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Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead

A fetuin and influenza virus technology, applied in the field of biomedicine, can solve the problems of complex preparation process of monoclonal antibody and high price of monoclonal antibody, and achieve the effect of low cost and simple operation.

Inactive Publication Date: 2013-09-04
MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of the monoclonal antibody coated with magnetic beads in this method is complicated, and the commercialized monoclonal antibody is expensive, and a monoclonal antibody can only specifically bind to one subtype of influenza virus. Only one subtype of influenza virus can be enriched

Method used

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  • Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead
  • Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead
  • Method for enriching influenza virus through utilizing asialofetuin-containing magnetic bead

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] (1) Use a vortex to disperse the carboxylated magnetic microspheres by shaking for 10 minutes. The magnetic microspheres were washed twice with sterile pH 7.6 PBS, then resuspended in sterile pH 7.6 PBS, and stored at 4°C for future use.

[0025] (2) Weigh 0.01g magnetic microspheres (about 0.5×10 6 ), use a magnetic separation rack to separate the magnetic beads from the solution, and resuspend the separated magnetic microspheres with 5ml sterile pH 4.5 PBS. Add 0.2 g of EDC to the suspension and react at 4° C. for 15 minutes, then add 0.05 g of NHS and react at 4° C. for 4 hours. After the reaction, the magnetic beads were separated from the liquid, washed three times with sterile pH 7.6 PBS, resuspended in 1ml of sterile pH 7.6 PBS, and stored at 4°C. Use 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (English abbreviation: EDC) and N-hydroxysuccinimide (English abbreviation: NHS) to carboxylate the carboxyl group of the magnetic beads Activate to obtain magnetic...

Embodiment 2

[0029] (1) Use a vortex to disperse the carboxylated magnetic microspheres by shaking for 8 minutes. The magnetic microspheres were washed twice with sterile pH 7.6 PBS, then resuspended in sterile pH 7.6 PBS, and stored at 4°C for future use.

[0030] (2) Weigh 0.01g magnetic microspheres (about 0.5×10 6 ), use a magnetic separation rack to separate the magnetic beads from the solution, and resuspend the separated magnetic microspheres with 5ml sterile pH 4.2 PBS. Add 0.22g of EDC to the suspension and react at 4°C for 15 minutes, then add 0.055g of NHS and react at 4°C for 4 hours. After the reaction, the magnetic beads were separated from the liquid, washed three times with sterile pH 7.6 PBS, resuspended in 1ml of sterile pH 7.6 PBS, and stored at 4°C. Use 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (English abbreviation: EDC) and N-hydroxysuccinimide (English abbreviation: NHS) to carboxylate the carboxyl group of the magnetic beads Activate to obtain magnetic bead...

Embodiment 3

[0034] (1) Use a vortex to disperse the carboxylated magnetic microspheres by shaking for 12 minutes. The magnetic microspheres were washed twice with sterile pH 7.6 PBS, then resuspended in sterile pH 7.6 PBS, and stored at 4°C for future use.

[0035] (2) Weigh 0.01g magnetic microspheres (about 0.5×10 6 ), use a magnetic separation rack to separate the magnetic beads from the solution, and resuspend the separated magnetic microspheres with 5ml sterile pH 4.5 PBS. Add 0.18 g of EDC to the suspension and react at 4° C. for 15 minutes, then add 0.45 g of NHS and react at 4° C. for 4 hours. After the reaction, the magnetic beads were separated from the liquid, washed three times with sterile pH 7.6 PBS, resuspended in 1ml of sterile pH 7.6 PBS, and stored at 4°C. Use 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (English abbreviation: EDC) and N-hydroxysuccinimide (English abbreviation: NHS) to carboxylate the carboxyl group of the magnetic beads Activate to obtain magneti...

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Abstract

The invention discloses a method for enriching an influenza virus through utilizing an asialofetuin-containing magnetic bead. The method comprises the following steps of: (1) utilizing a vortex instrument to shock and disperse carboxylation magnetic microspheres; (2) using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-H-hydroxy succinimide (NHS) to activate the carboxyls of the carboxylation magnetic microspheres so as to obtain the magnetic microspheres with carboxyl activity; (3) directly adding the dissolved fetuin protein into the activated magnetic microspheres of step (2) for covalent binding; and (4) directly adding the magnetic microspheres bonded with the fetuin protein in the step (3) into a liquid to be detected so as to enrich the virus. According to the method, the built fetuin protein magnetic microspheres is directly added into the liquid to be detected so as to enrich the virus, and the detection can be carried out after the enrichment without intermediate links such as centrifugation, precipitation and the like, so that time is saved, and no special instruments are needed; and the method has the advantages of rapid sensitivity, strong antijamming capability, convenience, economy, simplicity, practicability, no environmental pollution and the like.

Description

technical field [0001] The invention provides a method for enriching influenza virus with sialic acid-fetuin magnetic beads, and relates to a method for enriching influenza virus in liquid phase, especially capable of enriching large-volume and low-concentration subtypes of influenza Viruses belong to the field of biomedicine. Background technique [0002] Currently, a known method for enriching influenza virus is the method for enriching influenza virus from erythrocytes. Utilizing the characteristic that influenza virus can agglutinate red blood cells and dissociate from red blood cells under certain conditions, the red blood cells are agglutinated at 4°C after hydroformylation, and released at 37°C to achieve the enrichment of influenza virus the goal of. However, the enrichment efficiency of erythrocyte-enriched influenza virus is low. [0003] Another method is the immunomagnetic bead enrichment method for influenza virus. It uses magnetic beads as the carrier, and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/02C12R1/93
Inventor 夏志平张馨元李乾学李志萍
Owner MILITARY VETERINARY RES INST PLA MILITARY MEDICAL ACAD OF SCI
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