Cynoglossus semilaevis derived exosome sandwich ELISA (Enzyme-linked Immuno Sorbent Assay) detection method and kit

A semi-smooth tongue sole and detection method technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems that are not suitable for detection, cannot reflect the particle size distribution and overall concentration of exosomes, and achieve reliable identification results. highly specific effect

Inactive Publication Date: 2018-11-20
TIANJIN BOHAI SEA FISHERIES RES INST
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The resolution of the transmission electron microscope can reach 0.1-0.2nm, and the observed object can be magnified to millions of times, which is suitable for the observation of the ultrastructure of the exosome double-layer capsule; however, the transmission electron microscope cannot reflect all the exosomes in the sample The particle size distribution and overall concentration of the particles; the particle size that can be measured by flow cytometry is above 150nm, which is not suitable for detecting free exosome particles

Method used

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  • Cynoglossus semilaevis derived exosome sandwich ELISA (Enzyme-linked Immuno Sorbent Assay) detection method and kit
  • Cynoglossus semilaevis derived exosome sandwich ELISA (Enzyme-linked Immuno Sorbent Assay) detection method and kit
  • Cynoglossus semilaevis derived exosome sandwich ELISA (Enzyme-linked Immuno Sorbent Assay) detection method and kit

Examples

Experimental program
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Effect test

Embodiment 1

[0038] An exosome sandwich ELISA method derived from tongue sole semen, comprising the following steps:

[0039] 1. Collection of semen samples

[0040] Collect 0.5 mL of semen from sexually mature male tongue sole in a centrifuge tube.

[0041] 2. Extraction of exosomes

[0042] (1) Transfer 0.5mL of the semen sample to a 1.5mLEP tube, place it at 4°C, centrifuge at 1200g for 15 minutes to remove sperm cells, and centrifuge at 15000g at 4°C for 20 minutes to remove small cell impurities and debris. μm filter membrane for pre-filtration, and the filtrate is filtered through a 0.22μm filter membrane;

[0043] (2) Extract exosomes from the filtered samples using the Total Exosome Isolation Kit;

[0044] (3) Collect the dissolved sample as exosome suspension, subpackage, and store at -80°C for later use;

[0045] 3. Identification of exosomes: Hitachi H600IV transmission electron microscope observation (see figure 1 ).

[0046] 4. Store the obtained exosomes for future use....

Embodiment 2

[0060] A sandwich ELISA method for identification of exosomes derived from the blood of tongue sole, comprising the following steps:

[0061] 1. Collection of blood samples

[0062] Collect 0.5 mL of blood from sexually mature male tongue sole in a centrifuge tube, and centrifuge to obtain serum.

[0063] 2. Extraction of exosomes

[0064] Exosomes were extracted from serum samples using the Total Exosome Isolation Kit to obtain an exosome suspension.

[0065] 3. Identification of exosomes: Hitachi H600IV transmission electron microscope observation.

[0066] 4. Store the obtained exosomes for future use.

[0067] 5. Carry out ELISA detection (see principle figure 2 ),

[0068] (1) Dilute the mouse monoclonal antibody against CD63 with carbonate buffer, the carbonate buffer is 0.05M, pH=9.6, and coat it on a 96-well microtiter plate, 100 μL / well, 4 After overnight incubation at ℃, carefully remove the supernatant;

[0069] (2) Add 300 μL / well of blocking solution conta...

Embodiment 3

[0080] A sandwich ELISA identification kit for exosomes derived from semi-smooth tongue sole semen, the kit includes an ELISA plate coated with a mouse monoclonal antibody against CD63, standard exosomes, and horseradish peroxide Enzyme-labeled anti-HSP90 antibody 1:2000-1:8000, sample diluent, washing solution, stop solution and chromogenic solution; the washing solution is 0.01M phosphate buffer containing 0.05% Tween-20, pH=7.2 -7.4; the chromogenic solution is one-component TMB, and the stop solution is 1M HCl.

[0081] The preparation method of described test kit:

[0082] 1. The preparation method of the microtiter plate coated with the mouse monoclonal antibody against CD63 is as follows: (1) dilute the mouse monoclonal antibody against CD63 with carbonate buffer (0.05M, pH=9.6) to A certain concentration (1-5μg / mL), coated on a 96-well microplate, 100μL / well, after overnight incubation at 4°C, carefully remove the supernatant;

[0083] (2) Add 100-300 μL / well of bloc...

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Abstract

The invention discloses a cynoglossus semilaevis derived exosome sandwich ELISA (Enzyme-linked Immuno Sorbent Assay) detection method and a kit and belongs to the technical field of fish biology, particularly the field of application of marine fish exosome markers. According to the method disclosed by the invention, an anti-CD63 antibody serves as an capture antibody, an anti-HSP90 antibody servesas a detection antibody (an enzyme-labelled antibody), and a double-antibody sandwich ELISA method for detecting the cynoglossus semilaevis derived exosome is developed. The invention further provides a kit capable of identifying and quantifying, used for identifying marker proteins of exosome in cynoglossus semilaevis seminal fluid. The kit has important reference significances for identifying exosome of samples derived from other body fluid of fishes as well as other marker proteins. The method disclosed by the invention is high in specificity, high in efficiency and simple and convenient to operate, does not need large instruments or equipment and expensive chemical reagents, and has popularization value.

Description

technical field [0001] The invention belongs to the field of fish biotechnology, in particular to the application field of ELISA identification of exosomes derived from tongue sole. Background technique [0002] The diameter of exosomes is 30-120nm, which is the smaller type of exocrine vesicles, widely exists in animal body fluids, and contains different biomolecules, such as lipids, proteins and nucleic acids. A variety of cells can secrete exosomes under normal and pathological conditions. Exosomes have a wide range of application research value, and have a wide range of applications in tumor research, pathological analysis, and biomarker applications. [0003] There are many methods for the identification of exosomes, commonly used are transmission electron microscope morphology identification, flow cytometry identification, particle size distribution identification, and identification based on western blot of exosome marker proteins. The resolution of the transmission...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/535G01N21/78
CPCG01N21/78G01N33/535G01N33/577G01N33/6854
Inventor 张博赵娜高燕贾磊刘克奉鲍宝龙车金远高磊
Owner TIANJIN BOHAI SEA FISHERIES RES INST
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