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Method and kit for human influenza virus typing detection based on magnetic resolution and colorful quantum dot labelling

A human influenza virus, magnetic separation technology, applied in the field of medical detection, can solve the problems of numerous operation steps, high detection cost, low detection sensitivity, etc.

Active Publication Date: 2015-12-30
湖北诺美华抗体药物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the publicly reported methods for detecting human influenza virus antigens are mainly double-antibody sandwich ELISA, colloidal gold immunochromatography, indirect immunofluorescence, etc., but these methods may have many steps, long detection time, or detection Problems such as low sensitivity and high detection cost

Method used

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  • Method and kit for human influenza virus typing detection based on magnetic resolution and colorful quantum dot labelling

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Preparation of Anti-human Influenza Virus Immune Nanomagnetic Beads

[0073] 1. Optimization of reaction conditions for anti-human influenza virus polyclonal antibody-coupled magnetic beads:

[0074] Using magnetic beads coupled with anti-human influenza A virus NP protein polyclonal antibody as a solid phase carrier, and quantum dot-labeled anti-human influenza A virus NP protein monoclonal antibody as a detection antibody, the detection of human Influenza A virus antigen, observe the coupling of magnetic beads and polyclonal antibodies. A series of optimization options were carried out on the particle size of the magnetic beads, as well as the concentration of EDC / NHS activator, the concentration of conjugated antibody, the coupling time, and the type of blocking agent.

[0075] 1.1 Selection of magnetic bead size

[0076] Select carboxyl magnetic beads with a particle size of 50nm, 180nm, 350nm, 1150nm, and 3μm, add PBS buffer containing 4mg / mlEDC and 4mg...

Embodiment 2

[0089] Example 2 Preparation of anti-human influenza virus nanoprobes labeled with multicolor quantum dots

[0090] 1. Optimization of the reaction conditions for nanocarboxyl quantum dot-labeled mouse anti-human influenza A virus NP protein monoclonal antibody:

[0091] 1.1. Determination of the optimal labeling pH of the carboxyl quantum dot-labeled antibody probe

[0092] The pH of the phosphate buffer in the labeling reaction was set to 5, 6, 7, 8, and 9 respectively, and the fluorescence intensity of the labeled product was measured with a full spectrometer, and the influence of different pH values ​​on the coupling reaction was observed, and the quantum dot-labeled monoclonal antibody was determined. The optimum pH for the reaction is 7.0-8.0. This experiment chooses pH7.4.

[0093] 1.2. Determination of the optimal labeling amount of carboxy quantum dot-labeled antibody probes

[0094] Set the ratio of quantum dot molar concentration to monoclonal antibody concentrat...

Embodiment 3

[0101] Example 3 Optimization of immune capture conditions for human influenza virus antigens by immune nano-magnetic beads

[0102] Using immune nano-magnetic beads coupled with rabbit anti-human influenza A virus NP protein polyclonal antibody as the solid phase carrier, quantum dot-labeled mouse anti-human influenza A virus NP protein monoclonal antibody as the detection antibody, through the double-antibody sandwich Based on the principle of the method, a detection system for human influenza virus antigens was established. A series of optimization options were carried out on the dosage of immunomagnetic nano-magnetic beads in the detection system, capture time and other conditions.

[0103] 1. Selection of the amount of immune nano-magnetic beads added

[0104] Add 20 μl, 40 μl, 60 μl, 80 μl, 100 μl, 120 μl, 140 μl, 160 μl, 180 μl, 200 μl of immune nano-magnetic beads prepared in Example 1 to 0.5 ml human influenza A virus (ATCC No. VR-1743) sample treatment solution (8g...

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Abstract

The invention discloses a method and a kit for human influenza virus typing detection based on magnetic resolution and colorful quantum dot labelling. The kit comprises anti-human influenza virus immune nano-magnetic beads, anti-human influenza virus nanoprobes, quality control samples, a sample treating liquid and a PBST buffer solution, wherein the anti-human influenza virus immune nano-magnetic beads are rich in human influenza virus antigen; the anti-human influenza virus nanoprobes are labelled by colorful quantum dots; the quality control samples are a positive quality control sample and a negative quality control sample; the positive quality control sample is obtained by drying an inactived A-type human influenza virus and an inactived B-type human influenza virus, and combining the dried human influenza viruses on a swab; the negative quality control sample is a pharynx swab of a person whose detection results for the A-type human influenza virus and the B-type human influenza virus are both negative through clinical confirmation. The method and the kit are simple, convenient, fast, and high in sensitivity. The invention further provides a preparation method and a use method of the kit.

Description

technical field [0001] The invention relates to the technical field of medical detection, in particular to a rapid detection method and a detection kit for detecting human influenza virus antigens based on magnetic separation and multicolor quantum dot labeling, and methods for preparing and using the detection kit. Background technique [0002] Influenza virus, referred to as influenza virus, is an RNA virus that causes influenza in humans and animals. In taxonomy, influenza virus belongs to the Orthomyxoviridae family. It can cause acute upper respiratory tract infections and rapidly spread through the air. There are often periodic pandemics around the world. The virus was first discovered in 1933 by the Englishman Wilson Smith, who called it H1N1. H stands for hemagglutinin; N stands for neuraminidase. Numbers represent different types. [0003] Influenza virus is spherical or filamentous, with a diameter of 80-120nm. The virus consists of three layers, the inner laye...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/531
Inventor 胡征杨波董俊
Owner 湖北诺美华抗体药物技术有限公司
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