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A pretreatment method for influenza virus detection based on antibody magnetic bead capture

A technology of influenza virus and influenza A virus, which is applied in the field of pretreatment of influenza virus detection based on antibody magnetic beads capture, can solve the problem of low specificity and achieve the effect of strong specificity, low cost and high sensitivity

Active Publication Date: 2018-01-16
BEIJING CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can be used for various subtypes of viruses, but the specificity is not high [5]

Method used

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  • A pretreatment method for influenza virus detection based on antibody magnetic bead capture
  • A pretreatment method for influenza virus detection based on antibody magnetic bead capture
  • A pretreatment method for influenza virus detection based on antibody magnetic bead capture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Pretreatment of throat swab samples of influenza A (H1N109pdm) subtype or influenza A (H3N2) subtype

[0038] (1) Shake the carboxylated superparamagnetic beads with a vortex shaker (at 3000 rpm for 5 minutes) to completely disperse them into a homogeneous suspension.

[0039] (2) Add 4g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 0.2g superparamagnetic beads, EDC), reacted for 30 minutes at 4°C and 300 rpm.

[0040] (3) Add 0.4 g of N-Hydroxysuccinimide (NHS) to the above reaction system, and react at 4° C. and 300 rpm for 6 hours.

[0041] (4) Take 1 / 10 of the above reaction system, add 2 mg of influenza A virus M2e monoclonal antibody, use pH 7.4 PBS buffer to dilute to a total reaction volume of 2 mL, and react at 4 ° C (conditions 300 rpm, time 8 hours).

[0042] (5) After the reaction, use a magnetic stand to adsorb the magnetic beads, wash the antibody magnetic beads twice with 3 mL...

Embodiment 2

[0046] Pretreatment of Lower Respiratory Tract Lavage Fluid Samples for Influenza B Virus

[0047] (1) Shake the carboxylated superparamagnetic beads with a vortex shaker (at 3000 rpm for 5 minutes) to completely disperse them into a homogeneous suspension.

[0048] (2) Add 4g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 0.2g superparamagnetic beads, EDC), reacted for 30 minutes at 4°C and 300 rpm).

[0049] (3) Add 0.4 g of N-Hydroxysuccinimide (NHS) to the above reaction system, and react at 4° C. and 300 rpm for 6 hours.

[0050] (4) Take 1 / 10 of the above reaction system, add 2mg of influenza B virus M2 monoclonal antibody, use pH 7.4 PBS buffer to dilute to a total reaction volume of 2mL, and react at 4°C (condition 300rpm, time 8 hours).

[0051] (5) After the reaction, use a magnetic stand to adsorb the magnetic beads, wash the antibody magnetic beads twice with 3 mL of PBS buffer solution ...

Embodiment 3

[0055] Pretreatment of throat swab samples of unknown type influenza virus

[0056] (1) Shake the carboxylated superparamagnetic beads with a vortex shaker (at 3000 rpm for 5 minutes) to completely disperse them into a homogeneous suspension.

[0057] (2) Add 4g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to 0.2g superparamagnetic beads, EDC), reacted for 30 minutes at 4°C and 300 rpm).

[0058] (3) Add 0.4 g of N-Hydroxysuccinimide (NHS) to the above reaction system, and react at 4° C. and 300 rpm for 6 hours.

[0059] (4) According to the mass ratio of anti-influenza virus M2e monoclonal antibody:anti-influenza virus M2 monoclonal antibody=2:1, mix antibodies in PBS buffer.

[0060] (5) Take 1 / 10 of the activated carboxyl superparamagnetic beads, add 2mg of mixed monoclonal antibodies, use pH 7.4 PBS buffer to dilute to a total reaction volume of 2mL, and react at 4°C (conditions 300rpm, time 8 ho...

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Abstract

The invention discloses a pretreatment method for detecting an influenza virus based on antibody magnetic bead capture. The method comprises the following steps: activating carboxyl magnetic beads with 1-(3-dimethylamino propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy succinimide; covalently coupling an influenza virus A M2e monoclonal antibody and / or an influenza virus B M2 monoclonal antibody to activated carboxyl magnetic beads; and mixing the coupled antibody magnetic beads with a to-be-tested biological sample, and enriching influenza viruses. The method is simple and convenient to operate, fast, high in enrichment efficiency, good in selection specificity and suitable for a plurality of samples.

Description

technical field [0001] The invention relates to a pretreatment method for influenza virus detection, in particular to a pretreatment method for influenza virus detection based on antibody magnetic bead capture. Background technique [0002] Influenza virus is a representative species of Orthomyxoviridae, including human influenza virus and animal influenza virus. Among them, people infected with influenza A virus are highly contagious and have a high infection rate. Worldwide, annual influenza epidemics result in approximately 3 to 5 million severe cases and approximately 250,000 to 500,000 deaths. The rapid identification of influenza viruses is a key element in the response to and control of influenza pandemics. Identifying and clarifying the new influenza virus in the first place, and understanding the source and related characteristics of the virus will help to quickly assess the risk of virus transmission and health hazards, and provide important scientific support fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/6806C12Q1/70C12Q2523/308
Inventor 潘阳王全意杨鹏崔淑娟张代涛
Owner BEIJING CENT FOR DISEASE PREVENTION & CONTROL