cy 5-labeled BSA and incubated at room temperature for 2h with rotation. PBST is washed twice, PBS is washed once, fluorescence is detected by using a fluorescence microscope, and the coating efficiency of the protein is judged according to the fluorescence intensity. The results are shown in the figure.

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 trichloride hexahydrate is 0.7:1.

Example 4 other alternatives to synthetic magnetic beads (FIG. 4)

To 100mL of ethylene glycol, 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 were added, and after stirring uniformly, the mixture was reacted in a muffle furnace at 220℃for 8 hours to obtain magnetic beads ①.

The similar products are prepared by the same process by replacing ferric sulfate with ferric acetate, ferric nitrate, ferric phosphate, ferric citrate, ferric pyrophosphate and the like.

3.12G of ferric trichloride hexahydrate, 0.23g of poly (ethylene glycol) 2-mercaptoethyl ether acetic acid, 4.2g of sodium acetate and 2.4g of polyethylene glycol are added into 100mL of ethylene glycol, and the mixture is stirred uniformly and then reacted in a muffle furnace at 220 ℃ for 8 hours to obtain magnetic beads ②.

To 100mL of ethylene glycol were added 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, and after stirring uniformly, the mixture was reacted in a muffle furnace at 220℃for 8 hours to obtain magnetic beads ③.

3.12G of ferric trichloride 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 100mL of ethylene glycol, and after being stirred uniformly, the mixture is reacted for 10 hours at 200 ℃ in a muffle furnace, so as to obtain the magnetic beads ④. The urea is used for replacing trisodium citrate to prepare a similar product through the same process.

After judging whether the magnetic beads meet the requirements, washing the magnetic beads with ethanol, adding 0.01 mu mol/mL 4-maleimidobutyric acid-N-succinimidyl ester for reaction for 15min, washing, putting the magnetic beads into 10 mu M fluorescent dye Cy 5-labeled BSA, and incubating for 2h at room temperature in a rotating way. PBST is washed twice, PBS is washed once, fluorescence is detected by a fluorescence microscope, and the coupling efficiency of the prepared magnetic beads to proteins is judged according to the fluorescence intensity, and the result is shown in figure 4.

The results show that the above alternatives can all prepare magnetic beads meeting the requirements and can be coated with 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 with the best effect (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 quick and convenient.

(2) The existing method for affinity capture of exosomes by using magnetic beads requires a large amount of antibodies, and the high price of the antibodies leads to the very high cost of the magnetic bead method. In the case of Dynabeads ^TM MyOne^TM carboxilic Acid, cat No. 65012, the official instructions for antibodies andThe mass ratio of (2) is about 1:10. The mass ratio of the antibody to the magnetic beads is (0.01-1) to 1, but for the invention, the mass ratio of the antibody to the magnetic beads is (0.00005-0.005) to 1, so that the dosage of the antibody is greatly reduced, the preparation cost of the exosome affinity magnetic beads is greatly reduced, and the exosome capturing efficiency is not reduced.

The operation method is as follows:

Example 5 exosomes (magnetic beads of the invention) were captured in concentrated/pre-enriched cell supernatants and used for quantitative exosome detection. (FIG. 5, FIG. 6)

Adding exosome-free culture medium into a culture dish with H3122 cells (a lung cancer cell strain) density of about 80%, culturing for 3 days, collecting culture medium, centrifuging 300g for 10min, collecting supernatant, centrifuging 2000g for 30min, collecting supernatant; the supernatant was filtered through a 0.22 μm filter to obtain a cell-free, apoptotic body cell supernatant. 20mL of the above culture medium was concentrated to 130. Mu.L by ultrafiltration through a 30kDa ultrafiltration tube to give an exosome-pre-enriched culture medium.

To 130. Mu.L of the cell supernatant subjected to the exosome concentration/pre-enrichment treatment, 6 times of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG were added, and after mixing uniformly, exosome-capturing beads (CD 63: magnetic bead mass ratio: 0.0005:1) of the present invention were added at a content of 0.5. Mu. gCD63 (an exosome-specific antibody), and the mixture was rotated at 4℃overnight to obtain exosome-capturing magnetic beads.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM and the content of the housekeeping gene GAPDH was used to reflect the content of exosomes.

EXAMPLE 6 Capture of exosomes in concentrated/Pre-enriched cell supernatants(FIG. 5, FIG. 6)

The same batch of CD63 antibodies (similar products are obtained by using other exosome specific antibodies through the same process) is coated with the magnetic beads according to the invention, and the coating method is as followsIs an official specification of (3).

6 Volumes of 0.12% (w/v) BSA solution and 0.002% (w/v) murine IgG were added to two equal amounts of the exosome concentrated/pre-enriched 130. Mu.L cell supernatants, respectively, and after mixing, 4.5. Mu.g and 13.5. Mu.g CD63 antibodies (similar products were obtained by the same procedure with other exosome-specific antibodies) were added, respectivelyThe incubation was rotated at 4℃overnight to give magnetic beads capturing exosomes.

Will capture exosomesRNA was extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and reverse transcribed into cDNA, 8. Mu.L of reverse transcribed cDNA, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP) were added to each ddPCR well. After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM and the content of exosomes was reacted with the positive droplet count of the housekeeping gene GAPDH.

In combination with examples 5 and 6, it can be seen from FIGS. 5 and 6 that the exosome capture kit of the present invention has a specific ratio in the cell supernatantMuch higher exosome capture efficiency: the exosome capturing kit of the invention can achieve/>, with respect to the exosome capturing efficiency in cell supernatant, by consuming the same antibodyAbout 28 times; after 27 times of the antibody of the exosome capturing kit is consumed, the capturing efficiency of the exosome capturing kit on cell supernatant is still inferior to that of the exosome capturing kit.

Example 7 capturing exosomes (magnetic beads of the invention) in plasma (fig. 7, fig. 8)

Plasma 12000g was centrifuged for 10min to obtain supernatant, 1000. Mu.L of 0.14% BSA solution was added to 400. Mu.L of plasma, and then 0.002% (w/v) mouse IgG was added thereto, and after mixing uniformly, 0.5. Mu.g of the exosome-capturing beads of the present invention having a CD63 content (CD 63: magnetic beads mass ratio: 0.0005:1) were added thereto, and the mixture was incubated overnight at 4℃under rotation.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM and the content of exosomes was reacted with the positive droplet count of the housekeeping gene GAPDH.

EXAMPLE 8 capturing exosomes in plasma(FIG. 7, FIG. 8)

The supernatant was collected by centrifugation of 12000g of plasma for 10min, 1000. Mu.L of 0.14% BSA solution was added to 400. Mu.L of plasma, and 0.002% (w/v) murine IgG was added thereto, and after mixing, 0.5. Mu.g, 1.5. Mu.g, and 4.5. Mu.g of CD63 antibody (similar product was obtained by the same procedure as the other exosome-specific antibody) were added theretoIncubate overnight at 4 ℃.

Will capture exosomesRNA was extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and reverse transcribed into cDNA, 8. Mu.L of reverse transcribed cDNA, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (N0 dUTP) were added to each ddPCR well. After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM and the content of exosomes was reacted with the positive droplet count of the housekeeping gene GAPDH.

In combination with examples 7 and 8, it can be seen from FIGS. 7 and 8 that the exosome-trapping kit of the present invention has a specific ratio in plasmaMuch higher exosome capture efficiency: the exosome capturing kit of the invention can achieve/>, which can achieve the exosome capturing efficiency in plasmaAbout 240%; /(I)The capturing efficiency of the exosome capturing kit of the invention on plasma exosome is still inferior to that of the exosome capturing kit of the invention after the 9 times of antibody is consumed.

Example 9 exploration of the ratio of coating of magnetic beads with antibodies of the present invention on the premise of ensuring exosome Capture efficiency (FIG. 9)

6 Times of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG were added to 130. Mu.L of the cell supernatant subjected to the exosome concentration/pre-enrichment treatment, and after mixing uniformly, exosome trapping beads (the mass of the beads is 0.2mg, the mass ratio of the CD63 to the beads is 0.00005-0.005:1) were added to the mixture, and the mixture was spun overnight at 4℃to obtain exosome trapping beads.

6 Times of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG were added to two equal amounts of the exosome concentrated/pre-enriched 130. Mu.L cell supernatants, respectively, and after mixing, 20. Mu.g CD63 antibody (similar product obtained by the same procedure with other exosome specific antibodies) was added, respectively(Mass 0.2mg, CD63 andMass ratio of 0.1:1), and incubating overnight at 4deg.C to obtain magnetic beads with exosomes captured.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of exosomes was reacted with the positive droplet count of the housekeeping gene GAPDH, and the results are shown in FIG. 9.

The result shows that the mass ratio of the antibody to the magnetic beads in the magnetic beads is between 0.00005 and 0.005:1, so that a better exosome capturing effect can be obtained. The capturing effect of the exosome is better than that of the antibody and the magnetic bead with the mass ratio of 0.1:1The dosage of the antibody is greatly saved, and the capturing efficiency of exosomes is improved.

Example 10 explored the effect of different sized magnetic beads on exosome capture efficiency (FIG. 10)

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 are added into 100mL of ethylene glycol, and the mixture is stirred uniformly and then reacted for 8 hours at 220 ℃ in a muffle furnace, so that the 30-150nm magnetic beads are obtained.

The synthesis method of the magnetic beads with different sizes comprises the following steps: 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 are added into 100mL of ethylene glycol, and the mixture is stirred uniformly and then reacted for 8-72 hours at 200 ℃ in a muffle furnace to obtain the magnetic beads with the particle size of 200-800 nm. Wherein the size of the magnetic beads can be adjusted by controlling the reaction time, for example, a magnetic bead of 200nm is obtained for 8 hours and a magnetic bead of 800nm is obtained for 72 hours.

Plasma 12000g was centrifuged for 10min to obtain supernatant, 1000. Mu.L of 0.14% BSA solution was added to 400. Mu.L of plasma, and then 0.002% (w/v) mouse IgG was added thereto, and after mixing uniformly, 0.5. Mu.g of the exosome-trapping beads of the present invention having a CD63 content (the mass of each of the beads was 0.2 mg) were added thereto, and the mixture was incubated overnight at 4℃under rotation.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of the exosomes was reacted with the concentration of the housekeeping gene GAPDH, and the results are shown in FIG. 10.

The results showed that the particle size of the beads was reduced in the range of 800-30nm with the same amount of antibody added and the same mass of the beads. The capture efficiency of immunomagnetic beads for plasma exosomes increases gradually, probably due to the number of magnetic beads n=m/(4/3R ³ p), where m is the total mass of the beads, R is the bead radius, p is the bead density. It can be seen that in the case where m is constant, the number of beads is inversely proportional to R ³. 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) can improve the capturing efficiency of exosomes by more magnetic beads under the condition of the same total mass of the magnetic beads.

Example 11 explored the effect of different magnetic bead addition on exosome capture efficiency (FIG. 11, FIG. 12)

Adding exosome-free culture medium into a culture dish with H3122 cells (a lung cancer cell strain) density of about 80%, culturing for 3 days, collecting culture medium, centrifuging 300g for 10min, collecting supernatant, centrifuging 2000g for 30min, collecting supernatant; the supernatant was filtered through a 0.22 μm filter to obtain a cell-free, apoptotic body cell supernatant. 20mL of the above medium was concentrated to 130. Mu.L by ultrafiltration through a 30kDa ultrafiltration tube to give an exosome-pre-enriched medium.

To 130. Mu.L of the cell supernatant subjected to the exosome concentration/pre-enrichment treatment, 6 times of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG were added, and after mixing uniformly, exosome-capturing beads (the number of added beads was 1X 10 ⁶-1 x 10¹²) according to the present invention having a CD63 content (an exosome-specific antibody) were added, and incubated overnight at 4℃under rotation to obtain exosome-capturing beads.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of the exosomes was reacted with the positive droplet count of the content of the housekeeping gene GAPDH, and the results are shown in FIG. 11.

Plasma 12000g was centrifuged for 10min to obtain supernatant, 1000. Mu.L of 0.14% BSA solution was added to 400. Mu.L of plasma, and then 0.002% (w/v) mouse IgG was added thereto, and after mixing uniformly, 0.5. Mu.g of the exosome-trapping beads of the present invention having a CD63 content (CD 63: magnetic beads mass ratio: 0.0005:1) were added thereto, and the mixture was incubated overnight at 4℃under rotation.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). Following PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM and the content of exosomes was reacted using the positive droplet count of the housekeeping gene GAPDH, the structure is shown in FIG. 12.

As can be seen from fig. 11 and 12, for cell supernatants, the capture of the cell supernatant exosomes by the capture beads can be maximized when the number of capture beads is greater than 1 x10 ⁹; when the number of the trapping magnetic beads is larger than 1 x10 ¹¹ for plasma, the trapping of the trapping magnetic beads to the plasma exosomes can reach the maximum value. This result was consistent with the number of exosomes reported in the data (https：//www.thermofisher.com/cn/zh/home/life-science/cell-analysis/exosomes/exosomes-webinar-q-and-a.html), which was around 10 ⁹/mL in cells and around 10 ¹¹/mL in plasma.

The recommended number of beads per ml of sample is between 1 x 10 ⁹-1 x 10¹², considering cost effectiveness.

In summary, the magnetic beads of the present invention are consumed only in cell supernatant and plasma, respectivelyThe 1/27 and 1/9 antibodies of (C1/C2)Higher exosome capture efficiency; in cell supernatant and plasma, the magnetic beads of the invention andThe same amount of antibody was consumed, and the capturing efficiency for exosomes in cell supernatant and plasma could reach 28-fold and 2.4-fold, respectively. Therefore, the magnetic beads can effectively reduce antibody consumption and improve the exosome capturing efficiency. /(I)

(3) Existing magnetic bead affinity capture exosome kit and based on sameThe exosome affinity capture method of (2) is not efficient for capturing exosomes. The invention has the exosome capture kit compared with the control and is based onHas higher capture efficiency (in the example described herein, the control Exosome capture kit in cell supernatant is exoome-Human CD63 Isolation/Detection Reagent, invitrogen ^TM; and the control Exosome capture kit in plasma is ExoCap ^TM, JSR LIFE SCIENCES).

The operation method is as follows:

Example 12 exosomes (the magnetic beads of the invention and control exosome capture kit) were captured in concentrated/pre-enriched cell supernatants and characterized by Western Blot. (FIG. 13)

Adding exosome-free culture medium into a culture dish with H2228 cells (a non-small cell lung cancer cell strain) density of about 80%, culturing for 3 days, collecting culture medium, centrifuging for 10min at 300g, collecting supernatant, centrifuging for 30min at 2000g, and collecting supernatant; the supernatant was filtered through a 0.22 μm filter to obtain a cell-free, apoptotic body cell supernatant. 20mL of the above culture medium was concentrated to 130. Mu.L by ultrafiltration through a 30kDa ultrafiltration tube to give an exosome-pre-enriched culture medium.

The kit treatment method comprises the following steps: 6 times of 0.12% (w/v) BSA solution and 0.002% (w/v) mouse IgG were added to 130. Mu.L of the concentrated/pre-enriched cell supernatant, and after mixing, 0.5. Mu.g of the exosome-trapping beads of the present invention having a CD63 content (CD 63: magnetic bead mass ratio: 0.0005:1) were added thereto, and the mixture was incubated at 4℃overnight with rotation to obtain exosome-trapping beads.

Control exosome capture kit treatment method: the exosome capturing magnetic beads in 100 μl of the kit are washed 3 times with 0.1% BSA solution, 70 μl of 0.1% BSA solution is added to 130 μl of cell supernatant subjected to equivalent exosome concentration/pre-enrichment treatment, and the mixture is rotated at 4deg.C and incubated overnight to obtain exosome capturing magnetic beads.

Washing the magnetic beads with the captured exosomes twice by using PBST and once by using PBS; adding 40 μl of the lysate for cracking for 30min, adding 10 μl L Loading Buffer, and decocting at 95deg.C for 5min; taking 14 mu L of supernatant sample after magnetic separation; 80V electrophoresis was performed for 30min,120V electrophoresis was performed for 1h.

Transferring the membrane after electrophoresis, and then sealing with 5% (w/v) of PBST solution of milk powder at room temperature for 1h;

Rabbit anti-human EpCAM (epithelial cell adhesion molecule, a protein enriched in exosomes of tumor patients) was added at a ratio of 1:1000 and incubated overnight at 4 ℃;

Washing with WB (Western Blot) washes 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) wash solutions 3 times, development was performed by adding a developer solution. The results are shown in FIG. 13.

The results show that in the cell supernatant, the exosomes extracted by the exosome capture kit can show obvious bands when being detected by WB, while the exosomes extracted by the control exosome kit under the same conditions are very light 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 13 exosomes were captured in plasma (magnetic beads of the invention versus control exosome capture kit) and characterized by Western Blot. (FIG. 13)

The kit treatment method comprises the following steps: plasma 12000g was centrifuged for 10min to obtain supernatant, 1000. Mu.L of 0.14% BSA solution was added to 300. Mu.L of plasma, and then 0.002% (w/v) mouse IgG was added thereto, and after mixing uniformly, 0.5. Mu.g of the exosome-capturing beads of the present invention having a CD63 content (CD 63: magnetic beads mass ratio: 0.0005:1) were added thereto, and the mixture was incubated overnight at 4℃under rotation.

Control exosome capture kit treatment method: taking 500 mu L of avidin-coupled magnetic beads; magnetically separating, removing supernatant; resuspension with 1mL Washing Buffer (kit provided); mu.L (1. Mu.g/. Mu.L) biotin-CD63 was added; rotating at room temperature for incubation for 1h; magnetically separating, removing supernatant; washing 3 times with 0.5mL Washing Buffer; re-suspending with 0.5mL Washing Buffer for later use; taking 100 mu L of the magnetic beads, and magnetically separating and discarding the supernatant; add 300. Mu. L TREAMENT Buffer (kit provided), resuspend; add 300 μl of plasma; rotating at room temperature and incubating overnight; magnetically separating after incubation, and discarding supernatant; add 1mL Washing Buffer to resuspend transfer to new tube; magnetically separating, and discarding the supernatant; washing once again with 1mL Washing Buffer; 100. Mu.L of Washing Buffer was resuspended for use.

Centrifuging 12000g of plasma for 10min to obtain supernatant; 100. Mu.L of plasma was added to the above beads, and the beads were incubated overnight at 4℃with spin, thereby obtaining exosomes captured.

The results show that in plasma, the exosomes extracted by the exosome capture kit of the invention can show obvious bands when tested by WB, whereas the exosomes extracted by the control exosome kit under the same conditions are not tested 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 were captured in concentrated/pre-enriched cell supernatants (magnetic beads of the invention versus control exosome capture kit) and a comparison of capture efficiency was made with ddPCR. (FIG. 14)

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of the exosomes was reflected by the concentration of the housekeeping gene GAPDH, and the results are shown in FIG. 14.

The results show that the exosome capturing kit provided by the invention has a far better exosome capturing effect in cell supernatant than that of a control exosome capturing kit. This example demonstrates from a nucleic acid perspective that the exosome capture kit of the invention has a higher exosome capture efficiency in cell supernatant than the control exosome capture kit.

Example 15 exosomes were captured in plasma (magnetic beads of the invention versus control exosome capture kit) and a comparison of capture efficiency was made using ddPCR. (FIG. 15)

Control exosome capture kit treatment method: taking 500 mu L of avidin-coupled magnetic beads; magnetically separating, removing supernatant; resuspension with 1mL Washing Buffer (kit provided); mu.L (1. Mu.g/. Mu.L) biotin-CD63 was added; rotating at room temperature for incubation for 1h; magnetically separating, removing supernatant; washing 3 times with 0.5mL Washing Buffer; re-suspending with 0.5mL Washing Buffer for later use; taking 250 mu L of the magnetic beads, and magnetically separating and discarding the supernatant; add 300. Mu. L TREAMENT Buffer (kit provided), resuspend; add 300 μl of plasma; rotating at room temperature and incubating overnight; magnetically separating after incubation, and discarding supernatant; add 1mL Washing Buffer to resuspend transfer to new tube; magnetically separating, and discarding the supernatant; washing once again with 1mL Washing Buffer; 250 μL of Washing Buffer was resuspended for use.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu.L of GAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (No dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of the exosomes was reacted using the concentration of the housekeeping gene GAPDH, and the results are shown in FIG. 15.

The result shows that the exosome capturing kit of the invention has better exosome capturing effect in plasma than that of the control plasma exosome capturing kit.

Example 15 exosomes (magnetic beads of the invention) were captured in unconcentrated/pre-enriched cell supernatants and used for quantitative exosome detection. (FIG. 16)

Adding exosome-free culture medium into a culture dish with H2228 cells (a non-small cell lung cancer cell strain) density of about 80%, culturing for 3 days, collecting culture medium, centrifuging for 10min at 300g, collecting supernatant, centrifuging for 30min at 2000g, and collecting supernatant; the supernatant was filtered through a 0.22 μm filter to obtain a cell-free, apoptotic body cell supernatant.

0.4G BSA solution and 0.4ng mouse IgG are added into 20mL unconcentrated/pre-enriched H2228 cell (a non-small cell lung cancer cell strain) supernatant, the mixture is uniformly mixed, and then the exosome capturing magnetic beads (the mass ratio of CD63 to the magnetic beads is 0.0005:1) with the content of 0.5 mu gCD are added, and the mixture is rotated and incubated overnight at 4 ℃ to obtain the exosome capturing magnetic beads.

The results show that the magnetic beads of the invention are not only suitable for capturing exosomes in pre-enriched cell supernatants, but also in non-pre-enriched cell supernatants.

Example 16 exosomes were captured in plasma using the magnetic beads of the present invention and compared to a commercially available kit. (FIG. 17)

After the magnetic beads are washed by ethanol, 0.01 mu mol/mL of 4-maleimidobutyric acid-N-succinimidyl ester is added for reaction for 15min, and after washing, avidin is added according to the ratio of the magnetic beads to the avidin of 1:0.005, and the magnetic beads are incubated overnight to complete the coating of the magnetic bead antibodies.

The coated magnetic beads were blocked with 5% (w/v) serum albumin overnight, washed with PBS, and then biotin-labeled CD63 antibody (similar product obtained by the same procedure with other exosome-specific antibodies) was added in a ratio of 1:0.0005, and after incubation for 1h, washed with PBS, the exosome-capturing magnetic beads were prepared.

Control exosome capture kit treatment method: taking 500 mu L of avidin-coupled magnetic beads; magnetically separating, removing supernatant; resuspension with 1mL Washing Buffer (kit provided); mu.L (1. Mu.g/. Mu.L) biotin-CD63 was added; rotating at room temperature for incubation for 1h; magnetically separating, removing supernatant; washing 3 times with 0.5mLWashing Buffer; re-suspending with 0.5mL Washing Buffer for later use; taking 250 mu L of the magnetic beads, and magnetically separating and discarding the supernatant; add 300. Mu. L TREAMENT Buffer (kit provided), resuspend; add 300 μl of plasma; rotating at room temperature and incubating overnight; magnetically separating after incubation, and discarding supernatant; add 1mL Washing Buffer to resuspend transfer to new tube; magnetically separating, and discarding the supernatant; washing once again with 1mL Washing Buffer; 250 μL of Washing Buffer was resuspended for use.

The exosome-trapped magnetic beads were extracted with miRNeasy Serum/PLASMA ADVANCED KIT (QIAGEN RNA extraction kit) and the RNA was reverse transcribed into cDNA, 8. Mu.L of the reverse transcribed cDNA was added to each ddPCR well, 1. Mu. LGAPDH primer, 1. Mu.L of enzyme-free water, and 10. Mu.L of ddPCR ^TM Supermix for Probes (N0 dUTP). After PCR amplification, the housekeeping gene GAPDH in the exosomes was quantified using Bio-Rad QX200 ^TM, and the content of the exosomes was reacted using the concentration of the housekeeping gene GAPDH, and the results are shown in FIG. 17.

The results show that the exosome capturing kit of the invention has better exosome capturing effect in plasma by using the avidin antibody than that of the control plasma exosome capturing kit.

As described in the examples above, the exosome capture kit of the present invention demonstrates a higher exosome capture efficiency in both cell supernatant and serum than the control exosome capture kit, as well as a greater range of use (capturing exosomes in unconcentrated/pre-enriched cell supernatant) from both protein and nucleic acid perspectives. In summary, 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.

Without being limited by a particular theory, in the present invention, exosomes may be captured by modifying fewer antibodies on more thiol-based small magnetic beads (e.g., 1 x 10 ⁹-2 x 10¹² magnetic beads per milliliter of sample), since the number of exosomes in the sample is limited, more magnetic beads means fewer exosomes are captured per magnetic bead, thereby avoiding steric hindrance of exosomes on the magnetic beads, and improving the efficiency of exosomes capture; meanwhile, each magnetic bead only needs to capture a small amount of exosomes, so that the concentration of the antibody coating on the magnetic beads can be reduced, thereby saving the antibodies. Therefore, the invention provides a technical scheme with low antibody consumption and high exosome capturing efficiency.

In the cell supernatant and plasma, the magnetic beads of the invention are consumed onlyThe 1/27 and 1/9 antibodies of (C1/C2)Higher exosome capture efficiency; in cell supernatant and plasma, the magnetic beads of the invention are used in combination withThe same amount of antibody was consumed, and the capturing efficiency for exosomes in cell supernatant and plasma could reach 28-fold and 2.4-fold, respectively. Therefore, the magnetic beads can effectively reduce antibody consumption and improve the exosome capturing efficiency.

The exosome capture kit of the present invention is demonstrated herein from both a protein and nucleic acid perspective to have a higher exosome capture efficiency in cell supernatant as well as serum than the control exosome capture kit, as well as a greater range of use (capturing exosomes in unconcentrated/pre-enriched cell supernatant).

Claims

1. A method of isolating exosomes, the method comprising contacting a sample containing exosomes from a subject with nanomagnetic beads, thereby isolating exosomes from the sample, wherein the sample is derived from a cell supernatant or plasma, wherein the nanomagnetic beads are ferric salt nanomagnetic beads having surface-modified sulfhydryl groups introduced by a sulfhydryl-functional crosslinking agent, wherein the ferric salt is selected from ferric chloride, ferric sulfate, ferric acetate, ferric nitrate, ferric phosphate, ferric citrate, ferric pyrophosphate; the thiol-functionalized cross-linker is selected from methoxy-polyethylene glycol-polycaprolactone-cysteine ethyl ester, poly (ethylene glycol) 2-mercaptoethyl ether acetic acid, wherein the size of the nanomagnetic beads is in the range of 30-150nm, wherein the nanomagnetic beads have superparamagnetism, wherein the nanomagnetic beads have antibodies or linker proteins as conjugates, wherein the antibodies are antibodies to exosurface proteins, wherein the mass ratio of the antibodies to the magnetic beads or linker proteins to the magnetic beads is between 0.00005:1 and 0.005:1.

2. The method of claim 1, wherein the nanomagnetic beads have any one of the following properties: 1) Is formed by agglomerating Fe ₃O₄ nano particles with the particle size of 8-12 nm; 2) The addition amount of the magnetic beads is 1x 10 ⁹-2x 10¹² magnetic beads per milliliter of sample; 3) The size of the nano magnetic beads is selected from 30nm,40nm,50nm,60nm,70nm,80nm,90nm,100nm,110nm,120nm,130nm,140nm and 150nm; 4) The mass ratio of antibody to magnetic beads or adaptor protein to magnetic beads is selected from 0.00005:1,0.0005:1,0.005:1.

3. The method of claim 2, wherein the nanomagnetic beads have any one of the following properties: 1) The antibody is one or more antibodies selected from exosome surface proteins CD9,CD63,CD81,CD44,CD31,Rab5b,EpCAM,TSG101,HSP90,HSP70,ANXA5,FLOT1,ICAM1,ALIX,GM130,ICAM-1,SNAP,MHC I/II,HLA-G,Integrins,Claudins,Tim4; 2) The linker protein is avidin; 3) The amount of the magnetic beads added is an amount selected from the group consisting of: 1x 10 ⁹ magnetic beads, 1.5x10 ⁹ magnetic beads, 5x 10 ⁹ magnetic beads, 8x 10 ⁹ magnetic beads, 1x 10 ¹⁰ magnetic beads, 1.5x10 ¹⁰ magnetic beads, 5x 10 ¹⁰ magnetic beads, 8x 10 ¹⁰ magnetic beads, 1x 10 ¹¹ magnetic beads, 1.5x10 ¹¹ magnetic beads, 5x 10 ¹¹ magnetic beads, 8x 10 ¹¹ magnetic beads, 1x 10 ¹² magnetic beads, 1.5x10 ¹² magnetic beads, 2x 10 ¹² magnetic beads per milliliter of sample.

4. The method of claim 1, wherein the sample is from a concentrated/pre-enriched exosome-treated cell supernatant; or from cell supernatants not subjected to concentration.

5. The method of any one of claims 1-4, wherein the method comprises the following features: blocking agents are added to the sample and/or incubated to capture exosomes.

6. The method of claim 5, wherein the blocking agent is bovine serum albumin, human serum albumin or glycine and/or the incubation is 1-4 hours at 37 ℃, or 2-6 hours at room temperature, or overnight at 4 ℃.