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Method for detecting influenza virus antibody through agglutination reaction based on eukaryotic cell for recombinant expression on influenza virus hemagglutinin and neuraminidase

A neuraminidase and neuraminidase protein technology, applied in the biological field, can solve the problems of long detection time, poor stability, complex process, etc., and achieve the effect of extensive detection and fast reaction 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

[0007] The purpose of the present invention is to provide a fast, simple and stable influenza virus antibody based on recombinant expression according to the problems of complex process, long detection time and poor stability in the methods for detecting influenza virus antibodies in the prior art. Lectin and neuraminidase eukaryotic agglutination method for the detection of influenza virus antibodies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0024] Example 2 Preparation of Eukaryotic Cells Simultaneously Recombinantly Expressing Influenza Virus HA and NA

[0025] According to the instructions of the Lipofectamine 2000 kit, transfect 2.5 μg pcDNA-H5HA plasmid, 0.5 μg pcDNA-N1NA plasmid and 10 μl Lipofectamine 2000 transfection complex into a 35 mm diameter petri dish containing human cells with a growth density of about 80%-90% fusion rate. For embryonic kidney HEK-239 cells, the transfection complex was removed 6 hours after transfection, and fresh complete medium (DMEM medium containing 10% fetal bovine serum) was replaced.

[0026] After transfection, HEK-239 cells can transiently recombinantly express HA and NA, that is, cells that transiently recombinantly express HA and NA of influenza virus can be obtained.

[0027] For further optimization, cells with stable recombinant expression of HA and NA can be screened: after 72 hours of transfection, the transfected cells were blown and dispersed, and 1 / 20 of the ce...

Embodiment 3

[0028] Example 3 Treatment of Eukaryotic Cells Simultaneously Recombinantly Expressing Influenza Virus HA and NA

[0029] Transient or stable recombinant expression of the strain HA and NA cells, after adherent or suspension culture, the cells were blown and dispersed with PBS containing 2% BSA, the cell suspension was centrifuged, and the supernatant was removed and then washed with 2% BSA PBS Press 10 7 cells / ml for resuspension. Remove the supernatant, resuspend with an equal volume of PBS, wash by centrifugation twice, add an equal volume of PBS again, 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 cells were pelleted by 10 7 The concentration of each / ml is resuspended in PBS buffer containing 0.2% Nepalese gold ester, 2% BSA, 20% glycerol, and 0.5% heparin, so that it can be stored stably for a long time, that is, the recombinant expressed HA after treatment is obtained. an...

Embodiment 4

[0030] Example 4 Using co-recombined eukaryotic cells expressing influenza virus HA and NA to perform agglutination reaction to detect influenza virus antibodies

[0031] Add 1 drop (about 50 μl) of the eukaryotic cell suspension of the recombinant expression HA and NA antigen treated in Example 2 to a clean slide, then add 50 μl of the sample to be tested (serum, bronchial lavage fluid, Plasma, interstitial fluid, etc.), gently shake the slide back and forth or use a micropipette tip to mix it well, and observe whether cell agglutination occurs at room temperature for 1-3 minutes.

[0032] 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 and / or NA reactive with the strain HA and / or NA The antibody or antibody concentration is below the detection level achievable by the method.

[0033] When agglutinatio...

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Abstract

The invention relates to the technical field of biology, in particular to a method for detecting an influenza virus antibody through agglutination reaction based on an eukaryotic cell for recombinant expression on influenza virus hemagglutinin and neuraminidase. The method comprises the following steps of: taking the eukaryotic cell for the recombinant expression on the influenza virus hemagglutinin and the neuraminidase as an agglutination reaction matrix, and detecting the influenza virus hemagglutinin antibody and the neuraminidase antibody in a sample to be detected through the recombinant expression of the influenza virus hemagglutinin and the neuraminidase in the agglutination reaction way. In the invention, the influenza virus hemagglutinin and the neuraminidase are co-expressed on the surface of the eukaryotic cell, thereby effectively avoiding self-coagulation to affect the agglutination reaction due to the combination of the hemagglutinin and a cell receptor; at the same time, the invention can more widely and rapidly detect the influenza virus antibody in different serotypes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting influenza virus antibodies based on recombinantly expressed influenza virus hemagglutinin and neuraminidase eukaryotic cell agglutination reaction. 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. [0...

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N15/44C12N15/55C12N15/85C12N15/79
Inventor 王革非李蕊李康生
Owner SHANTOU UNIV MEDICAL COLLEGE
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