Rapid detection method of influenza virus neuraminidase antibody

A technology of neuraminidase and neuraminidase protein, which is applied in botany equipment and methods, biochemical equipment and methods, measuring devices, etc., can solve the problems of heavy workload and long time consumption, and achieve fast response time, The effect of extensive testing

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

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a fast and simple eukaryotic cell expressing influenza virus neuraminidase based on race according to the shortcomings of large workload and time-consuming in the detection of influenza virus neuraminidase antibody in the prior art Rapid detection method of influenza virus neuraminidase antibody in agglutination reaction

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of the eukaryotic cell of embodiment 1 recombinant expression influenza virus neuraminidase (NA)

[0022]In this example, an H5N1 subtype strain A / Chicken / Guangdong / 1 / 2005 (H5N1) was taken as an example to prepare eukaryotic expression cells for recombinantly expressing influenza virus neuraminidase NA. 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 NA can be obtained on the public database GenBank, and the sequence 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 NA, a pair of primers for NA full-length gene cloning is designed, and the primer sequence is as follows: upstream primer N 1NA-...

Embodiment 2

[0027] The treatment of the eukaryotic cell of embodiment 2 recombinant expression influenza virus neuraminidase (NA)

[0028] Transiently or stably recombinantly express the cells of the strain NA, after culture, treat the cells with disodium EDTA (0.02%) at room temperature for about 2 minutes, centrifuge the cell suspension, remove the supernatant and wash with PBS containing 2% BSA Perform resuspension to obtain EDTA-treated cell suspension. Add an equal volume of 10% cold formaldehyde and fix at 4°C for 2 hours. Wash 3 times by centrifugation and resuspension in PBS. Finally, the cell pellet was resuspended at a concentration of 107 cells / ml in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 15% glycerol, and 0.5% heparin, so that it could be stored stably for a long time, and obtained Treated recombinant eukaryotic cells expressing NA antigen.

Embodiment 3

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

[0030] Add 1 drop (about 50 μl) of the treated recombinant eukaryotic cell suspension expressing NA antigen in Example 2 to a clean glass slide, and then add 50 μl of samples 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 NA antibodies reactive with the strain NA or the antibody concentration is lower than that of the method. Achievable detection concentration.

[0032] When agglutination occurs in the...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a rapid detection method of an influenza virus neuraminidase antibody. The detection method comprises the following steps of: coning the influenza virus neuraminidase gene and then transfecting eukaryotic cells so that influenza virus neuraminidase protein is in recombination expression on the surface of eukaryotic cells, combining with the influenza virus neuraminidase antibody in a sample to be detected, performing agglutination reaction and judging whether the influenza virus neuraminidase antibody exists in the sample to be detected according to whether the agglutination reaction happens or not. The method is simple, rapid, stable and highly sensitive, and has wide application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rapid detection method for influenza virus neuraminidase antibody. Background technique [0002] Antibodies to influenza virus neuraminidase 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 judge, diagnose or confirm whether the person or animal is infected (including early infection, latent infection or post-infection recovery, etc.) with the specific serotype of influenza virus, or has been vaccinated against influenza Virus vaccines, or evaluation of their resistance to specific serotypes of influenza viruses, etc. [0003] There are mainly two kinds of glycoprotein spikes with strong antigenicity on the surface of influen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N15/55C12N15/44C12N15/85
Inventor 李蕊李康生王革非李卫中蔡汉杰吴嘉伟吴彬冰孟燕萍黄秀梅
Owner SHANTOU UNIV MEDICAL COLLEGE
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