27 results about "CD81" patented technology

The invention provides a method for detecting an external vesicle marker through high-flux nano plasma exciting light immune color development, and belongs to the technical field of human extracellular vesicles. After a serum sample is subjected to centrifugal treatment, the serum sample and a CD81 capture antibody substrate are incubated, and the particle size distribution of the vesicles is monitored in real time; a zwitterionic polymer modified gold nanoparticle LAM detection probe and a polyethylene glycol modified silver nanoparticle LprG detection probe are prepared to be specifically combined with a vesicle surface antigen, a chromogenic enhancement solution is adopted to induce a plasma resonance signal, and full-hole scanning imaging is carried out; and constructing a double-layer game optimization model to cooperatively optimize the detection sensitivity and the signal stability, carrying out weighted summation on normalized signals of the particle size subgroups to obtain comprehensive detection signal intensity, comparing the comprehensive detection signal intensity with a threshold value, and outputting a final judgment result. The technical problem that quantitative accuracy is affected by signal intensity deviation caused by vesicle particle size difference in outer vesicle marker detection is solved.

The invention discloses an EV membrane protein single molecule detection method based on droplet microfluidics and application thereof. The method comprises the following steps: firstly, constructing an Ab-oligo compound which is formed by sequentially connecting an antibody, Linker, Linker connection DNA, RNA capable of being specifically cut by RNA enzyme and template DNA for starting signal amplification; then capturing the extracellular vesicles in the plasma by using the CD9 / CD63 / CD81 functionalized magnetic beads; the method comprises the following steps: adding an Ab-oligo compound and a sealing buffer solution into extracellular vesicles, incubating, removing the uncombined Ab-oligo compound, adding RNA enzyme for incubating and enzyme digestion, collecting template DNA for starting signal amplification, and performing microdroplet digital PCR analysis to realize single molecule analysis of EV membrane protein. According to the invention, the one-to-one correspondence relationship between the antibody and oligonucleotide is established through Linker for the first time, single protein information is converted into single nucleic acid chain information, and EV membrane protein single molecule detection is realized. The method is simple to operate, can qualitatively and quantitatively detect the single EV membrane protein, and is beneficial to further research on the relationship between the EV membrane protein and diseases.

The invention discloses a method for screening an active matter for protecting skin cell telomeres, and relates to the technical field of biotechnology and cosmetic development, and the method is technically characterized by comprising the following steps: a) providing stem cells derived from an exosome which carries at least one microRNA for regulating and controlling a telomere protection factor CTC1; b) acting a candidate active matter on the stem cells, and separating to obtain a treated exosome; the technical effects are as follows: the combination of miR-150-5p and miR-181c is delivered through the exosome, the dual-miRNA coordinated regulation is realized, the limitation of the traditional single-target detection is broken through, the CD63 / CD81 protein on the membrane surface of the exosome can be specifically combined with the skin cell membrane, and the Franz diffusion experiment verifies that the micro-fluidic chip integrates the collagen scaffold and the dynamic shear force.

The invention discloses a plasma or serum exosome separation method which comprises the following steps: firstly, coupling an antibody for resisting four transmembrane protein CD81 on the surface of an exosome by using a magnetic bead to form an immunomagnetic bead; pretreating a serum / plasma sample, enriching exosomes, co-incubating with the immunomagnetic beads, washing the magnetic beads after incubation capture is completed, and washing off uncombined impurities; after being captured, the exosome can be directly cracked, the magnetic beads are removed for downstream experiments, or the magnetic beads are eluted to obtain the complete exosome. The exosome affinity antibody is matched with the magnetic beads, the plasma / serum sample exosome is separated, the unique affinity separation technology not only can remove high-abundance protein in the serum / plasma sample, but also can eliminate other vesicles with similar membrane structures with the exosome, so that the purity of the separated exosome is far higher than that of the exosome obtained by other separation methods.

Disclosed are a specific marker combination of mesenchymal stem cell-derived small extracellular vesicles and the use thereof. The characteristic marker combination comprises aminopeptidase N (CD13), integrin β-1 (CD29), membrane glycoprotein THY-1 (CD90), and markers CD9, CD63 and CD81. By using the characteristic marker combination of mesenchymal stem cell-derived small extracellular vesicles for nano-flow cytometry analysis at the single-vesicle level, mesenchymal stem cell-derived small extracellular vesicles can be identified. On the basis of the provided characteristic marker combination, specific modification and engineering on the basis of small extracellular vesicle membrane proteins can further be realized.

The application discloses a nucleic acid free extraction reagent of plasma exosome and a direct PCR amplification kit. The nucleic acid free extraction reagent comprises 0.001-0.1% polidocanol and 1-100 mM Tris-HCl in mass volume concentration, and the solvent is water. The direct PCR amplification kit provided by the application comprises the nucleic acid free extraction reagent and an immunomagnetic bead suspension, a washing liquid, a free extraction reagent and a PCR Mix solution; the surface of the immunomagnetic bead is simultaneously coated with three exosome specific antibodies of CD9, CD63 and CD81, so that the recovery rate of the exosome can be greatly improved during the extraction and purification of the plasma exosome; the PCR Mix solution contains an anti-inhibitory component, can effectively neutralize the inhibitors such as proteins in the PCR system, and ensures the amplification efficiency of the PCR. The nucleic acid free extraction reagent of the application can realize the amplification detection of the exosome nucleic acid free extraction, and simplifies the operation process of the PCR detection.

The present invention provides a method for preparing a platelet lysate rich in extracellular vesicles and its application. The platelet lysate prepared by intermittent ultrasonic lysis does not require the exogenous addition of biochemical components such as thrombin, collagen, or calcium ions, making it simpler and safer. By setting reasonable ultrasonic process parameters, the prepared platelet lysate is not only rich in various factors (such as bFGF, VEGF, PDGF-BB, and IL-6, IL-15, IL-17, and INF-γ) but also has a high content of bioactive proteins. Furthermore, the extracellular vesicles are not only high in content but also small in diameter, with an average diameter of as low as 79.9 nm. The proportion of exosomes is relatively large, and the extracellular vesicles have high expression of the transmembrane protein CD9 and low expression of CD81.

The application discloses an affinity peptide targeting human CD81 protein and application thereof. The amino acid sequence of the affinity peptide provided by the application is shown as SEQ ID NO: 7. The affinity peptide provided by the application has high affinity and high selectivity with a small loop fragment of an extracellular CD81 protein, can recognize and combine with the CD81 protein in free and living cells, can be used for high-sensitivity detection and in-situ imaging of the CD81 protein, and can be further used as a selective inhibitor of a migration process of tumor cells expressing the CD81 protein. The affinity peptide provided by the application can specifically target and recognize the CD81 protein, is a specific probe for analysis and detection of the CD81 protein and functional research, and has application prospects in detection of diseases such as malignant tumors and development of targeted drugs. The application has important application value.

The invention discloses an extraction method and application of membranous small vesicles. The extraction method comprises the following steps: collecting a biological sample, and selecting at least one of blood, urine and cell culture supernatant; centrifuging the sample at 10,000 xg for 20 minutes, and removing cell debris and apoptotic bodies to obtain a pretreated sample; adding 20% by volume of sample separation liquid and CD63 / CD81 antibody modified magnetic beads into the pretreated sample, and incubating and capturing target vesicles; freezing the captured vesicles with liquid nitrogen for 15-35 minutes, and dissolving the vesicles with a 33-43 DEG C water bath; and cracking the dissolved vesicles with 0.5 W / cm < 2 > ultrasonic waves to finally obtain the intact vesicles of 30-70 nm and soluble protein factors. Original plasma or urine can be directly treated, dilution is not needed, and loss of a target object is avoided; a low-cost and degradable biomimetic polymer is developed to replace an imported kit, and the cost is reduced by 75%; the whole process lt; the time is 2.5 hours, which is superior to that of an ultracentrifugation method; and the extracted membranous small vesicles have good integrity and are suitable for liquid biopsy and drug carrier construction.

The invention relates to the technical field of biological medicines, and provides a high VEGF protein / miR-210 extracellular vesicle induced by a serum-free combined culture medium and application of the high VEGF protein / miR-210 extracellular vesicle. According to the invention, high VEGF protein / miR-210 extracellular vesicles are obtained based on induction of a serum-free culture medium; the serum-free culture medium comprises a basic culture medium component combination: sugar-free RPMI1640 and MCDB131 in a volume ratio of 4: 1; the metabolic regulation additive is prepared by synergistically adding sodium pyruvate, cysteine and a proline hydroxylase inhibitor and taking alanine glutamine as an energy substitute; the growth factor is a combination of polyvinyl alcohol, 4-hydroxyethylpiperazine ethanesulfonic acid, ITS-X, epidermal growth factors and basic fibroblast growth factors, wherein the ITS-X, the epidermal growth factors and the basic fibroblast growth factors are added before use. CD63 and CD81 are specifically expressed on the surface of the prepared high VEGF protein / miR-210 extracellular vesicle, the high VEGF protein / miR-210 extracellular vesicle is rich in VEGF protein and miR-210, and the high VEGF protein / miR-210 extracellular vesicle is applied to targeted therapy of allergic rhinitis by inhibiting Th2 type immunoreaction.

The present invention relates to exosomes prepared from placenta mesenchymal stem cells and their uses. On the one hand, the present invention relates to exosomes isolated and extracted using placenta mesenchymal stem cells, which have an average particle size of 50-200 nm, and which express membrane protein CD9 and membrane protein CD81. The positive expression rate of the exosome membrane protein CD9 is greater than 65%, and the positive expression rate of the membrane protein CD81 is greater than 75%. The preparation method of the exosomes is as follows: inoculate mesenchymal stem cells into a culture flask, culture to make the cells adhere to the wall, and then add IL-1β to the culture medium for culture; after replacing the complete culture medium, culture in an incubator at 37°C, 2% O2, and 5% CO2; subject the cells to differential centrifugation to obtain exosomes in the form of a suspension. The present invention also relates to the use of the prepared exosomes in the preparation of drugs for treating inflammatory diseases. The method of the present invention has the advantages of high exosome yield, high activity, etc.

The invention relates to a nucleic acid aptamer combined with CD81 protein. The nucleic acid aptamer has a nucleotide sequence as shown in SEQ ID NO.1; or a nucleotide sequence which has at least 30% of homology with SEQ ID NO.1 and is combined with CD81 protein; 1, or an RNA sequence transcribed by a nucleotide sequence as shown in SEQ ID NO. 1. By improving screening conditions, the nucleic acid aptamer which is stable in chemical property, easy to store and mark and capable of being combined with CD81 protein with high affinity and high specificity is obtained through screening; the structure is relatively stable, simple, easy to modify, capable of being artificially synthesized in a short time, stable in chemical property, easy to store and mark and wide in application prospect.

The application discloses a chimeric antigen receptor with a CD81 costimulatory domain and application thereof. The chimeric antigen receptor with the CD81 costimulatory domain comprises, from N-terminus to C-terminus: (i) an extracellular antigen binding domain; (ii) a transmembrane domain; (iii) a costimulatory domain, wherein the costimulatory domain is a CD81 costimulatory domain; and (iv) a signaling domain. The application connects the C-terminal intracellular domain of the CD81 molecule as the costimulatory domain of the CAR with the signaling domain, successfully constructs a novel CD19-CAR-T cell (81z) with the CD81 as the costimulatory signal. Compared with the existing CD28 or 4-1BB costimulatory domain CAR-T, the CAR-T cell with the CD81 costimulatory domain obviously reduces the release of toxic cytokines after tumor cell stimulation, reduces the release of mononuclear cell inflammatory factors IL-1beta and IL-6 mediated, and has good targeted killing CD19 + tumor cell ability.

This invention relates to a stem cell culture method using a roller bottle for improving the yield and quality of separation and purification of extracellular vesicles, and to a method for separating stem cell-derived extracellular vesicles. When exosomes or extracellular vesicles are separated from a culture medium obtained by culturing stem cells using the roller bottle method, the yield is greatly increased, and high-quality extracellular vesicles with high CD9 and CD81 expression can be obtained. As a result, stem cells can be cultured in large quantities using the stem cell culture method using the roller bottle of the present invention, and exosomes and extracellular vesicles can be separated and purified with high yield.

The invention discloses an antibody combination for detecting minimal residual disease and application thereof. The antibody combination comprises a CD45 antibody, a CD19 antibody, a CD33 antibody, a CD10 antibody, a CD20 antibody, a CD38 antibody, a CD34 antibody, a CD58 antibody, a CD66c antibody, a CD73 antibody, a CD81 antibody, a CD123 antibody and a CD304 antibody. A specific antibody combination for detecting the minimal residual disease is designed, a specific gate setting strategy is combined, 13-color flow analysis is realized, and target cells can be accurately locked and normal cells and differential cells can be effectively distinguished by combining composition and development process analysis of CD19 + cells and leukemia cell related immunophenotype (LAIP) analysis; the detection rate, the accuracy and the sensitivity are obviously improved.

The invention discloses a kit and a method for detecting exosomes based on flow-type fluorescence coded microspheres. The kit comprises a first solution, a second solution, a third solution and a fourth solution. The first solution is a microsphere solution coupled with an antibody and comprises a microsphere solution coupled with a CD9 antibody, a microsphere solution coupled with a CD63 antibody and a microsphere solution coupled with a CD81 antibody. The microspheres coupled with the CD63 antibody are provided with amino modified functional groups. The microsphere solution coupled with the CD9 antibody and the microsphere solution coupled with the CD81 antibody both have carboxyl modified functional groups. The second solution is a solution for marking a CD63 antibody. The third solution comprises a solution for marking a CD9 antibody and a solution for marking a CD81 antibody. The fourth solution is a washing buffer solution. According to the present invention, the exosome can be detected through the conventional flow cytometry, the detection of multiple indexes can be performed by one sample, the cost is saved, the detection time is shortened, the detection efficiency is improved, the repeatability is good, and the good application prospect is provided.

The present invention relates to a reagent panels for the measurement of minimal residual disease (MRD) associated with pediatric B-cell precursor acute lymphoblastic leukemia (B- ALL) by multiparametric flow cytometry. The reagent panel of the invention comprises a combination of antibodies directed against markers, wherein the combination of antibodies comprises i) antibodies targeting markers CD45, CD20, CD34, CD38, CD10, CD58, CD66c, CD73, CD81, CD123, CD304, CD44, CD86, CD99 and CD371, and ii) antibodies targeting markers CD19 and / or CD22, wherein the antibodies are conjugated with fluorochromes. The invention further relates to the use of said panels for detecting MRD associated with B-ALL and / or for identifying a subject at risk of developing B-ALL relapse. The invention also relates to methods of detecting MRD associated with B-ALL. Key advantages of the invention include achieving high MRD detection sensitivity (e.g., 10⁻⁵ or 0.001%) and high specificity.

The utility model discloses a three-chromatography-channel cancer early screening test strip based on exosome protein. The three-chromatography-channel cancer early screening test strip comprises a drainage sheet, the first chromatography channel, the second chromatography channel and the third chromatography channel are arranged around the drainage piece; the first chromatography channel, the second chromatography channel and the third chromatography channel respectively comprise a sample pad, a chromatography film, a water absorption pad and a bottom plate; the chromatographic membrane is coated with the colloidal gold pad, the detection line and the quality control line; the colloidal gold pads of the three chromatography channels are coated with three kinds of gold-labeled antibodies containing different exosome proteins; the detection line is coated with an antibody which is specifically combined with a substance to be detected; and the quality control lines are coated with antibodies which are specifically combined with the corresponding colloidal gold particles. The test strip can be used for detecting three special transmembrane proteins CD63, CD81 and CD9 in the exosome, and further can be used for identifying circulating tumor cells such as gastric cancer, lung cancer, lymph cancer and colorectal cancer. Early screening and early discovery of specific cancer cells can be realized, postoperative rehabilitation monitoring is also facilitated, and the method has a great clinical application space.

The invention relates to an exosome derived from glioma internal cystic fluid and application of the exosome in preparation of anti-glioma drugs, and belongs to the technical field of biology. The glioma endocystic fluid source exosome is prepared to solve the treatment difficulty caused by the fact that a blood-brain barrier hinders drug delivery and glioma is high in invasiveness and drug resistance in the prior art. The particle size of the exosome is 60-130 nm, the exosome specifically expresses CD63, CD81 and TSG101 proteins, and a four-stage gradient centrifugation method (300 g to 3000g to 10000g to 100000g) is adopted for separation and extraction. Experiments prove that the exosome inhibits glioma cell invasion and proliferation by regulating and controlling tumor cell ribosome functions. Compared with a traditional treatment medicine, the exosome has the advantages of efficient targeting property, low toxicity and low cost, and further development of a new medicine is facilitated.

This study of the regulatory T phenotype of Sézary cells led to the discovery of overexpression of CD81 by Sézary cells. CD81 has also been shown to be a relevant therapeutic target in the treatment of Sézary syndrome, NK / T lymphoma, hepatosplenic T-cell lymphoma, and acute T-cell leukemia. CD81 is therefore seen as a diagnostic marker and therapeutic target in T-cell malignancies. The present invention therefore implicates CD81 as a biomarker and biotarget in T-cell malignancies.

The invention relates to the technical field of biological medicine detection, in particular to a CD81 monoclonal antibody, a detection kit and application of the CD81 monoclonal antibody. According to the detection kit disclosed by the invention, the expression level of the CD81 protein in peripheral blood can be detected with high sensitivity by utilizing the independently researched and developed CD81 monoclonal antibody and the biotin-labeled nucleic acid aptamer; and the CD81 monoclonal antibody and the nucleic acid aptamer have high specificity, so that the interference of non-specific binding is reduced, and the detection accuracy is improved. According to the kit, by detecting the expression level of the CD81 protein in peripheral blood, early prediction and diagnosis can be carried out before preeclampsia clinical symptoms appear, and precious time is provided for clinical intervention. In addition, the kit is simple and convenient in operation flow and easy to popularize and apply in clinical laboratories.

The present invention relates to amniotic mesenchymal stem cell-derived exosomes and their uses. On the one hand, the present invention relates to the use of exosomes isolated and extracted from amniotic mesenchymal stem cells in the preparation of a medicament for treating inflammatory diseases, wherein the exosomes have an average particle size of 50 to 200 nm, the positive expression rate of the exosome membrane protein CD9 is greater than 70%, and the positive expression rate of the membrane protein CD81 is greater than 80%. The exosomes of the present invention are obtained by treating amniotic mesenchymal stem cells with IL-1β and culturing them under low oxygen partial pressure conditions. It also relates to a method for regulating the level of TNF-α secretion by peripheral blood mononuclear cells (PBMC) using exosomes, as well as exosomes isolated and extracted using amniotic mesenchymal stem cells and their preparation method. The exosomes of the present invention have biological regulatory properties.

The invention discloses a special high-concentration lysate for exosome protein extraction, and belongs to the technical field of exosome lysate. The reagent is mainly prepared by mixing a reagent SDS (Sodium Dodecyl Sulfate), NP-40, sodium deoxycholate, TritonX-100 and NaCl. The specific preparation method comprises the following steps: taking 10ml of 50mM. Tris-HCl buffer solution, adding 1g. SDS, 1g. NP-40, 0.1 g of sodium deoxycholate, 100. Mu l. TritonX-100 and 0.58 g. NaCl, and fully blowing, beating and uniformly mixing. A subversive exosome protein extraction solution is provided by organically integrating the two functional modules of'mild splitting 'and'synchronous concentration' into a whole. According to the formula of the lysate, exosomes can be mildly and specifically lysed under the condition that the concentration of effective components is extremely high, and the integrity of key membrane proteins such as CD9, CD63 and CD81 is perfectly protected; meanwhile, due to the built-in concentration capacity, a high-concentration protein sample can be obtained without relying on tedious steps such as super-separation in the cracking process. The special high-concentration lysate for exosome protein extraction achieves the effect of improving the efficiency and yield of exosome protein extraction.

The invention provides an exosome staining solution and an exosome staining method, and the exosome staining solution comprises the following components in percentage by volume: 1-5% of a stabilizer; 1-5% of a fluorescent coloring agent; 1-5% of an auxiliary coloring agent; 70-80% of a buffer agent; and the balance of water. Wherein the stabilizer comprises lycium barbarum polysaccharide, the fluorescent staining agent comprises a CD81 immunofluorescent antibody and propidium iodide, the auxiliary staining agent comprises saponin, and the buffering agent comprises trihydroxymethyl aminomethane and sodium chloride. According to the exosome staining solution, through the synergistic effect of all the components in the formula, non-specific binding with non-exosome particles (such as lipoprotein and cell debris) can be remarkably reduced, false positive signals are reduced, high-specificity fluorescence labeling is achieved, fluorescence stability is effectively enhanced, and fluorescence quenching can be delayed.

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of serum exosomes after electroacupuncture, and the method comprises the following steps: establishing a rat myocardial ischemia reperfusion injury model; applying electroacupuncture stimulation with current of 1mA, frequency of 2Hz and sparse and dense waves to'neiguan 'acupoints on two sides of the rat; serum is collected, and exosomes are extracted through a differential centrifugation method and an ultracentrifugation method; identifying form, particle size and surface marker proteins CD9 and CD81 of the extracted exosome by using a transmission electron microscope and a nanoflow detection technology; the exosome can be internalized by H9c2 myocardial cells, the survival rate of hypoxia / reoxygenation damaged myocardial cells is improved, and the levels of lactic dehydrogenase, creatine kinase and creatine kinase isoenzyme in cell supernatant are reduced. According to the scheme provided by the invention, a novel post-acupuncture serum exosome for treating myocardial ischemia reperfusion injury is provided, and the exosome effectively plays an acupuncture-like role in a treatment process, so that an effect of relieving myocardial ischemia reperfusion is achieved.

The invention relates to a method, a system and a composition for delivering macromolecules to marine fish sperms based on exosomes, and belongs to the crossing field of biotechnology and reproductive engineering.According to the method, the exosomes displaying CD81 protein serve as a carrier, and the macromolecules are delivered to marine fish sperms through specific binding of the CD81 protein and a receptor ligand of CD9 protein on a fish sperm cell membrane. And delivering the biological macromolecules loaded in the exosome into the marine fish sperms. Targeting and tracing are integrated, the targeting can be enhanced through the potential interaction effect of CD81 and sperm surface protein CD9, and real-time tracing of the delivery process can be achieved through EGFP fluorescence.

The invention discloses a chimeric antigen receptor with a CD81 costimulatory structural domain and application of the chimeric antigen receptor. A chimeric antigen receptor having a CD81 costimulatory domain comprising, from N-terminus to C-terminus: (i) an extracellular antigen binding domain; (ii) a transmembrane domain; (iii) a co-stimulatory domain, where the co-stimulatory domain is a CD81 co-stimulatory domain; and (iv) a signaling domain. According to the invention, a C-terminal intracellular domain of a CD81 molecule is used as a costimulatory structural domain of CAR to be connected with a signal transduction structural domain, and a novel CD19-CAR-T cell (81z) taking CD81 as a costimulatory signal is successfully constructed. Compared with the existing CAR-T of the CD28 or 4-1BB costimulatory domain, the CAR-T cell with the CD81 costimulatory domain has the advantages that the toxic cytokine release is obviously reduced after the CAR-T cell is stimulated by tumor cells, the release of mediated mononuclear inflammatory factors IL-1beta and IL-6 is reduced, and the CAR-T cell has good capability of targeted killing of CD19 + tumor cells.