Immunoassay reagent for assaying influenza A H1N1 virus antigen

An immunodetection, type A technology, applied in antiviral immunoglobulins, antiviral agents, and serum immunoglobulins, etc., can solve the problems of undetectable and low virus content, and achieve high affinity, good specificity, The effect of increasing sensitivity

Active Publication Date: 2011-05-04
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The HA-targeted influenza typing detection kit for influenza A H1N1 has not yet been reported, and the existing literature work is only limited to antibody preparation. The main problem with the current detection reagents is that due to the limitations of ELISA reagents, clinical samples ( Mainly throat swabs) The virus content is extremely low, below the detection limit of ELISA reagents, and cannot be detected

Method used

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  • Immunoassay reagent for assaying influenza A H1N1 virus antigen
  • Immunoassay reagent for assaying influenza A H1N1 virus antigen
  • Immunoassay reagent for assaying influenza A H1N1 virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] [Example 1] Preparation of DNA vaccine

[0023] The HA gene sequence of H1N1 influenza virus A / California / 04 / 2009 is shown in SEQ ID NO.1. The HA sequence was synthesized by Shanghai Sangong and inserted into the multiple cloning site of pCDNA3.1(+) vector after double enzyme digestion (XhoI / ApaI) Point, constructed into pCDNA3.1 / SF-HA vector.

[0024] Large-scale preparation of plasmid DNA

[0025] According to the steps of the QIAGEN Plasmid Maxi Kit produced by QIAGEN Company, the recombinant plasmid DNA was extracted, the gained DNA was dissolved in TE solution, and the OD260 value and OD280 value of the prepared plasmid DNA were measured on a UV spectrophotometer, according to the calculated DNA purity and The concentration formula calculates the purity and concentration of DNA. According to its initial concentration, it was diluted to a final concentration of 1 μg / μl for electrophoresis identification, and the rest were stored in a -20°C refrigerator for later...

Embodiment 2

[0026] [Example 2] Preparation of mouse anti-H1N1 virus antigen monoclonal antibody

[0027] 1. Immunization Methods and Procedures :

[0028] The immunization route adopts the intramuscular injection method of the quadriceps femoris. The amount of DNA injected into each 6-8-week-old BALB / c mouse is 50 μg. , the electric shock time is 50ms), and a booster immunization was carried out 3 weeks after the initial immunization.

[0029] 2. Monoclonal antibody preparation:

[0030] 2.1 Cell Fusion:

[0031] 2.1.1 The mice were killed, and the spleen was removed under aseptic operation. The spleen was placed in a 5ML RPMI-1640 culture dish (without adding serum), cut into two halves, and then the cells were squeezed out with sterilized equipment.

[0032] 2.1.2 Aspirate the cells into a sterile plastic tube, let it stand for 5 minutes to allow the tissue pieces to settle down, transfer the cells to another sterile test tube and centrifuge at 300XG for 10 minutes.

[0033] 2.1...

Embodiment 3

[0045] [Example 3] Preparation of rabbit anti-H1N1 virus antigen polyclonal antibody

[0046] 1. Rabbit polyclonal antibody preparation: Rabbits were immunized by intramuscular injection of 3-month-old rabbits into the quadriceps femoris muscle. The amount of DNA injected was 100 μg. After injection, 3 times of positive and negative electric shocks (100V Voltage, electric shock time is 50ms), and a booster immunization was carried out 3 weeks after the initial immunization. Ten days after the last booster, the rabbits were killed and bled, and the separated serum was stored at -20°C.

[0047] 2. Purification of rabbit polyclonal antibody: rough extraction by salting out method, and then purified by DEAE-cellulose ion exchange chromatography. The purity of the antibody is identified by SDS-PAGE. It is required that two clear bands appear at 25KD and 50KD of the loaded protein, and the purity is above 90%.

[0048] 3. Rabbit polyantibody biotin labeling: Biotin was purchased f...

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Abstract

The invention provides an influenza A H1N1 virus hemagglutinin (HA) protein deoxyribonucleic acid (DNA) sequence-containing recombinant expression vector and a DNA vaccine expressed by the recombinant expression vector aiming at an influenza A H1N1 virus antigen. A synthesized influenza A H1N1 virus HA gene is inserted into a plasmid vector to construct the influenza A H1N1 virus HA gene sequence-containing recombinant expression vector, and recombinant plasmid DNA is extracted to prepare an influenza A H1N1 virus DNA vaccine; and the influenza A H1N1 virus DNA vaccine is used for immunizing a mouse to prepare a monoclonal antibody. The invention also provides an immunological method and an immunoassay reagent for assaying the influenza A H1N1 virus antigen. In the invention, a monoclonal antibody cell strain and a polyclonal antibody with high specificity and affinity are prepared; and the prepared immunoassay reagent has high sensitivity and high specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA vaccine prepared by genetic engineering technology, a monoclonal antibody and an immunodetection reagent prepared therefrom. Background technique [0002] Hemagglutinin (HA) is the main outer membrane protein of influenza virus. HA is immunogenic and can make the human body produce protective antibodies, but it is easy to mutate, which is the cause of influenza epidemic. The preparation of specific monoclonal antibodies and polyclonal antibodies against influenza virus hemagglutinin protein is the basis for the detection of influenza virus protein typing. Currently, conventional protein immunization methods are used for the preparation of antibodies against HA. Among them, Wang Huijuan et al. 【1】 Expressing the HA protein of A-H1N1 in insect baculovirus for immunizing antigens and screening antigens. Zhang Xiangbin and others 【2】 (Chinese Veterinary Science 2008.38(11): 962-9...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/44C12N5/20C07K16/10C07K16/06A61K48/00A61P31/16G01N33/577G01N33/569C12R1/91
Inventor 严兵陈建军
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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