H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use

A technology of monoclonal antibody and avian influenza virus, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems that cannot meet the needs of clinical testing

Inactive Publication Date: 2006-08-09
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
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  • H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use
  • H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use

Examples

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Embodiment 1

[0042] Embodiment 1: Preparation of anti-H5 subtype avian influenza virus HA gene monoclonal antibody

[0043] 1) Antigen preparation:

[0044] Nine-day-old fertilized chicken embryos were inoculated with the H5N1-Yu22 strain virus, incubated at 30°C for 2 days, and the chicken embryo liquid was collected to obtain the amplified H5N1-Yu22 strain virus. Collect the live virus and inactivate it with 0.03% formalin at 4°C. The inactivated virus is detected by HA to determine the titer of the inactivated virus solution (Note: For the specific methods of HA titer determination and HI detection, refer to WHO operation guideline, we chose HA=1024, which was provided by the Department of Microbiology, University of Hong Kong).

[0045] 2) White mice: female, 6-week-old Balb / c mice were purchased from the Anticancer Center of Xiamen University, and were bred in the center for experiments.

[0046] 3) Preparation of hybridoma:

[0047] We use standard in vivo immunization methods and...

Embodiment 2

[0060] Example 2: Preparation of H5 Subtype Influenza Virus HA Recombinant Antigen

[0061] 1) Bacmid-H5HA recombinant virus obtained

[0062] Select the HA full-length cDNA gene of the avian influenza AIV-H5N1-Yu22 virus strain as a template (provided by the Department of Microbiology, University of Hong Kong), design a pair of primers (synthesized by Shanghai Boya), set BamHI at the 5' end, and EcoRI at the 3' end, and carry out PCR amplification, using gel recovery kit (Hua Shun company) to recover the PCR product, to obtain a DNA fragment of about 1.7kb, and then clone it into the pMD18-T vector (Dalian Bao Biological Company), and obtain the correct DNA fragment by enzyme digestion and sequencing. Cloning of plasmid pMD18-T-H5HA. Then the target fragment was digested with BamHI and EcoRI from pMD18-T-H5HA to obtain the H5HA gene, and then ligated into the pFastBacl vector, and the ligated material was transformed into CaCl 2 The sensitized E.coli.DH5α was identified to ...

Embodiment 3

[0068] Example 3: Assembly of H5 subtype influenza virus HA antigen detection kit (enzyme-linked immunoassay, ELISA).

[0069] 1) Principle:

[0070] This kit uses the double-antibody sandwich method to detect the HA antigen of the H5 subtype influenza virus in the specimen.

[0071] Firstly, the monoclonal antibody against H5 influenza virus HA gene is pre-coated on the polyethylene microwell plate strip of the kit. When the cleaved H5 influenza virus HA antigen is added to the microwell, the pre-coated monoclonal antibody It can be captured, and then the enzyme-labeled monoclonal antibody added can also bind to it, and then the result can be judged by the degree of color development of the enzyme-catalyzed substrate. When the specimen does not contain influenza virus antigen or H5 influenza virus, the substrate will not develop color. Detectable specimens include excreta, oral and nasal secretions, whole virus or lysed virus cultured in chicken embryos, etc.

[0072] 2) P...

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Abstract

This invention relates to a monoclonal antibody capable of combining with H5 subtype avian influenza virus HA protein specifically, the hybridoma cell line secreting said antibody and a preparing method. The invention also relates to a serial test kit for testing H5 subtype avian influenza virus by the antibody and a bit of said antibody in the test sample of the virus and its usage in treatment.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of specifically binding H5 subtype avian influenza virus hemagglutinin (HA) protein, a hybridoma cell line secreting the monoclonal antibody, and a method for preparing the monoclonal antibody. The present invention also relates to a series of detection kits for detecting H5 subtype avian influenza virus in a sample prepared by using the monoclonal antibody, and the detection kit and treatment of the H5 subtype avian influenza virus in a sample by the monoclonal antibody the use of. Background of the invention [0002] Since H5 avian influenza first broke out in geese on farms in Guangdong Province in my country in 1996 (Xu X et al, 1999, Virology), another strain of H5 virus derived from this virus has spread to poultry farms in Hong Kong (1997). April) and the market (November 1997) broke out, which caused the first direct human-to-human transmission of avian influenza virus in history. There wer...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/44C12N15/63G01N33/577
Inventor 夏宁邵张军罗文新陈毅歆葛胜祥管轶陈鸿霖
Owner XIAMEN UNIV
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