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Influenza virus antibody detection method based on eukaryotic cell agglutination reaction of recombined expression influenza virus hemagglutinin

A technology of influenza virus and eukaryotic cells, applied in the biological field, can solve the problems of heavy workload, long detection time, poor stability, etc., and achieve the effect of extensive detection and fast response time

Active Publication Date: 2014-04-02
SHANTOU UNIV MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of influenza virus based on the eukaryotic cell agglutination reaction of recombinantly expressed influenza virus hemagglutinin according to the problems of large workload, poor stability, and long detection time in the existing influenza virus antibody detection technology. Antibody detection method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation of the eukaryotic cell of embodiment 1 recombinant expression influenza virus hemagglutinin (HA)

[0021]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 sequence of its HA can be obtained on the public database GenBank, and its sequence number is EU874899 .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 cloning was designed. 3 protective bases, Hind III restriction site, KOZAK sequence, start codon and pair...

Embodiment 2

[0025] Example 2 Recombinant treatment of eukaryotic cells expressing influenza virus hemagglutinin (HA)

[0026] Transiently or stably recombinantly expressing the cells of the strain HA, after culturing, gently digest the cells with low concentration of trypsin (0.05%) for about 5 minutes, add a medium containing fetal bovine serum to terminate the reaction, and centrifuge the cell suspension. After removing the supernatant, resuspend with PBS containing 2% BSA to obtain the cell suspension after trypsin treatment.

[0027] In order to prevent cell self-agglutination that may be caused by HA, neuraminidase (also known as neuraminidase, Neuraminidase) was added to the cell suspension after trypsin treatment to eliminate the interaction between the recombinantly expressed HA and the cell's own receptors. function to prevent cell agglutination. That is, after the cell suspension was centrifuged, the cells were pressed in 10 mM sodium citrate (pH 6.0) buffer solution. 7 cells / ...

Embodiment 3

[0029] Example 3 Using eukaryotic cells recombinantly expressing influenza virus hemagglutinin to perform agglutination reaction to detect influenza virus antibodies

[0030] Add 1 drop (approximately 50 μl) of the treated recombinant eukaryotic cell suspension expressing the HA antigen in Example 2 to a clean glass slide, and then add 50 μl of the sample to be tested (serum, bronchial lavage fluid, plasma, tissue fluid, etc.), gently shake the slide back and forth or use a micropipette tip to mix it, and observe whether there is cell agglutination at room temperature for 1-3 minutes.

[0031] Set up positive serum and negative serum controls. If the control sample meets the expected results and the sample to be tested does not agglutinate, it means that the sample to be tested does not contain influenza virus HA antibodies reactive with the strain HA or the antibody concentration is lower than that of the method. Achievable detection concentration.

[0032] When agglutinatio...

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Abstract

The invention relates to the field of biotechnology, particularly an influenza virus antibody detection method based on the eukaryotic cell agglutination reaction of recombined expression influenza virus hemagglutinin. In the detection method, influenza virus hemagglutinin genes are cloned and transfect eukaryotic cells, so that the surfaces of the eukaryotic cells express the influenza virus hemagglutinin protein in a recombined mode; the protein is combined with the influenza virus hemagglutinin antibody in the sample to be detected to produce agglutination reaction; and the influenza virus antibody in the sample to be detected is detected according to whether the agglutination reaction occurs. The detection method is quick and stable, and can be widely used for quickly detecting different serotypes of influenza virus antibodies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting influenza virus antibodies based on eukaryotic cell agglutination of recombinantly expressed influenza virus hemagglutinin. 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 met...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N15/44C12N15/85C12N15/79
Inventor 王革非李康生李蕊
Owner SHANTOU UNIV MEDICAL COLLEGE
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