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Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof

The technology of monoclonal antibody and hemagglutinin protein is applied in the field of animal virus and animal epidemiology detection technology, and can solve the problems of damping the enthusiasm of farmers and herdsmen for breeding, high incidence of infectious diseases, economic loss of Tibetan chicken breeding industry, etc. problems, to achieve the effect of wide detection sample range, strong specificity and short detection time

Inactive Publication Date: 2013-04-24
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of comprehensive and systematic chicken disease prevention and control technology, the incidence of various infectious diseases in chickens is very high, especially the outbreak and prevalence of global influenza, which has brought great economic losses to the Tibetan chicken breeding industry and seriously damaged agricultural production. Enthusiasm of pastoralists

Method used

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  • Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof
  • Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof
  • Monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The preparation of embodiment 1 H9 subtype influenza virus

[0049] 1. Isolation of H9 subtype influenza virus

[0050] 1. Separation process: Collect sick chicken samples with sterilized throat swabs, place in sterilized phosphate buffered saline (abbreviated as PBS, formula: Na 2 CO 3 1.59g, NaHCO 3 2.93g, with ddH 2 O to 1000ml), and then 0.2ml of the throat swab sample solution was inoculated into 9-day-old specific pathogen-free (Specific pathogen Free, SPF) chicken embryos (purchased from Beijing Meria Weitong Experimental Animal Technology Co., Ltd.), and placed at 35 After incubating at ℃ for 72 hours, the allantoic fluid was harvested for qualitative hemagglutination test, and the samples that were positive in the hemagglutination test were then finalized by RT-PCR test.

[0051] 2. Identification basis: RT-PCR tested positive samples, amplified HA, sent to Shanghai Sangon Bioengineering Co., Ltd. for sequencing, and compared the obtained DNA sequence wit...

Embodiment 2

[0067] Example 2 Preparation of monoclonal antibody against H9 subtype influenza hemagglutinin protein

[0068] 1. Use the purified and inactivated H9 subtype influenza virus A / Chicken / Tibet / S1 / 2009 original strain as the antigen, emulsified with Freund's adjuvant (freund's complete adjuvant is used for the first immunization, Freund's complete adjuvant is used for the second and third immunizations) 5-8 week old BALB / c mice (purchased from Hubei Experimental Animal Research Center). The first immunization dose was 20 μg / mouse, and each mouse was injected subcutaneously at multiple points on the back of the neck. Two weeks later, a booster immunization was given with the same dose, and a booster immunization was given again two weeks later, with a dose of 40 μg / mouse. Two weeks later, a small amount of mouse blood was collected by docking the tail, and the serum was collected, and the antibody titer of the mouse was detected by the hemagglutination inhibition method. After th...

Embodiment 3

[0096] Example 3 Identification of Anti-H9 Subtype Influenza Hemagglutinin Protein Monoclonal Antibody

[0097] 1. Identification of HI potency

[0098] The H9 subtype influenza virus isolated by the invention is used as an antigen to measure the potency of hybridoma cell culture supernatant and mouse ascites by hemagglutination inhibition method. The results are shown in Table 1.

[0099] Table 1: Determination of titers of hybridoma cell culture supernatant and mouse ascites by hemagglutination inhibition method (HI)

[0100] cell culture supernatant mouse ascites Hemagglutination inhibition (HI) titer 2 9 2 18

[0101] 2. Identification of Ig subclasses (types) of monoclonal antibodies

[0102] The monoclonal antibody obtained in the present invention was identified with the Mouse Mab Isotyping Test Kit (Mouse Mab Isotyping Test Kit, purchased from THERMO Company), and it was determined that the monoclonal antibody prepared in the present inve...

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Abstract

The invention discloses a monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein, which is secreted by hybridoma cell strain 4D10 with the preserving number of CCTCC No: C2012152. The invention further discloses a double antibody sandwich ELISA test kit of H9 subtype flue virus and a detection method. The monoclonal antibody of anti-H9 subtype flu virus haemagglutinin protein is used as a primary antibody, a monoclonal antibody marked by horseradish peroxidase is used as a second antibody, and a separation, augmentation, inactivation and purification method of H9 subtype flue virus and a preparation and purification method of anti-H9 subtype flu virus haemagglutinin monoclonal antibody are disclosed. According to the test kit and the detection method disclosed by the invention, the H9 subtype flue virus can be detected directly, and the monoclonal antibody has the characteristics of being high in specificity, high in sensitivity, short in detection time, wide in detection sample range and the like.

Description

technical field [0001] The invention belongs to the technical field of detection of animal viruses and zoonotic diseases. Specifically, the invention relates to a monoclonal antibody against H9 subtype influenza virus hemagglutinin protein, and the invention also relates to anti-H9 subtype influenza virus hemagglutinin protein The application of the monoclonal antibody in the preparation of the H9 subtype influenza virus double antibody sandwich ELISA kit and the double antibody sandwich ELISA kit and detection method. Background technique [0002] In 1975, Kohler G and Milstein C published the cell fusion method to establish hybridoma technology in Nature magazine, creating an epoch-making hybridoma monoclonal antibody technology. After cloning, high-purity antibodies with identical structures and various characteristics are produced, which are called monoclonal antibodies (Monoclonal Antibody, McAb), or monoclonal antibodies for short. The discovery and use of monoclonal ...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/577G01N33/569G01N33/543
Inventor 周红波金梅林陈焕春程艳青但汉并郭学波张艳黄慧敏刘小坤
Owner HUAZHONG AGRI UNIV
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