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KR-20260067528-A - A method to analyze surface proteins of extracellular vesicles by clustering between extracellular vesicles

KR20260067528AKR 20260067528 AKR20260067528 AKR 20260067528AKR-20260067528-A

Abstract

The present invention relates to a method for analyzing surface proteins of extracellular vesicles, specifically comprising: (a) a step of isolating extracellular vesicles from a biological sample; (b) a step of preparing a nanocomplex by labeling the surface proteins of the extracellular vesicles with a biotin-conjugated antibody; (c) a step of preparing a nanocomplex by combining the biotin-conjugated antibody with the extracellular vesicles; (d) a step of clustering the nanocomplex by adding an avidin-like protein to the nanocomplex through binding between the biotin-conjugated antibody and the avidin-like protein; and (d) a step of detecting the surface proteins of the extracellular vesicles; thereby enabling the analysis or detection of surface proteins of extracellular vesicles without impurities, which can be widely utilized for early diagnosis of diseases, improvement of analysis accuracy, and research on vesicle-based therapies.

Inventors

  • 이원종
  • 이소예

Assignees

  • 인천대학교 산학협력단

Dates

Publication Date
20260513
Application Date
20241106

Claims (13)

  1. (a) A step of isolating extracellular vesicles from a biological sample; (b) a step of preparing a nanocomplex by labeling the surface protein of the extracellular vesicle with a biotin-conjugated antibody; (c) adding an avidin-like protein to the nanocomplex to cluster the nanocomplex through binding between the biotin-bound antibody and the avidin-like protein; and (d) a step of detecting surface proteins of the extracellular vesicles; A method for analyzing surface proteins of extracellular vesicles, comprising
  2. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in claim 1, the detection in step (d) is performed using a flow cytometer.
  3. A method for analyzing a surface protein of an extracellular vesicle, characterized in that, in claim 1, the surface protein of the extracellular vesicle is tetraspanin or a tumor antigen.
  4. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in paragraph 3, the tetraspanin is one or more proteins selected from the group consisting of CD9, CD37, CD53, CD63, CD81, and CD82.
  5. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in claim 3, the tumor antigen is one or more proteins selected from the group consisting of PD-L1 (Programmed Death-Ligand 1), B7-H3 (Cluster of Differentiation 276; CD276), TIM-3 (T-cell Immunoglobulin and Mucin-domain containing-3), CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4; CD263), LAG-3 (Lymphocyte Activation Gene 3), NKG2D-ligand (Natural Killer Group 2, Member D Ligands), ER (Estrogen receptor), HER2 (human epidermal growth factor receptor 2), PR (progesterone receptor), and Trop2 (Tumor-associated calcium signal transducer 2).
  6. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in claim 1, the avidin-like protein is one or more proteins selected from the group consisting of streptavidin, neutravidin, and avidin.
  7. A method for analyzing surface proteins of extracellular vesicles according to claim 1, wherein the biological sample is one or more selected from the group consisting of urine, mucus, saliva, tears, blood, plasma, serum, sputum, cerebrospinal fluid, pleural fluid, nipple aspirate, lymph fluid, airway fluid, serous fluid, genitourinary fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intra-organ fluid, ascites, cystic tumor fluid, amniotic fluid, cell tissue fluid, and cell culture fluid.
  8. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in claim 1, the ratio of the extracellular vesicle to the antibody bound to biotin is 1:1 to 1:1000.
  9. A method for analyzing surface proteins of extracellular vesicles, characterized in that, in claim 1, the ratio of extracellular vesicles to avidin-like proteins is 1:1 to 1:1000.
  10. A disease screening method comprising a method for analyzing surface proteins of extracellular vesicles according to claim 1.
  11. A disease screening method according to claim 11, characterized in that the disease is a tumor.
  12. In paragraph 12, the tumor is lung cancer, ovarian cancer, skin cancer, colon cancer, cervical cancer, gastric cancer, breast cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, colorectal cancer, small intestine cancer, rectal cancer, proanal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, lymphoma, bladder cancer, gallbladder cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renopelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, brainstem A disease screening method characterized by having one or more selected from the group consisting of gliomas and pituitary adenomas.
  13. An extracellular vesicle surface protein analysis kit comprising an antibody that specifically binds to extracellular vesicle surface proteins, biotin, and an avidin-like protein.

Description

A method to analyze surface proteins of extracellular vesicles by clustering between extracellular vesicles The present invention relates to a method for analyzing surface proteins of extracellular vesicles through clustering of extracellular vesicles, a disease screening method, and a kit for analyzing surface proteins of extracellular vesicles. Extracellular vesicles (EVs) are nano-sized particles containing a lipid bilayer that are secreted from cells into bodily fluids such as blood, urine, and saliva. They contain various substances exhibiting biological activity, such as proteins, nucleic acids, and lipids, and reflect the state of the cell from which they originated. DNA, RNA, and other substances contained within extracellular vesicles are not degraded by external stimuli, making them a focus of attention as stable biomarkers. Non-invasive liquid biopsy is a diagnostic method that isolates cell-free nucleic acids (cfNA), circulating tumor cells (CTCs), and extracellular vesicles (EVs) present in a patient's liquid specimen and utilizes them for disease diagnosis, prognosis, and monitoring the effectiveness of treatments. Unlike conventional tissue biopsies, which involve directly inserting tools such as endoscopes into specific sites to collect tissue, it has the advantages of lower risk and simpler analysis methods. A flow cytometer is a device that classifies and analyzes cells or particles present in a liquid based on their physical and chemical properties. It can analyze not only single cells but also nano-sized particles and can process multiple particles simultaneously at high speeds. However, since extracellular vesicles are nano-sized particles, they are difficult to detect directly in a flow cytometer. Furthermore, the presence of lipoproteins, which are similar in size to the extracellular vesicles in the sample, may lead to contamination during the separation process, potentially affecting purity. To address these issues, methods using magnetic beads or antibody-coated beads are being developed; however, these methods have the disadvantage of requiring a washing process, which complicates the steps and may result in sample loss. According to prior art, Patent Document 1 discloses a composition for separating extracellular vesicles based on PEG, alcohol, and salt content; according to Patent Document 2, a method for separating and concentrating extracellular vesicles using a highly absorbent resin; and according to Patent Document 3, a method for analyzing surface proteins and nucleic acids of extracellular vesicles by clustering extracellular vesicles into phospholipid complexes. However, there is a lack of research on methods to separate only extracellular vesicles without the influence of impurities such as lipid proteins in the sample. Figure 1 is a schematic diagram showing the process of extracellular vesicle-specific cluster formation and detection using extracellular vesicles, CD-63 antibody, biotin, and streptavidin. Figure 2 shows the results of measuring the size of vesicles according to the order in which extracellular vesicles, CD-63 antibody, biotin, and streptavidin are combined. Figure 3 shows the results of measuring the size of extracellular endoplasmic reticulum versus endoplasmic reticulum prepared with different streptavidin concentrations. Figure 4 shows the results of measuring the size of extracellular vesicles versus vesicles prepared with different antibody-biotin concentrations. Figure 5 shows the results of confirming the presence or absence of cluster formation of serum-derived extracellular vesicles depending on the presence or absence of CD-63 antibody-biotin and streptavidin. Figure 6 shows the results confirming that extracellular vesicles specifically form clusters through changes in the size of extracellular vesicles and liposomes. Figure 7 shows the results of confirming the presence or absence of cluster formation of extracellular vesicles derived from MDA-MB-231 (breast cancer cell line) in the presence or absence of CD-63 antibody-biotin and streptavidin. Figure 8 shows the change in the fluorescence signal of the surface protein CD63 of extracellular vesicles depending on the presence or absence of cluster formation of serum-derived extracellular vesicles. Figure 9 shows the change in the fluorescence signal of the surface protein CD81 of extracellular vesicles depending on the presence or absence of cluster formation of serum-derived extracellular vesicles. Figure 10 shows the change in the fluorescence signal of the surface protein PD-L1 of the extracellular vesicles depending on the presence or absence of cluster formation of extracellular vesicles derived from MDA-MB-231. The present invention will be described in detail below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which the present invention pertains. In general, the nomenclature use