Methods for enriching exosomes and conjugates for use in the methods

Abstract

This invention broadly relates to the field of bioassay technology, and more specifically, to a method for enriching exosomes and the conjugates used in this method. The conjugates are formed by conjugating a CD63 antibody or its antigen-binding fragment with a matrix support; wherein the heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the CD63 antibody or its antigen-binding fragment are shown in SEQ ID NO: 1-3, and the light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO: 4-6. The conjugates provided by this invention have high affinity and high purification efficiency, and show great promise for application in exosome purification.

Description

Technical Field

[0001] This invention generally relates to the field of bioassay technology, and more specifically, to a method for enriching exosomes and the conjugates used in the method. Background Technology

[0002] Exosomes are nanoscale membrane vesicles containing mRNAs, miRNAs, proteins, and lipids, and also possess signaling molecules (CD63, CD9, etc.) on their membranes. Currently, commonly accepted proteins in exosomes include heat shock proteins, the tetraspanic membrane protein family, multivesicular endosome production-related proteins (ALIX, TSG101, etc.), and the GTPase RAB family; these components are largely involved in exosome formation and origin. Exosomes are particularly abundant in the tumor microenvironment and are closely related to tumor development, immune escape, and microenvironment establishment; furthermore, they play a crucial role in tissue damage repair, immune antigen presentation, and neurodegenerative diseases. Therefore, in clinical practice, exosomes isolated from specific sites in patients are often used to detect specific diseases, especially cancer, cardiovascular diseases, and neurological disorders. Simultaneously, research on exosomes as drug delivery carriers for therapeutic purposes is continuously developing.

[0003] Exosomes are widely distributed in body fluids (serum, cerebrospinal fluid, etc.), secretions (saliva, breast milk, semen, etc.), and excretions (urine, etc.). Isolating and identifying exosomes from various samples is crucial for subsequent experimental research.

[0004] Existing methods for exosome isolation mainly include differential ultracentrifugation, precipitation, ultrafiltration, and affinity chromatography. Among these, affinity chromatography can conveniently obtain high-purity exosomes and other extracellular vesicles from cell culture media and body fluids from samples such as serum. Over the past two decades, several tetraspan membrane proteins, especially CD63, CD81, and CD9, have been used as markers for exosomes in affinity chromatography due to their accumulation in small EVs compared to whole-cell lysates, and the steady-state accumulation of CD63 in MVBs. However, their presence in other EVs has recently been observed. By capturing EVs specifically carrying CD63, CD9, or CD81, and then analyzing their protein composition and endosome marker enrichment, it has been suggested that EVs containing only CD9 or CD81 but not CD63 may not form in endosomes (and are therefore ectosomes), while those carrying CD63 along with one or two other tetraspan membrane proteins may correspond to endosome-derived exosomes. This observation suggests that CD63 may be a more specific exosome marker. However, existing methods for affinity purification of exosomes based on CD63 antibodies still suffer from poor affinity and therefore cannot be well applied to exosome purification. Summary of the Invention

[0005] This invention covers the following technical solutions:

[0006] In one aspect, this invention relates to conjugates for exosome purification, which are formed by conjugating CD63 antibody or its antigen-binding fragment with a matrix support.

[0007] The heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the CD63 antibody or its antigen-binding fragment are shown in SEQ ID NO:1 to 3, respectively, and the light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO:4 to 6, respectively.

[0008] Another aspect of the present invention relates to a chromatography separation apparatus containing the conjugates described above.

[0009] Another aspect of the invention relates to a kit containing the conjugate as described above.

[0010] Another aspect of the present invention relates to a method for enriching exosomes, comprising:

[0011] i) Co-incubate the composition containing exosomes with the conjugate as described above; and

[0012] ii) Wash away non-specifically bound impurities.

[0013] The conjugate provided by this invention uses a CD63 antibody with high affinity and high purification efficiency, and has good application prospects in exosome purification. Attached Figure Description

[0014] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0015] Figure 1 The image shows the ELISA results comparing the EXS4 antibody with a commercial monoclonal antibody.

[0016] Figure 2 The nanoflow cytometry results of the purified exosomes;

[0017] Figure 3 Electron micrograph of the purified exosomes. Detailed Implementation

[0018] Reference will now be made to detailed embodiments of the present invention, one or more of which are described below. Each example is provided for explanation and not for limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the invention without departing from its scope or spirit. For example, features described or illustrated as part of one embodiment may be used in another embodiment to produce further embodiments.

[0019] Unless otherwise stated, all terms used to disclose this invention (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Further guidance is provided below for a better understanding of the teachings of this invention. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0020] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0021] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0022] The terms “containing,” “comprising,” and “including” as used in this invention are synonyms and are inclusive or open-ended, not excluding additional, uncited members, elements, or method steps.

[0023] In this invention, the numerical range represented by endpoints includes all numerical values ​​and fractions contained within that range, as well as the endpoints mentioned.

[0024] When this document uses the term "about" to refer to a value or parameter, it includes (and describes) an implementation of the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

[0025] This invention relates to concentration values, which include fluctuations within a certain range. For example, fluctuations are allowed within a corresponding precision range. For instance, 2% may fluctuate within ±0.1%. For larger values ​​or values ​​that do not require overly precise control, even greater fluctuations are permitted. For example, 100mM may fluctuate within ranges of ±1%, ±2%, ±5%, etc. Regarding molecular weight, fluctuations of ±10% are allowed.

[0026] As used herein, unless otherwise indicated, the singular forms of the articles “a,” “an,” and “the” include plural referents.

[0027] In this invention, the terms "multiple" or "various" are used unless otherwise specified, referring to a quantity of 2 or more.

[0028] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0029] In this invention, terms such as "preferred," "better," "more suitable," and "ideal" merely describe implementation methods or embodiments with better effects and should be understood not to limit the scope of protection of this invention. In this invention, terms such as "optionally," "optionally," and "optional" mean that something is optional, that is, selected from either "with" or "without" a parallel solution. If multiple "optional" statements appear in a technical solution, unless otherwise specified and without contradiction or mutual constraint, each "optional" statement is independent.

[0030] This invention relates to a conjugate for exosome purification, which is formed by conjugating a CD63 antibody or its antigen-binding fragment with a matrix support.

[0031] The heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the CD63 antibody or its antigen-binding fragment are shown in SEQ ID NO:1 to 3, respectively, and the light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO:4 to 6, respectively.

[0032] In this invention, the technical term "antibody" refers to a full-length antibody, while "antigen-binding fragment" broadly refers to any functional fragment of an antibody containing a complementarity-determining region (CDR). The term "full-length antibody" includes both polyclonal and monoclonal antibodies, and the term "functional antibody fragment" is a substance containing part or all of the antibody's CDR, lacking at least some amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. In some instances, the term "antigen-binding fragment" includes antigen-compound binding fragments of these antibodies, including Fab, F(ab')2, Fd, Fv, scFv, the smallest antibody recognition unit, and single-chain derivatives of these antibodies and fragments, such as scFv-Fc.

[0033] The term "complementarity-determining region" or "CDR" refers to a highly variable region of the heavy and light chains of an immunoglobulin, as defined in this invention by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy-chain CDRs and three light-chain CDRs. Here, depending on the context, the term "CDR" is used to refer to a region containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to an antigen or epitope it recognizes. In another specific embodiment, the CDR region or CDR refers to a highly variable region of the heavy and light chains of an immunoglobulin as defined by the IMGT.

[0034] Variants of antibodies or their antigen-binding fragments are also within the scope of this invention. The antibodies or their antigen-binding fragments of this invention comprise heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. The sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, compared to their respective corresponding sequences in the complementarity-determining region combinations shown in SEQ ID NO: 1–6, each contain a mutation of up to 3 amino acids (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids, or any combination thereof); preferably, the mutation is a conserved mutation. In some embodiments, the sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the antibody or its antigen-binding fragment, compared to any one of the complementarity-determining region combinations shown in SEQ ID NO: 1–3, each contain a substitution of up to 3 amino acids (e.g., substitution of 1, 2, or 3 amino acids).

[0035] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region HCVR and a light chain variable region LCVR, wherein the amino acid sequences of said HCVR and LCVR have at least 80% identity with the sequences shown in SEQ ID NO:7 or 8, respectively. In some embodiments, the amino acid sequence of the HCVR has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the HCVR sequence shown in SEQ ID NO:7; in some embodiments, the amino acid sequence of the LCVR has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the LCVR sequence shown in SEQ ID NO:8. In some embodiments, the CD63 antibody or its antigen-binding fragment has a heavy chain variable region HCVR as shown in SEQ ID NO:7 and a light chain variable region LCVR as shown in SEQ ID NO:8.

[0036] In some cases, antibody variants include at least one heavy chain and one light chain, while in others, the variant forms contain two identical light chains and two identical heavy chains (or sub-parts thereof). In some cases, variants are obtained by performing conserved mutations (e.g., conserved substitutions or modifications) on the antibody sequence provided by this invention. A "conserved mutation" is a mutation that maintains the normal function of the protein, preferably a conserved substitution.

[0037] "Conservative substitution" refers to the substitution of amino acids in a protein with other amino acids that have similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity), so that the protein can be frequently modified without changing its biological activity.

[0038] Substitutions generally considered conserved are substitutions between aliphatic amino acids Ala, Val, Leu, and Ile; interchange of hydroxyl residues Ser and Thr; exchange of acidic residues Asp and Glu; substitution between amide residues Asn and Gln; exchange of basic residues Lys and Arg; and substitution between aromatic residues Phe and Tyr. Those skilled in the art will recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0039] As used in this invention, the term "matrix support" refers to and includes any solid-phase (including gel-type) support capable of binding the affinity reagents disclosed herein. In some embodiments, well-known matrix supports include glass, agar, agarose, agarose derivatives, magnetic beads, silica, titanium dioxide, alginate, cellulose, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum arabic, tragacanth gum, arabic gum, locust bean gum, xanthan gum, pectin, mucin, hepatitin and gelatin, silicon, ceramics, glass, polyurethane, polystyrene, polystyrene-divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinylpyrrolidone, copolymers or mixtures thereof. The support material can have virtually any structural configuration, as long as the coupled affinity reagent is capable of binding CD63 antibody or its antigen-binding fragment. Therefore, the support structure can be an SPE solid-phase extraction column, a centrifuge tube with a separation membrane, an EP tube, a separation membrane, a microplate with recesses, microspheres, a column, a sheet, or a multi-well plate. Those skilled in the art will recognize many other suitable matrix supports for binding affinity reagents, or will be able to identify such matrix supports using routine experiments.

[0040] Furthermore, the matrix support may be non-porous or include one or more pores. In particular, it may be a porous resin.

[0041] In some embodiments, the matrix support is (agarose) microspheres. The particle size range of the microspheres is preferably 0.1 μm to 1 mm, for example 0.1 μm, 0.5 μm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 500 μm, and 700 μm.

[0042] Furthermore, the substrate support may be modified with polymers as needed, or its surface may be coated with a polymer film. In some embodiments, the polymer is selected from amino, aldehyde, epoxy resin, thiol, and polysaccharide groups. In some embodiments, the polymer film is selected from polyvinyl alcohol film, agarose film, or polyvinyl alcohol-agarose composite film.

[0043] CD63 antibodies or their antigen-binding fragments can be directly coupled to a matrix support, or indirectly coupled to a matrix support via a polymer; the coupling group can be one or more of the amino, carboxyl, thiol, and hydroxyl groups on the CD63 antibody or its antigen-binding fragment.

[0044] According to another aspect of the invention, a chromatographic separation apparatus containing the conjugate as described above is also involved.

[0045] Chromatographic separation apparatus may be a chromatographic column, resin, membrane, filter, or other structures known to those skilled in the art.

[0046] According to another aspect of the invention, a kit is also provided which contains the conjugate as described above.

[0047] The term “kit” refers to any article (e.g., packaging or container) that includes at least one device, and a kit may further include instructions for use, supplementary reagents and / or components or parts used in the methods or steps described herein.

[0048] In some implementations, the kit may also include components such as washing buffer and elution solution.

[0049] According to another aspect of the invention, a method for enriching exosomes is also disclosed, comprising:

[0050] i) Co-incubate the composition containing exosomes with the conjugate as described above; and

[0051] ii) Wash away non-specifically bound impurities.

[0052] In some embodiments, the composition is derived from animals, preferably humans.

[0053] In some embodiments, the composition is derived from tumor cells or cells infected by pathogens.

[0054] Exemplary tumors include, but are not limited to, lung cancer, breast cancer, ovarian cancer, stomach cancer, pancreatic cancer, laryngeal cancer, esophageal cancer, testicular cancer, liver cancer, parotid gland cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, uterine cancer, endometrial cancer, kidney cancer, bladder cancer, prostate cancer, thyroid cancer, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, glioblastoma, neuroblastoma, etc. Samples derived from melanoma, colorectal cancer, lung cancer, pancreatic cancer, liver cancer, prostate cancer, breast cancer, and ovarian cancer are commonly used in these examples.

[0055] Exemplary pathogens include, but are not limited to, viruses, bacteria, parasites, and fungi.

[0056] The concept of pathogens can also be understood as organic macromolecules, organic small molecules, or inorganic molecules that have pathogenicity / immune-activating functions.

[0057] In some embodiments, the composition is selected from cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspiration fluid, bronchoalveolar lavage fluid, urine, semen, vaginal secretions, mucus, saliva, sputum, or a clear lysate obtained from a biological tissue sample.

[0058] The composition can be fresh or pre-frozen and then thawed.

[0059] In some embodiments, the composition is isolated under conditions that substantially do not destroy the morphological or functional characteristics of exosomes or cell surface antigens.

[0060] As mentioned above, the exosomes in the composition should retain their original antigenic profiles so that they are "antigenically intact" so that the detected exosomes can be used to analyze their concentration / particle size.

[0061] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or other experimental methods known in the art, or follow the conditions recommended by the manufacturer.

[0062] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0063] Example 1: Preparation of CD63 antibody

[0064] 1. Antigen Immunization and Monoclonal Antibody Preparation Process

[0065] In this embodiment, recombinant human CD63 protein from Sino Biological was used as the immunogenicity. BALB / c mice were injected intraperitoneally with 150 μg / 200 μl of recombinant human CD63 protein once a week for four weeks. Afterward, tail blood was collected weekly, and the expression level of CD63 antibody in the serum was detected using dot blot technology. Mice with high CD63 antibody expression levels were selected, and their spleen cells were extracted and fused with SP2 / 0 myeloma cells to form fusion molecules. This fusion process was performed using 50% PEG (molecular weight 1450) as a fusion agent, following standard cell fusion methods.

[0066] 2. Culture of hybridoma cells

[0067] After reviving the hybridoma cell line, we cultured it until the cell number increased to approximately 1 × 10⁻⁶. 9 Then, the cells were centrifuged at 1000 rpm for 5 minutes to collect them for subsequent experiments.

[0068] 3. Extraction of cellular RNA

[0069] In a clean bench environment, we first added 1 mL of Trizol reagent to the centrifuged cells and let it stand for 5 minutes. Next, we added 2 mL of chloroform, shook vigorously for 15 seconds, and then let it stand at room temperature for 3 minutes. Following this, we centrifuged at 12000 rpm for 15 minutes and carefully transferred the supernatant to a new EP tube. We then added 0.5 mL of isopropanol, let it stand at room temperature for 10 minutes, and centrifuged again at 12000 rpm for 10 minutes. After removing the supernatant, we added 1 mL of 75% ethanol and centrifuged at 7500 rpm for 5 minutes. After the precipitate dried, we added 50 μL of double-distilled water. Finally, we identified and quantified the purity of the extracted RNA by agarose gel electrophoresis and stored it at -70°C for later use.

[0070] 4. Reverse transcription to generate cDNA

[0071] RNA from hybridoma cells was successfully converted into cDNA using the HiFi Script cDNA Synthesis Kit (from Cwbiotech, product number: CW2569). The specific steps were as follows: 1 μL of total cellular RNA was added to 6 μL of RNase-Free ddH2O, 0.5 μL of Oligo dT Primer, 0.5 μL of PRIME Script RT Enzyme Mix I, and 2 μL of 5×PrimeScript Buffer. After thoroughly mixing these components, the mixture was incubated at 37°C for 15 minutes, followed by a rapid increase to 85°C and a holding time of 5 seconds to complete the reverse transcription process.

[0072] 5. cDNA amplification and antibody gene sequence determination

[0073] Next, using the obtained cDNA as a template, the variable regions of the heavy and light chains of the antibody were amplified by PCR using degenerate primers. The PCR amplification products were then ligated into a T / A vector and transformed into DH5α competent cells. After plating and overnight incubation at 37°C, single clones were selected from the culture plates for further amplification. Subsequently, plasmids were extracted, and the antibody gene sequence was determined.

[0074] Through this series of operations, we successfully obtained a new antibody, named EXS4. Sequencing revealed the amino acid sequence of the antibody's heavy chain variable region (HCVR): EVQLQ QSGKWIYTHGASVRISCKASGDHYSTDNVHWVKQHDFDQDEWIGYKIA NNQHYGYNQKFKSKADDVRCVSSNTAYMDCDLLYCEDSAVYFCARDACQWDWIQVWGILFWFTVSA (SEQ ID NO:7); and the amino acid sequence of the antibody's light chain variable region (LCVR): DIVMDYKTKFMSTSVGDRVDIYNKALHRY DVAVAWYQQHILMSPKSAEYSASLWYFGVPDRKRLFGSGTDFTFTQWIQ MSEDLAVIYCQLWGPAMITFSGSYKPELKR (SEQ ID NO:8). Analysis confirmed that this antibody is of IgG type.

[0075] Example 2 Antibody Activity Assay

[0076] 1. Soak the surface-coupled Protein A probe in 300 μL of buffer K (PBS + 0.002% Tween 20 + 0.02% BSA) for 10 minutes;

[0077] 2. Prepare a working solution of antibody at 5 μg / mL using bufferK;

[0078] 3. Prepare working solutions with gradient concentrations of recombinant human CD63 protein (Sino Biological) using buffer, with concentrations of 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.625 μg / mL, and 0 μg / mL.

[0079] 4. Add reagents according to the instructions of the Gator label-free analyzer (Starchild Medical Technology, CAT#:Gator) to detect antibody affinity.

[0080] The test shows that K in EXS4 D The value is 3.24 × 10 -11 (M).

[0081] Example 3: Comparison of activity with commercially available CD63 antibody using ELISA method

[0082] The newly prepared EXS4 antibody and the control commercial antibody were diluted using serial dilution methods, and their binding ability to recombinant human CD63 protein was evaluated. The experimental procedure is as follows:

[0083] First, recombinant human CD63 protein was diluted to a concentration of 1 μg / mL using ELISA coating buffer, and then coated in 96-well plates overnight at 4°C. The next day, the liquid in the wells was removed, and each well was washed three times with 300 μL of PBS. Next, each well was blocked at room temperature for 30 minutes with PBS containing 2% FBS.

[0084] After blocking, EXS4 antibody and commercially available anti-human CD63 antibody (from Santa Cruz, catalog number sc-365604) were added. To compare binding capacity at different concentrations, serial dilutions were used, setting multiple concentration gradients including 5, 2.5, 1.25, 0.625, 0.03, 0.015, 0.08, 0.04, 0.02, and 0.01 μg / ml. 100 μL of antibody solution was added to each well and incubated at room temperature for 1 hour.

[0085] After incubation, the supernatant was removed, and each well was washed three times with 300 μL of PBS. Then, 100 μL of 1:1000 diluted HRP-labeled rabbit anti-mouse IgG antibody (from Sigma) was added to each well, and the wells were incubated at room temperature for 1 hour.

[0086] After cleaning the wells, add 100 μL of ELISA developer (from SolarBio) to each well and react at room temperature in the dark for 15 minutes. Finally, add 100 μL of ELISA stop solution (also from SolarBio) and measure the absorbance of each well at 450 nm.

[0087] Experimental results are as follows Figure 1 As shown in the figure, it is clear that at most concentrations, the EXS4 antibody exhibits superior affinity compared to commercially available CD63 antibodies. This finding indicates that our newly prepared EXS4 antibody possesses high affinity efficacy in binding recombinant human CD63 protein, providing a powerful tool for future research and applications.

[0088] Example 4: Preparation of CD63 antibody-conjugated magnetic beads

[0089] The magnetic beads used are special magnetic beads for fixing disulfide bonds (purchased from Chongqing Bolanying Biotechnology Co., Ltd.).

[0090] 1. Mix the MSP-MBA-F1 magnetic beads thoroughly by shaking. Take 3.3 mg of the magnetic beads into a 1.5 mL centrifuge tube, retain the magnetic beads by magnetic force, and discard the supernatant. Wash 3 times with 4 CME buffer (pH 6.0), vortexing 200 μL each time.

[0091] 2. Remove the supernatant using magnetic force, quickly add 50 μL of MES, vortex vigorously to resuspend the magnetic beads, add 100 μL of EXS4 antibody solution (the antibody concentration is determined according to the ratio of approximately 10 μg of antibody required for 1.0 mg of MSP-MBA-F1 magnetic beads saturated with conjugation), and vortex to mix; then add 50 μL of reducing agent, gently vortex to mix, and incubate at 25°C for 45 min.

[0092] 3. Add 200 μL of blocking solution, mix gently to keep the magnetic beads suspended; continue the gentle mixing reaction at room temperature for about 30 minutes.

[0093] 4. After blocking the antibody beads with magnetic retention, discard the supernatant; wash three times each time with 200μL of washing buffer using a pipette.

[0094] 5. Magnetically retain the antibody beads, remove the supernatant, add 330 μL of preservation solution, mix gently, and store at 2-8℃ for later use.

[0095] Example 5: Purification and Identification of Exosomes

[0096] Mature 293 cells were cultured, digested, and transferred to a new T75 culture flask. 20 mL of exosome-free medium was added for culturing. After 48 hours, the medium was collected into centrifuge tubes to obtain exosome-rich medium. A conventional low-temperature centrifuge and an ultracentrifuge were pre-cooled to 4°C. The cells were centrifuged at 2000g for 10 minutes to remove dead cells and debris. Larger vesicles were removed by filtration through a 0.22 μm filter. The supernatant was transferred to an ultracentrifuge tube, balanced with PBS, and centrifuged at 120,000g for 4 hours. The supernatant was discarded, and residual liquid on the tube wall was carefully aspirated with sterile filter paper. The liquid at the bottom of the tube was resuspended with 1 mL of PBS and mixed with 20 μL of the magnetic beads prepared in Example 4. The mixture was incubated at 4°C with shaking for 2 hours. After complete adsorption of the magnetic beads, PBS was added for washing 3–6 times. The washings were discarded, and IDES enzyme was added to cleave the target product. The supernatant collected after gentle centrifugation was the purified exosome.

[0097] The purified exosomes were analyzed using nanoflow cytometry, and the results are as follows: Figure 2 As shown, the structure examined by electron microscopy is as follows Figure 3As shown in the figure. The results indicate that 20 μL of magnetic beads (corresponding to 2 μg of antibody) can purify 1.33 × 10⁻⁶ antibodies. 12 Exosomes with a particle / mL concentration exhibit very high purification efficiency.

[0098] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A conjugate for exosome purification, characterized in that, It is formed by the conjugation of CD63 antibody or its antigen-binding fragment with a matrix support; The CD63 antibody or its antigen-binding fragment has the heavy chain variable region HCVR shown in SEQ ID NO:7 and the light chain variable region LCVR shown in SEQ ID NO:8, wherein: SEQ ID NO:7 is: EVQLQQSGKWIYTHGASVRISCKASGDHYSTDNVHWVKQHDFDQDEWIGYKIANNQHYGYNQKFKSKADDVRCVSSNTAYMDCDLLYCEDSAVYFCARDA CQWDWI QVWGILFWFTVSA; SEQ ID NO:8 is DIVMDYKTKFMSTSVGDRVDIYNKALHRYDVAVAWYQQHILMSPKSAEYSASLWYFGVPDRKRLFGSGTDFTFTQWIQMSEDLAVIYCQLWGPAMITFSGSYKPELKR.

2. The exosome purified conjugate according to claim 1, characterized in that, The matrix support includes any one of glass, agar, agarose, agarose derivatives, magnetic beads, silica, titanium dioxide, alginate, cellulose, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum arabic, tragacanth gum, arabic gum, locust bean gum, xanthan gum, pectin, mucin, hepatitin and gelatin, silicon, ceramics, polyurethane, polystyrene, polystyrene divinylbenzene, polymethyl methacrylate, polyacrylamide, polyethylene terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, and polyvinylpyrrolidone, or copolymers or mixtures thereof.

3. The exosome purified conjugate according to claim 2, characterized in that, The matrix support is an SPE solid-phase extraction column, a centrifuge tube with a separation membrane, an EP tube, a separation membrane, a micro-reaction plate with concave holes, microspheres, a column, a sheet, or a porous plate.

4. A chromatography separation apparatus, characterized in that, Contains the conjugate according to any one of claims 1 to 3.

5. A reagent kit, characterized in that, Contains the conjugate according to any one of claims 1 to 3.

6. A method for enriching exosomes, characterized in that, include: i) Co-incubate the composition containing exosomes with the conjugate according to any one of claims 1 to 3; as well as ii) Wash away non-specifically bound impurities.

7. The method according to claim 6, characterized in that, The composition is derived from animals.

8. The method according to claim 7, characterized in that, The composition is derived from humans.

9. The method according to claim 6, characterized in that, The composition is derived from tumor cells or cells infected by pathogens.

10. The method according to claim 6, characterized in that, The composition is selected from cell culture supernatant, whole blood, serum, plasma, ascites, cerebrospinal fluid, bone marrow aspiration fluid, bronchoalveolar lavage fluid, urine, semen, vaginal secretions, mucus, saliva, sputum, or a clear lysate obtained from biological tissue samples.

11. The method according to any one of claims 6 to 10, characterized in that, The composition was isolated under conditions that substantially did not destroy the morphological or functional characteristics of exosomes or cell surface antigens.

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