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Method for detecting exosome GPCI protein

An exosome and protein technology, applied in the field of biotechnology detection, can solve the problems of a large number of samples, unsuitability for clinical promotion, and high price of flow cytometry, and achieve the effect of small sample size, low equipment cost, and high sensitivity

Active Publication Date: 2016-09-28
领航医学科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Exosome GPC1 protein detection methods currently include traditional molecular assays (such as Western blot, ELISA) and flow cytometry. Traditional molecular assays require a large number of samples and cannot be implemented clinically. Flow cytometry is expensive and inappropriate. in clinical promotion

Method used

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  • Method for detecting exosome GPCI protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Magnetic force-electrochemical sensor device

[0040] (1) Device introduction

[0041] The device has 8 independent channels ( figure 1 A). Each channel has a potentiostat capable of measuring current in the ±7.5µA range. The input signal is conditioned by a low-pass filter (cutoff frequency 5Hz) to suppress high-frequency noise. Eight potentiostats are connected to a digital-to-analog converter for potential control, an analog-to-digital converter for signal digitization, a multiplexer for channel selection, and a microcontroller unit for system operation ( figure 1 B). The card edge connector is used to connect the electrode box. The whole magnetic frame has eight cylindrical magnets placed under the electrode box. These magnets can enrich the magnetic beads to the sensor surface. The device can quickly read the data from each channel (each channel Time-consuming 50ms), all data can be detected and analyzed by specially designed software.

[0042] (...

Embodiment 2

[0044] Example 2 Preparation of Immunomagnetic Beads

[0045] 5 mg of magnetic beads coated with epoxy groups (M-270 epoxy resin magnetic beads, diameter 2.7 μm, purchased from Invitrogen Company) were added to 1 mL of 0.1 M sodium phosphate solution, and stirred at room temperature for 10 min. Apply an external magnetic field to separate the magnetic beads, discard the solution, then add 100 μL of 0.1 M sodium phosphate solution, add 100 μg of anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody or anti-Flotillin-1 antibody (when necessary to prepare double antibody immunomagnetic For beads, add 50 μg of each of the two antibodies; when it is necessary to prepare three-antibody immunomagnetic beads, add 33.3 μg of each of the three antibodies) and mix well (add IgG to the immunomagnetic beads as a negative control and mix well), add 100 μL Ammonium sulfate solution at a concentration of 3M, and the mixture was incubated overnight at 4°C with slow tilting rotation. The m...

Embodiment 3

[0046] Example 3 Biotin Labeling of Anti-GPC1 Antibody

[0047] Take 100 μL of anti-GPC1 antibody with a concentration of 1 mg / mL, dilute it 10 times with PBS, then add 5 μL of sulfo-NHS-biotin solution with a concentration of 10 mM, and incubate at room temperature for 2 h. After the reaction is completed, pass through a Zeba desalting column (7K MWCO (purchased from Thermo Scientific) to remove excess sulfo-NHS-biotin to obtain 80 μg of biotin-labeled anti-GPC1 antibody. The biotin-labeled anti-GPC1 antibody was stored at 4°C for future use.

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PUM

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Abstract

The invention discloses a method for detecting an exosome GPC1 protein. The method comprises 1, taking a sample to be detected, 2, adding exosome specific antibody-modified immunomagnetic beads into the sample to be detected, 3, orderly adding a biotin-labeled anti-GPC1 antibody, a streptavidin-labeled horse radish peroxidase and a horse radish peroxidase substrate into the immunomagnetic beads and 4, carrying out detection through a magnetic-electrochemical sensor device. The method has advantages of an immunomagnetic bead technology and an electrochemical sensor detection technology and has high sensitivity and a small sample amount. The detection device has a low cost, a small volume and high flux, specifically detects a specific exosome for GPC1 protein expression and has an important meaning for cancer early stage diagnosis and treatment detection.

Description

technical field [0001] The invention relates to the field of biotechnology detection, in particular to a method for detecting exosome GPC1 protein. Background technique [0002] Exosomes are extracellular nanoscale vesicles formed by cells through a series of regulatory processes such as "endocytosis-fusion-efflux", with a diameter of about 50-150nm. Exosomes can carry proteins, transport RNA, and play an important role in intercellular material and information transduction. Exosomes may affect tumor progression by regulating immune function, promoting tumor angiogenesis and tumor metastasis, and acting directly on tumor cells. Exosomes can be used in the diagnosis of tumors. Tumor exosomes are exosomes secreted by tumor cells in the body that contain specific markers. The detection of tumor exosomes can provide accurate information for the detection of the entire tumor. Because exosomes are abundant in body fluids (such as serum, ascites, urine, and cerebrospinal fluid),...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531G01N27/26
CPCG01N27/26G01N33/531G01N33/689
Inventor 相双红宋小慧叶慧
Owner 领航医学科技(深圳)有限公司
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