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Fluorescence micro cell agglutination method for detecting influenza virus antibody

A technology of influenza virus and micro cells, which is applied in the biological field, can solve the problems of limited throughput and large cell consumption, and achieve the effects of less cell consumption, extensive detection, and fast response time

Active Publication Date: 2011-04-20
SHANTOU UNIV MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortcomings of limited throughput and large cell consumption in this method, a method suitable for high-throughput detection, with less cell consumption, simple and fast, relatively simple and easy-to-control process, and capable of simultaneously detecting influenza virus HA and NA antibody detection method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 The method for the recombinant expression of influenza virus HA and NA eukaryotic cells labeled with green fluorescent protein

[0025] In this example, an H5 subtype strain A / Chicken / Guangdong / 1 / 2005 (H5N1) was taken as an example to prepare eukaryotic cells expressing recombinant influenza virus hemagglutinin HA. A / Chicken / Guangdong / 1 / 2005 (H5N1) is an H5N1 subtype influenza virus isolated from chickens in Guangdong Province in 2005. The gene sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. Viral RNA was extracted using Viral RNA Miniprep Kit after the strain virus was propagated, and reverse transcription was performed with Uni-12 primer (5'-AgCAAAgCAgg-3') and SuperScript III or M-MLV reverse transcriptase to obtain viral cDNA. According to the gene sequence of the strain HA, a pair of primers for HA full-length gene cloning was designe...

Embodiment 2

[0034] Embodiment 2 Fluorescein CFSE (carboxyfluorescein diacetate succinimidyl ester)-labeled method for eukaryotic cells expressing influenza virus HA and NA

[0035]In this example, an H5 subtype strain A / Chicken / Guangdong / 1 / 2005 (H5N1) was taken as an example to prepare eukaryotic cells expressing recombinant influenza virus hemagglutinin HA. A / Chicken / Guangdong / 1 / 2005 (H5N1) is an H5N1 subtype influenza virus isolated from chickens in Guangdong Province in 2005. The gene sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. Viral RNA was extracted using Viral RNA Miniprep Kit after the strain virus was propagated, and reverse transcription was performed with Uni-12 primer (5'-AGCAAAAGCAGG-3') and SuperScript III or M-MLV reverse transcriptase to obtain viral cDNA. According to the gene sequence of the strain HA, a pair of primers for HA full-length gene clonin...

Embodiment 3

[0043] Example 3 Green Fluorescent Protein and Red Fluorescent Protein Labeling Method for Separately Recombined Eukaryotic Cells Expressing Influenza Virus HA and NA

[0044] In this example, an H5-type strain A / Chicken / Guangdong / 1 / 2005 (H5N1) was taken as an example to prepare eukaryotic cells expressing recombinant influenza virus hemagglutinin HA. A / Chicken / Guangdong / 1 / 2005 (H5N1) is an H5N1 subtype influenza virus isolated from chickens in Guangdong Province in 2005. The gene sequences of its HA and NA can be obtained on the public database GenBank, and the sequence of HA The number is EU874899.2, and the serial number of NA is EU874900.2. Viral RNA was extracted using Viral RNA Miniprep Kit after the strain virus was propagated, and reverse transcription was performed with Uni-12 primer (5'-AGCAAAAGCAGG-3') and SuperScript III or M-MLV reverse transcriptase to obtain viral cDNA. According to the gene sequence of the strain HA, a pair of primers for HA full-length gene c...

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Abstract

The invention relates to the biological technology field, in particular to a fluorescence micro cell agglutination method for detecting influenza virus antibody. The method in the invention comprises the following steps: hemagglutinin gene and neuraminidase gene of influenza virus are cloned and then are transfected to a eukaryotic cell; the hemagglutinin and neuraminidase are recombined and expressed on the surface of the eukaryotic cell; the recombinant expression cell is marked by fluorescence; the hemagglutinin and neuraminidase which are recombined and expressed on the surface of the cell are combined with hemagglutinin antibody and neuraminidase antibody of the influenza virus to carry out agglutination reaction; and pictures are taken by a fluorescence microscope, whether influenza virus antibody exists in a to-be-detected sample is judged according to whether the fluorescence area is increased. In the detecting method of the invention, the cell marked by fluorescence is used, the cell using amount is reduced, high-throughput detection can be carried out, and quick detection, early detection, on-spot detection and evaluation of the influenza virus antibody can be realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fluorescent micro-cell agglutination method for detecting influenza virus antibodies. Background technique [0002] Antibodies to influenza virus hemagglutinin will appear in body fluids (such as serum, bronchial lavage fluid, plasma, tissue fluid, etc.) of specific serotype influenza virus infection, recovery after infection, latent infection, and vaccination of humans or animals, By detecting antibodies to a specific serotype of influenza virus, it is possible to determine, diagnose or confirm whether the person or animal is infected (including early infection, latent infection or recovery after infection, etc.) with the specific serotype of influenza virus, or has been inoculated with influenza virus Vaccines, or evaluation of their resistance to specific serotypes of influenza viruses, etc. [0003] At present, the method for detecting whether there is a certain serotype of in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569C12N15/44C12N15/55C12N15/85C12N15/79
Inventor 李康生王革非李卫中李蕊蔡汉杰黄秀梅孟燕萍吴彬冰吴嘉伟
Owner SHANTOU UNIV MEDICAL COLLEGE
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