CN114958743A - Separation detection method and application of neutrophil exosome - Google Patents
Separation detection method and application of neutrophil exosome Download PDFInfo
- Publication number
- CN114958743A CN114958743A CN202210543349.4A CN202210543349A CN114958743A CN 114958743 A CN114958743 A CN 114958743A CN 202210543349 A CN202210543349 A CN 202210543349A CN 114958743 A CN114958743 A CN 114958743A
- Authority
- CN
- China
- Prior art keywords
- neutrophil
- exosomes
- magnetic beads
- antibody
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000001808 exosome Anatomy 0.000 title claims abstract description 113
- 210000000440 neutrophil Anatomy 0.000 title claims abstract description 91
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 238000000926 separation method Methods 0.000 title claims abstract description 28
- 210000002966 serum Anatomy 0.000 claims abstract description 39
- 208000005718 Stomach Neoplasms Diseases 0.000 claims abstract description 24
- 206010017758 gastric cancer Diseases 0.000 claims abstract description 24
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 24
- 239000011886 peripheral blood Substances 0.000 claims abstract description 24
- 201000011549 stomach cancer Diseases 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000013399 early diagnosis Methods 0.000 claims abstract description 9
- 239000011324 bead Substances 0.000 claims description 113
- 239000000243 solution Substances 0.000 claims description 48
- 239000006228 supernatant Substances 0.000 claims description 34
- 239000006185 dispersion Substances 0.000 claims description 31
- 238000011534 incubation Methods 0.000 claims description 31
- 102100025470 Carcinoembryonic antigen-related cell adhesion molecule 8 Human genes 0.000 claims description 25
- 101000914320 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 8 Proteins 0.000 claims description 25
- 241000283707 Capra Species 0.000 claims description 19
- 239000007853 buffer solution Substances 0.000 claims description 18
- 102100025222 CD63 antigen Human genes 0.000 claims description 17
- 101000934368 Homo sapiens CD63 antigen Proteins 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 17
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000003153 chemical reaction reagent Substances 0.000 claims description 15
- 239000012228 culture supernatant Substances 0.000 claims description 15
- 238000002156 mixing Methods 0.000 claims description 15
- 102000034287 fluorescent proteins Human genes 0.000 claims description 14
- 108091006047 fluorescent proteins Proteins 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- 230000009471 action Effects 0.000 claims description 13
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 210000004369 blood Anatomy 0.000 claims description 8
- 239000008280 blood Substances 0.000 claims description 8
- 238000010790 dilution Methods 0.000 claims description 7
- 239000012895 dilution Substances 0.000 claims description 7
- 238000005199 ultracentrifugation Methods 0.000 claims description 7
- 238000001085 differential centrifugation Methods 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 239000003822 epoxy resin Substances 0.000 claims description 3
- 239000004005 microsphere Substances 0.000 claims description 3
- 229920000647 polyepoxide Polymers 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000007885 magnetic separation Methods 0.000 abstract description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 37
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 30
- 229940098773 bovine serum albumin Drugs 0.000 description 30
- 238000005119 centrifugation Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 7
- 238000012512 characterization method Methods 0.000 description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 210000001772 blood platelet Anatomy 0.000 description 4
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 102100037904 CD9 antigen Human genes 0.000 description 3
- 101710163595 Chaperone protein DnaK Proteins 0.000 description 3
- 101710178376 Heat shock 70 kDa protein Proteins 0.000 description 3
- 101710152018 Heat shock cognate 70 kDa protein Proteins 0.000 description 3
- 101000738354 Homo sapiens CD9 antigen Proteins 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000000439 tumor marker Substances 0.000 description 3
- 238000000035 BCA protein assay Methods 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 108010056891 Calnexin Proteins 0.000 description 1
- 102000034342 Calnexin Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N2015/1486—Counting the particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids
- G01N2446/86—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids the coating being pre-functionalised for attaching immunoreagents, e.g. aminodextran
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Organic Chemistry (AREA)
- Dispersion Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a separation detection method and application of a neutrophil exosome, and relates to the technical field of exosome separation detection. The invention provides a separation and detection method of neutrophil exosomes, which can realize magnetic separation of human peripheral blood or serum neutrophil exosomes, can quickly detect the abundance of the neutrophil exosomes by a simple flow cytometer, and has higher sensitivity and specificity. The method is applied to the serum samples of the gastric cancer patients and normal people, finds that the abundance of the neutrophil exosomes in the serum of the gastric cancer patients is obviously higher than that of the normal people, and can use the neutrophil exosomes as potential serum tumor markers for early diagnosis of the gastric cancer.
Description
Technical Field
The invention belongs to the technical field of exosome separation and detection, and particularly relates to a separation and detection method and application of neutrophil exosomes.
Background
Gastric Cancer (GC) is a significant cause of cancer death in the world. Due to the lack of diagnostic markers with high sensitivity and specificity, patients are often diagnosed at an advanced stage, leading to a poorer prognosis. The currently commonly used gastric cancer serum tumor markers include AFP, CEA, CA199, CA125, CA724 and the like, and although the markers are helpful for early diagnosis of gastric cancer, the markers still have the problems of false positive, false negative and the like. Compared with the traditional serum tumor marker, the exosome shows a plurality of advantages in tumor diagnosis. Neutrophils are important players in the development of cancer and have been shown to promote carcinogenesis, tumor growth metastasis, angiogenesis and immunosuppression in a variety of cancers. However, neutrophil exosomes are rarely studied for tumor diagnosis. Therefore, the method for separating and detecting the neutrophil exosomes is constructed, the separation and detection of the neutrophil exosomes in the serum of the gastric cancer patients and normal people are realized, and the method has important significance for early diagnosis of the gastric cancer.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for separating and detecting neutrophil exosomes and an application thereof, which can achieve magnetic separation of neutrophil exosomes in different blood samples, can perform rapid detection of neutrophil exosome abundance by a simple flow cytometer, has high sensitivity and specificity, and can be used as a potential early diagnosis biomarker for gastric cancer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a separation and detection method of a neutrophil exosome, which comprises the following steps:
(1) mixing exosomes extracted from different blood samples with immunomagnetic bead dispersion liquid coated by a CD66b antibody for incubation, enriching the magnetic beads under the action of magnetic force, discarding supernatant, and washing the magnetic beads by using PBS buffer solution to obtain magnetic beads for capturing the exosomes of the neutrophils;
(2) utilizing the BSA solution to resuspend the magnetic beads for capturing the exosomes of the neutrophils in the step (1), dividing the magnetic beads after the BSA solution is resuspended into an experimental group and a control group, mixing and incubating the experimental group and a mouse anti-human CD63 primary antibody, adding the BSA solution with the same amount as the experimental group primary antibody into the control group, mixing and incubating, then enriching the magnetic beads under the action of magnetic force, discarding the supernatant, and utilizing a PBS buffer solution to wash the magnetic beads;
(3) mixing and incubating the washed magnetic beads in the step (2) with a goat anti-mouse IgG fluorescent secondary antibody marked by fluorescent protein by using a BSA solution for resuspension, enriching the magnetic beads under the action of magnetic force, discarding the supernatant, dispersing the magnetic beads in a PBS buffer solution, and performing flow detection;
(4) the abundance of neutrophil exosomes is indicated as the relative fluorescence intensity of the experimental versus the control group.
Preferably, the blood sample of step (1) comprises peripheral blood and/or serum.
Preferably, the method for extracting exosomes from peripheral blood neutrophil culture supernatant comprises: separating and extracting human peripheral blood neutrophils, and collecting a neutrophil culture supernatant; concentrating by using a 100kDa MWCO ultrafiltration centrifugal tube after differential centrifugation, and extracting peripheral blood neutrophil culture supernatant exosomes by an ultracentrifugation method;
a method of extracting exosomes from serum, comprising: removing platelets and large molecular weight proteins in the serum sample, sucking upper layer transparent serum, diluting with PBS buffer solution, centrifuging at 100000g for 3h, and precipitating to obtain serum exosome.
Preferably, the preparation method of the CD66b antibody-coated immunomagnetic bead dispersion in step (1) comprises mixing rabbit anti-human CD66b antibody with superparamagnetic antibody
And performing sufficient rotary incubation on the M-270 epoxy resin microspheres to form CD66b antibody-coated immunomagnetic bead dispersion.
Preferably, the ratio of the rabbit anti-human CD66b antibody to the magnetic beads is 6 μ g antibody/1 mg magnetic beads; the rotary incubation comprises incubation at 37 ℃ for 18 h; the concentration of the immunomagnetic bead dispersion liquid is 5-15 mg/mL.
Preferably, the mass-to-volume ratio of the exosomes in the step (1) to the CD66b antibody-coated immunomagnetic bead dispersion is 2 μ g: 15 mu L of the solution;
the mixed incubation included incubation at 4 ℃ for 16 h.
Preferably, the volume percentage of the BSA solution in step (2) is 2%;
the volume ratio of the BSA solution to the mouse anti-human CD63 primary antibody is 50: 1, and the dilution ratio of the mouse anti-human CD63 primary antibody is 1: 50.
preferably, the mass percentage of the BSA solution in the step (3) is 2%;
the volume ratio of the BSA solution to the goat anti-mouse IgG fluorescent secondary antibody marked by the fluorescent protein is 100: 1, and the dilution ratio of the fluorescent protein-labeled goat anti-mouse IgG fluorescent secondary antibody is 1: 100.
preferably, the fluorescent protein of step (3) comprises FITC.
The invention also provides application of the neutrophil exosome obtained by the separation detection method in preparation of an early diagnosis reagent for gastric cancer.
Has the advantages that: the invention provides a separation and detection method of neutrophil exosomes, which can realize the rapid magnetic separation of human peripheral blood or serum neutrophil exosomes, can perform rapid detection of the abundance of the neutrophil exosomes by a simple flow cytometer, and has higher sensitivity and specificity. The method is applied to the serum samples of the gastric cancer patients and normal people, finds that the abundance of the neutrophil exosomes in the serum of the gastric cancer patients is obviously higher than that of the normal people, and can use the neutrophil exosomes as a potential serum tumor marker for early diagnosis of the gastric cancer.
Drawings
FIG. 1 is a fluorescent microscope and flow chart of successful coupling of magnetic beads and antibody;
FIG. 2 is a fluorescent microscope representation of successful immunomagnetic bead separation of neutrophil exosomes;
FIG. 3 is a diagram showing the characterization and identification of the exosomes captured by immunomagnetic beads;
FIG. 4 is a diagram of a methodological evaluation of a neutrophil exosome detection method;
FIG. 5 is a flow chart of results of detecting the abundance of neutrophil exosomes from gastric cancer and normal human serum.
Detailed Description
The invention provides a separation and detection method of a neutrophil exosome, which comprises the following steps:
(1) mixing exosomes extracted from different blood samples with immunomagnetic bead dispersion liquid coated by a CD66b antibody for incubation, enriching the magnetic beads under the action of magnetic force, discarding supernatant, and washing the magnetic beads by using PBS buffer solution to obtain magnetic beads for capturing the exosomes of the neutrophils;
(2) utilizing the magnetic beads for capturing the neutrophil exosomes in the BSA solution resuspension step (1), dividing the magnetic beads subjected to the BSA solution resuspension into an experiment group and a control group, performing mixed incubation on the experiment group and a mouse anti-human CD63 primary antibody, adding a BSA solution with the same amount as the experiment group primary antibody into the control group, performing mixed incubation, then enriching the magnetic beads under the action of magnetic force, discarding a supernatant, and washing the magnetic beads by utilizing a PBS buffer solution;
(3) mixing and incubating the washed magnetic beads in the step (2) with a goat anti-mouse IgG fluorescent secondary antibody marked by fluorescent protein by using a BSA solution for resuspension, enriching the magnetic beads under the action of magnetic force, discarding the supernatant, dispersing the magnetic beads in a PBS buffer solution, and performing flow detection;
(4) the abundance of neutrophil exosomes is indicated as the relative fluorescence intensity of the experimental versus the control group.
The invention mixes and incubates exosomes extracted from different blood samples and immune magnetic bead dispersion liquid coated by CD66b antibody, enriches magnetic beads under the action of magnetic force, discards supernatant, and washes the magnetic beads by PBS buffer solution to obtain the magnetic beads for capturing the neutrophil exosomes. The blood sample according to the invention preferably comprises peripheral blood and/or serum.
The method for extracting the peripheral blood neutrophil culture supernatant exosomes preferably comprises the following steps: separating and extracting human peripheral blood neutrophils, and collecting a neutrophil culture supernatant; concentrating by using a 100kDa MWCO ultrafiltration centrifugal tube after differential centrifugation, and extracting peripheral blood neutrophil culture supernatant exosomes by an ultracentrifugation method. The method for extracting, separating and extracting the human peripheral blood neutrophils is not particularly limited, and the conventional separation and extraction method in the field can be used. The differential centrifugation according to the invention preferably comprises: centrifuging for 10min at 300g, 20min at 2000g and 30min at 10000 g. The exosome can be extracted by performing ultrafiltration concentration after differential centrifugation and then performing ultracentrifugation, wherein the ultracentrifugation comprises 100000g centrifugation for 70 min.
The method for extracting the serum exosomes preferably comprises the following steps: removing platelets and large molecular weight proteins in the serum sample, sucking upper layer transparent serum, diluting with PBS buffer solution, centrifuging at 100000g for 3h, and precipitating to obtain human serum total exosome. The platelets of the invention are preferably removed by centrifugation, which comprises centrifugation at 1500g for 10 min. The present invention preferably removes large molecular weight proteins by centrifugation at 12000g for 20 min. The present invention is preferably carried out at 4 ℃ when the centrifugation is carried out.
The preparation method of the CD66b antibody-coated immunomagnetic bead dispersion liquid preferably comprises the steps of mixing a rabbit anti-human CD66b antibody with superparamagnetic particles
And performing sufficient rotary incubation on the M-270 epoxy resin microspheres to form CD66b antibody-coated immunomagnetic bead dispersion. The present invention preferably utilizes a kit for the preparation of the dispersion, preferably a magnetic bead-antibody coupling kit (Thermo Fisher corporation, cat. No. 14311D). In the preparation of the dispersion, the ratio of the rabbit anti-human CD66b antibody to the magnetic beads is preferably 6 μ g antibody/1 mg magnetic beads; the rotary incubation preferably comprises incubation at 37 ℃ for 18 h; the concentration of the immunomagnetic bead dispersion is preferably 10 mg/mL. The invention is not limited to other parameters of the rotary incubation, and can be carried out by ensuring sufficient mixing, for example, M is used in the examplesACS vortex mixer.
The dispersion liquid and the supernatant of the human peripheral blood neutrophil granulocyte culture or the serum exosome are mixed and incubated, and the mass-volume ratio of the supernatant of the human peripheral blood neutrophil culture or the serum exosome to the dispersion liquid is preferably 2 mu g: 15 mu L of the solution; the mixed incubation preferably comprises incubation at 4 ℃ for 16 h. In a specific embodiment of the present invention, it is preferable to mix and incubate the human peripheral blood neutrophil culture supernatant or serum exosome in an EP tube by adding the above dispersion and diluting the solution to a final volume of 500. mu.L with PBS buffer.
Preferably, the magnetic beads are enriched in the incubation liquid under the action of magnetic force, the supernatant is discarded, and the magnetic beads are washed by PBS buffer solution to obtain the magnetic beads for capturing the exosomes of the neutrophils. According to the invention, the magnetic beads are preferably enriched on a magnetic frame, the supernatant is discarded after the magnetic beads are enriched on the tube wall, and the magnetic beads with the captured neutrophil exosomes are obtained after washing with PBS for 2 times. In the invention, for the magnetic beads with the captured neutrophil exosomes, 3.75mg/mL glycine solution and 1mol/L TBS eluent can be used for eluting the neutrophil exosomes from the surfaces of the magnetic beads, and the supernatant is taken after magnetic separation to obtain the corresponding neutrophil exosome suspension.
After obtaining the magnetic beads for capturing the exosomes of the neutrophil granulocytes, the magnetic beads for capturing the exosomes of the neutrophil granulocytes are resuspended by using a BSA solution, the magnetic beads after the resuspension of the BSA solution are divided into an experimental group and a control group, the experimental group is mixed and incubated with a mouse anti-human CD63 primary antibody, the control group is added with the BSA solution which is equal to the experimental group primary antibody for mixing and incubation, then the magnetic beads are enriched under the action of magnetic force, the supernatant is discarded, and the magnetic beads are washed by using a PBS buffer solution. The volume percentage of the BSA solution of the invention is preferably 2%; and the volume ratio of the BSA solution to the mouse anti-human CD63 primary antibody is preferably 50: 1, and the dilution ratio of the mouse anti-human CD63 primary antibody is preferably 1: 50. the mixed incubation is preferably carried out for 1h under the room temperature condition (18-22 ℃) in a rotating way. According to the invention, the EP tube after mixed incubation is preferably placed on a magnetic frame, the supernatant is discarded after the magnetic beads are enriched on the tube wall, and the washing is carried out for 2 times by PBS.
After the washed magnetic beads are obtained, the washed magnetic beads are resuspended by using BSA solution, mixed and incubated with goat anti-mouse IgG fluorescent secondary antibody marked by fluorescent protein, the magnetic beads are enriched under the action of magnetic force, the supernatant is discarded, and the magnetic beads are dispersed in PBS buffer solution for flow detection. The invention aims at the experimental group and the control group, and needs to add a goat anti-mouse IgG fluorescent secondary antibody marked by fluorescent protein, wherein the fluorescent protein preferably comprises FITC. The mass percentage content of the BSA solution is preferably 2%; the volume ratio of the BSA solution to the goat anti-mouse IgG fluorescent secondary antibody marked by the fluorescent protein is preferably 100: 1, and the dilution ratio of the fluorescent protein-labeled goat anti-mouse IgG fluorescent secondary antibody is preferably 1: 100. the mixed incubation of the invention is preferably rotary incubation for 1h at room temperature. After the mixed incubation, preferably placing an EP tube on a magnetic frame, discarding supernatant after magnetic beads are enriched on the tube wall, and washing with PBS for 2 times; the magnetic beads were dispersed in 400. mu.L of PBS buffer for flow assay. The method of the present invention for flow detection is not particularly limited, and conventional methods in the art may be used.
After the flow assay, the present invention indicates the abundance of neutrophil exosomes as the relative fluorescence intensity of the experimental versus the control group.
By utilizing the separation detection method, the relative fluorescence intensity of FITC of the gastric cancer patient is obviously higher than that of a normal person through verification, namely the abundance of the neutrophil exosomes in serum of the gastric cancer patient is obviously higher than that of the normal person, and the FITC can be used as a potential serum tumor marker for early diagnosis of the gastric cancer.
The invention also provides application of the human serum neutrophil exosome obtained by the separation detection method in preparation of an early diagnosis reagent for gastric cancer.
The following examples are provided to illustrate the method and application of the present invention for the isolation and detection of human neutrophil exosome, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation and characterization of immunomagnetic bead dispersion
Preparing reagents by using immunomagnetic beads: magnetic bead-antibody coupling kit (Thermo Fisher corporation, cat # 14311D), vortex mixer (MACS corporation), magnetic frame (Millipore corporation, usa), rabbit anti-human CD66b antibody (abcam corporation), FITC-labeled goat anti-rabbit IgG fluorescent secondary antibody (Biolegend), carbon dioxide incubator (Forma corporation), inverted microscope (Nikon corporation, japan).
The implementation steps are as follows:
preparing an immunomagnetic bead dispersion liquid: weighing 2mg of magnetic bead powder, washing with 1mL of C1 reagent, and blowing and mixing. The EP tube is placed on a magnetic frame for 1min, and the supernatant is discarded after the magnetic beads adhere to the wall. Adding 100 mu L C1 reagent and 12 mu g CD66b antibody, adding 110 mu L C2 reagent after the mixture is blown and mixed evenly, placing an EP tube in a vortex mixer, and carrying out rotary incubation for 18h at 37 ℃. Discard the supernatant, add 400 μ L of HB reagent, wash, blow and mix well. Discard the supernatant, add 400 μ L LB reagent to wash, blow and mix well. Discard the supernatant, add 400 μ L SB reagent to wash, blow and mix well, repeat the operation 2 times. Discard the supernatant, add 400 μ L SB reagent to wash, blow and mix well, place EP tube in vortex mixer, rotate for 15min at room temperature. The supernatant was discarded, and 200. mu.L of SB reagent was added to obtain a CD66b antibody-coated immunomagnetic bead dispersion at a concentration of 10mg/mL, which was stored at 4 ℃.
Characterization of immunomagnetic beads: mu.L of immunomagnetic bead dispersion (6. mu.L of non-antibody-coupled naked magnetic bead dispersion as a control group) was taken, washed with PBS, and then resuspended in 200. mu.L of PBS. Adding 1 μ L FITC labeled goat anti-rabbit IgG fluorescent secondary antibody, and incubating for 30min on ice in dark. The EP tube was placed on a magnetic frame for 1min, the supernatant was discarded after the magnetic beads were attached to the wall, washed 3 times with PBS, and resuspended in 100. mu.L of PBS. 10 μ L of the dispersion was dropped on a glass slide, covered with a cover glass, and observed with an inverted fluorescence microscope. Another 10. mu.L of the dispersion was added to 200. mu.L of PBS for flow detection. In FIG. 1, A shows that the surface of the magnetic beads of the experimental group is coated with the CD66b antibody, which emits strong green fluorescence, while the control group does not. Flow results in fig. 1, B shows that the fluorescence intensity of FITC in the experimental group is significantly enhanced compared to the control group, indicating a very high antibody coating efficiency.
Example 2
Neutrophil exosome isolation, characterization and identification
Separating and identifying the reagent: PolymorphPrep isolate, high speed centrifuge (Eppendorf), 0.22 μm sterile filter (Millipore corporation, usa), ultracentrifuge (beckman corporation, usa), vortex mixer (MACS corporation), magnetic rack (Millipore corporation, usa), rabbit anti-human CD9 antibody (CST corporation), mouse anti-human CD63 antibody (abcam), mouse anti-human HSP70 antibody (CST corporation), rabbit anti-human CD66b antibody (abcam), horseradish peroxidase (HRP) -labeled goat anti-rabbit/anti-mouse IgG secondary antibody (beijing corporation, century), HRP chemiluminescent substrate, NTA analyzer (Zetaview), transmission electron microscope (FEI Tecnai 12, Philips corporation).
The implementation steps are as follows:
1) separating and extracting human peripheral blood neutrophils with polymorphocyte leukocyte separation solution (Polymorphprep separation solution), culturing at 37 deg.C for 48h, collecting the culture supernatant of neutrophils, and performing differential centrifugation (300g centrifugation for 10min, 2000g centrifugation for 20min, 10000g centrifugation for 30 min). Concentrating the supernatant to 40mL by a 100kDa MWCO ultrafiltration centrifugal tube, and centrifuging for 70min by an ultracentrifuge at 100000g to obtain the exosome of the human peripheral blood neutrophil culture supernatant. And extracting the exosome protein to perform BCA protein assay, and indicating the exosome concentration and content by protein concentration.
2) Neutrophil exosome isolation: 2 mu.g of the exosome of the human peripheral blood neutrophil culture supernatant is taken in an EP tube, 15 mu.L of immunomagnetic bead dispersion liquid is added, the final volume of the solution is diluted to 500 mu.L by PBS buffer solution, and the solution is incubated for 16h in a rotating way at 4 ℃. The EP tube is placed in a magnetic frame, and the supernatant is discarded after the magnetic beads are enriched on the tube wall. The beads with the neutrophil exosomes captured were obtained by washing 2 times with PBS.
3) Exosome capture characterization: mu.L of magnetic bead dispersion liquid with the captured neutrophil exosomes was taken, 50. mu.L of 2% BSA was added for blocking for 2h, the EP tube was placed on a magnetic frame, and 100. mu.L of LPBS was washed 3 times. DIO fluorescent dye (1: 25) was added, and the mixture was incubated for 30min on ice in the dark, and 50. mu.L of 2% BSA was used to stop the reaction, and the EP tube was placed on a magnetic frame, washed 3 times with 100. mu.L of PBS, and the magnetic beads were resuspended in 50. mu.L of PBS. 10 μ L of the solution was dropped on a glass slide, covered with a cover glass, and observed with an inverted fluorescence microscope. FIG. 2 shows that the surface of the magnetic beads is enriched with a variable number of membrane vesicles.
4) Detecting the surface marker protein of the neutrophil exosome by western blot: and (3) directly and fully cracking the neutrophil exosomes captured by the magnetic beads by using a protein lysate, centrifuging for 15min at 12000g, discarding the magnetic beads at the bottom of the tube, and absorbing the upper layer of transparent protein lysate. 1/3 volumes of 4 XSDS loading buffer were added and boiled for 8 min. Loading the sample with 150 μ g total protein, electrophoresis, transferring the protein to PVDF membrane by electrotransfer (350mA, 2h), blocking with 25g/L skimmed milk for 1h, incubating overnight at 4 deg.C with rabbit anti-human CD9 antibody, mouse anti-human CD63 antibody and mouse anti-human HSP70 antibody (1: 500), washing the membrane 3 times with TBS/0.5% Tween 20 the next day, incubating with HRP-labeled goat anti-rabbit/anti-mouse IgG secondary antibody at room temperature for 2h, washing the membrane 3 times with TBS/0.5% Tween 20, adding premixed HRP chemiluminescence substrate, and exposing to detection with chemiluminescence gel imaging system. As shown in fig. 3A, the magnetic bead-captured neutrophil exosomes highly expressed CD9, CD63, HSP70, CD66b, and did not express Calnexin.
5) Transmission electron microscopy and NTA characterization of exosome morphological characteristics: sucking 20 mu L of magnetic bead dispersion liquid capturing human peripheral blood neutrophil exosomes, dripping the mixture on a sample-carrying copper net after mixing uniformly, standing for 5min, sucking residual liquid by using filter paper, reversely buckling the copper net on 30g/L phosphotungstic acid (pH 6.8) liquid drops, carrying out negative dyeing at room temperature for 5min, drying under an incandescent lamp, and observing and taking a picture under a transmission electron microscope. FIG. 3B shows that exosomes are adsorbed on the surface of the magnetic bead, and the exosomes have a typical vesicle-like structure; FIG. 3C shows exosomes after glycine/TBS elution. FIG. 3D shows that the particle size of the neutrophil exosomes after elution by NTA detection is about 140 nm.
Example 3
Neutrophil exosome abundance detection and methodological evaluation
Reagent: PolymorphpRep separation, high speed centrifuge (Eppendorf), ultracentrifuge (Beckmann, USA), vortex Mixer (MACS), magnetic rack (Millipore, USA), mouse anti-human CD63 antibody (abcam), FITC-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend), carbon dioxide incubator (Forma, USA), and flow cytometer (Beckmann, USA).
Magnetic beads having neutrophil exosomes captured therein were obtained in the same manner as in example 2. 50 μ L of 2% BSA solution the magnetic beads with captured neutrophil exosomes were resuspended, 1 μ L of mouse anti-human CD63 primary antibody (control added with the same amount of BSA solution as the experimental primary antibody) was added, and incubated for 1h at room temperature with rotation. The EP tube is placed on a magnetic frame, supernatant is discarded after magnetic beads are enriched on the tube wall, and PBS is used for washing for 2 times. mu.L of 2% BSA solution was used to resuspend the beads, and 1. mu.L of FITC-labeled goat anti-mouse IgG fluorescent secondary antibody was added and incubated at room temperature for 1h with rotation. The EP tube is placed on a magnetic frame, the supernatant is discarded after the magnetic beads are enriched on the side wall of the tube, and the tube is washed for 2 times by PBS. The magnetic beads were dispersed in 400. mu.L PBS buffer for flow detection, and the abundance of neutrophil exosomes was indicated by the change in FITC fluorescence intensity (relative fluorescence intensity) of the experimental versus the control group.
And (3) sensitivity detection: the method is implemented by taking human peripheral blood neutrophil culture supernatant exosomes extracted by an ultracentrifugation method as a standard substance, performing PBS (phosphate buffer solution) gradient dilution, drawing a standard curve by using the relative fluorescence intensity of FITC and the lg value of exosome concentration, and calculating a linear range and a detection limit. FIG. 4A shows a linear range of 10 6 -10 12 particles/mL, detection sensitivity 1.1X 10 5 particles/mL。
And (3) specificity detection: similar to the extraction method of the exosome from the human peripheral blood neutrophil culture supernatant, the human peripheral blood mononuclear cell separation solution and the ultracentrifugation method are used for extracting the human peripheral blood lymphocyte exosome which does not express CD66b as a negative control, and the relative fluorescence intensities of the neutrophil exosome and the lymphocyte exosome under the same conditions are compared. Figure 4B shows no significant fluorescence enhancement of human peripheral blood lymphocyte exosomes, indicating that the method has good specificity.
Example 4
Detection and application of neutrophil exosome
Detection reagent: high speed centrifuge (Eppendorf), Bovine Serum Albumin (BSA), murine anti-human CD63 antibody (abcam), FITC-labeled goat anti-mouse IgG fluorescent secondary antibody (Biolegend), vortex Mixer (MACS), magnetic frame (Millipore, USA), flow cytometer (Beckmann, USA).
The implementation steps are as follows:
1) collecting human serum samples of gastric cancer patients and normal persons, centrifuging at 1500g for 10min to remove blood platelets, and centrifuging at 12000g for 20min to remove macromolecular weight proteins. The supernatant clear serum was aspirated using a 1mL syringe, diluted to 40mL with PBS buffer and filtered through a 0.22 μm sterile filter. Centrifuging at 100000g for 3h at 4 deg.C to obtain precipitate as human serum exosome. Serum exosome proteins were extracted for BCA protein assay, and exosome concentration and content were indicated as protein concentration.
2) 2 mu g of gastric cancer/normal human serum exosome is taken to be put in an EP tube, 15 mu L of immunomagnetic bead dispersion liquid is respectively added, the final volume of the solution is diluted to 500 mu L by PBS buffer solution, and the solution is incubated for 16h in a rotating way at 4 ℃. The EP tube is placed in a magnetic frame, and the supernatant is discarded after the magnetic beads are enriched on the tube wall. The beads were washed 2 times with PBS to obtain magnetic beads with neutrophil exosomes captured.
3)50 μ L of 2% BSA solution the magnetic beads with captured neutrophil exosomes were resuspended, 1 μ L of mouse anti-human CD63 primary antibody (control added with the same amount of BSA solution as the experimental primary antibody) was added, and incubated for 1h at room temperature with rotation. The EP tube is placed on a magnetic frame, supernatant is discarded after magnetic beads are enriched on the tube wall, and PBS is used for washing for 2 times. mu.L of 2% BSA solution was used to resuspend the beads, and 1. mu.L of FITC-labeled goat anti-mouse IgG fluorescent secondary antibody was added and incubated at room temperature for 1h with rotation. The EP tube is placed on a magnetic frame, the supernatant is discarded after the magnetic beads are enriched on the side wall of the tube, and the tube is washed for 2 times by PBS. The magnetic beads were dispersed in 400. mu.L of PBS buffer for flow detection, and the abundance of neutrophil exosomes was indicated by the change in FITC fluorescence intensity (relative fluorescence intensity) of the experimental group versus the control group. Fig. 5 shows that the relative fluorescence intensity of FITC of the gastric cancer patient is significantly higher than that of a normal person, namely the abundance of the neutrophil exosomes in serum of the gastric cancer patient is significantly higher than that of the normal person.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A method for separating and detecting neutrophil exosomes is characterized by comprising the following steps: (1) mixing exosomes extracted from different blood samples with immunomagnetic bead dispersion liquid coated by a CD66b antibody for incubation, enriching the magnetic beads under the action of magnetic force, discarding supernatant, and washing the magnetic beads by using PBS buffer solution to obtain magnetic beads for capturing the exosomes of the neutrophils;
(2) utilizing the magnetic beads for capturing the neutrophil exosomes in the BSA solution resuspension step (1), dividing the magnetic beads subjected to the BSA solution resuspension into an experiment group and a control group, performing mixed incubation on the experiment group and a mouse anti-human CD63 primary antibody, adding a BSA solution with the same amount as the experiment group primary antibody into the control group, performing mixed incubation, then enriching the magnetic beads under the action of magnetic force, discarding a supernatant, and washing the magnetic beads by utilizing a PBS buffer solution;
(3) mixing and incubating the washed magnetic beads in the step (2) with a goat anti-mouse IgG fluorescent secondary antibody marked by fluorescent protein by using a BSA solution for resuspension, enriching the magnetic beads under the action of magnetic force, discarding the supernatant, dispersing the magnetic beads in a PBS buffer solution, and performing flow detection;
(4) the abundance of neutrophil exosomes is indicated as the relative fluorescence intensity of the experimental versus the control group.
2. The separation detection method according to claim 1, wherein the blood sample in step (1) comprises peripheral blood and/or serum.
3. The separation detection method according to claim 2, wherein the method for extracting exosomes from peripheral blood neutrophil culture supernatant comprises: separating and extracting human peripheral blood neutrophils, and collecting a neutrophil culture supernatant; concentrating by using a 100kDaMWCO ultrafiltration centrifugal tube after differential centrifugation, and extracting peripheral blood neutrophil culture supernatant exosomes by an ultracentrifugation method;
a method of extracting exosomes from serum, comprising: removing platelets and macromolecular proteins in the serum sample, then sucking upper layer transparent serum, diluting with PBS buffer solution, centrifuging at 100000g for 3h, and precipitating to obtain serum exosome.
4. The separation detection method according to claim 1, wherein the preparation method of the CD66b antibody-coated immunomagnetic bead dispersion in step (1) comprises mixing rabbit anti-human CD66b antibody with superparamagnetic property
And performing sufficient rotary incubation on the M-270 epoxy resin microspheres to form CD66b antibody-coated immunomagnetic bead dispersion.
5. The separation and detection method of claim 4, wherein the ratio of the rabbit anti-human CD66b antibody to the magnetic beads is 6 μ g antibody/1 mg magnetic beads; the rotary incubation comprises incubation at 37 ℃ for 18 h; the concentration of the immunomagnetic bead dispersion liquid is 5-15 mg/mL.
6. The separation detection method according to claim 1, wherein the mass-to-volume ratio of the exosomes in step (1) to the CD66b antibody-coated immunomagnetic bead dispersion is 2 μ g: 15 mu L of the solution;
the mixed incubation included incubation at 4 ℃ for 16 h.
7. The separation detection method of claim 1, wherein the volume percentage of the BSA solution in step (2) is 2%;
the volume ratio of the BSA solution to the mouse anti-human CD63 primary antibody is 50: 1, and the dilution ratio of the mouse anti-human CD63 primary antibody is 1: 50.
8. the separation and detection method according to claim 1, wherein the BSA solution in step (3) is 2% by mass;
the volume ratio of the BSA solution to the goat anti-mouse IgG fluorescent secondary antibody marked by the fluorescent protein is 100: 1, and the dilution ratio of the fluorescent protein-labeled goat anti-mouse IgG fluorescent secondary antibody is 1: 100.
9. the separation detection method according to claim 1, wherein the fluorescent protein of step (3) comprises FITC.
10. Use of the neutrophil exosome obtained by the isolation and detection method according to any one of claims 1 to 9 in preparation of an early diagnosis reagent for gastric cancer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210543349.4A CN114958743A (en) | 2022-05-18 | 2022-05-18 | Separation detection method and application of neutrophil exosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210543349.4A CN114958743A (en) | 2022-05-18 | 2022-05-18 | Separation detection method and application of neutrophil exosome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114958743A true CN114958743A (en) | 2022-08-30 |
Family
ID=82985960
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210543349.4A Pending CN114958743A (en) | 2022-05-18 | 2022-05-18 | Separation detection method and application of neutrophil exosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114958743A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115197912A (en) * | 2021-04-08 | 2022-10-18 | 南京大学 | Application of TREM2 protein antibody coated magnetic beads in extraction of microglial cell-derived exosomes in serum |
| CN116718536A (en) * | 2023-04-27 | 2023-09-08 | 河北意和医学检验实验室有限公司 | Quantitative detection method and kit for active exosomes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
| CN110699320A (en) * | 2019-11-26 | 2020-01-17 | 江苏大学 | Human peripheral blood neutrophil exosome and extraction method and application thereof |
| CN113186161A (en) * | 2021-03-24 | 2021-07-30 | 南京医科大学 | Method for quickly separating ultra-high purity human blood neutrophils |
| CN114231523A (en) * | 2022-01-12 | 2022-03-25 | 西安交通大学 | Exosome separation method based on CD63 antibody coupling magnetic beads |
-
2022
- 2022-05-18 CN CN202210543349.4A patent/CN114958743A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105388055A (en) * | 2015-12-11 | 2016-03-09 | 浙江省肿瘤医院 | Method for separating tumor cell derived-exosomes from urine |
| CN110699320A (en) * | 2019-11-26 | 2020-01-17 | 江苏大学 | Human peripheral blood neutrophil exosome and extraction method and application thereof |
| CN113186161A (en) * | 2021-03-24 | 2021-07-30 | 南京医科大学 | Method for quickly separating ultra-high purity human blood neutrophils |
| CN114231523A (en) * | 2022-01-12 | 2022-03-25 | 西安交通大学 | Exosome separation method based on CD63 antibody coupling magnetic beads |
Non-Patent Citations (4)
| Title |
|---|
| KR GENSCHMER等: "Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung", 《CELL》, vol. 176, no. 1, 31 January 2019 (2019-01-31), pages 6 - 7 * |
| 庞伟等: "免疫磁珠法及密度梯度离心法分离纯化人外周血中性粒细胞的比较", 《军医进修学院学报》, vol. 31, no. 11, 31 December 2010 (2010-12-31), pages 1090 - 1092 * |
| 张贺龙等: "外泌体在胃癌中作用的研究进展", 《肿瘤》, vol. 39, 31 March 2019 (2019-03-31), pages 215 - 222 * |
| 赵磊: "胃癌细胞源性外泌体对正常人中性粒细胞的趋化和极化作用及其机制", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, no. 8, 15 August 2020 (2020-08-15), pages 1 - 96 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115197912A (en) * | 2021-04-08 | 2022-10-18 | 南京大学 | Application of TREM2 protein antibody coated magnetic beads in extraction of microglial cell-derived exosomes in serum |
| CN116718536A (en) * | 2023-04-27 | 2023-09-08 | 河北意和医学检验实验室有限公司 | Quantitative detection method and kit for active exosomes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114958743A (en) | Separation detection method and application of neutrophil exosome | |
| TWI577389B (en) | Methods and kits for the detection of circulating tumor cells in pancreatic patients using polyspecific capture and cocktail detection reagents | |
| CN106635995B (en) | Negative enrichment method for circulating tumor cells | |
| JP5223676B2 (en) | Magnetic immunodiagnostic method, especially for the demonstration of the presence of blood group antibody / antigen complexes | |
| CN107356744B (en) | Method for sorting and/or enriching circulating tumor cells and kit thereof | |
| CN111826351B (en) | Magnetic red blood cell cluster for enriching circulating tumor cells based on magnetic separation method | |
| WO2018172384A1 (en) | Methods and kits for exosome isolation and quantification | |
| CN111826349B (en) | Erythrocyte cluster based on size filtration method for enriching circulating tumor cells | |
| CN106701684A (en) | High-efficiency circulating tumor cell enrichment method | |
| CN109161530B (en) | Separation method of circulating tumor cells based on phenylboronic acid | |
| CN114231523A (en) | Exosome separation method based on CD63 antibody coupling magnetic beads | |
| CN112034170A (en) | Reagent card for quantitatively detecting helicobacter pylori antibody by fluorescence chromatography and detection method | |
| CN215856071U (en) | Centrifugal tube for separating extracellular vesicles and kit for extracting extracellular vesicles | |
| CN107655879B (en) | For detecting the micro-fluidic chemiluminescence detection system of the magnetic particle of sexual gland series | |
| JP7376021B2 (en) | Method for separating and analyzing microvesicles from human blood | |
| CN115558629A (en) | Exosome extraction affinity magnetic bead and extraction kit thereof | |
| CN111549091A (en) | Test method for basophil activation and degranulation | |
| CN117330481B (en) | Flow detection method for exosomes and application thereof | |
| CN116731962B (en) | Kit and method for separating red blood cells from whole blood | |
| CN111724954B (en) | Graphene oxide magnetic bead, antibody-coupled graphene oxide magnetic bead and application of graphene oxide magnetic bead in cell sorting | |
| CN112852725A (en) | Preparation method and application for extracting and purifying stem cell exosome by using protein cross-linked nano affinity microspheres | |
| CN111175488A (en) | Method for detecting specificity of platelet antibody by using flow cytometry and detection kit | |
| CN110927369A (en) | Kit for identifying circulating tumor cells by combining TCPP (TCPP) with CEP (cytokine induced apoptosis protein) probe and application | |
| Wu et al. | Positively charged magnetic nanoparticles for capture of circulating tumor cells from clinical blood samples | |
| CN110133294A (en) | A kind of catching method and detection method of peripheral blood CTC |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |