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Anti-H9 subtype avian influenza virus monoclonal antibody, hybridoma cell strain and application of monoclonal antibody

A hybridoma cell line, monoclonal antibody technology, applied in the field of immunochemistry, to achieve good specificity and sensitivity, good application prospects

Active Publication Date: 2017-05-31
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no method for specifically detecting H9 subtype AIV-specific antibodies. In order to better understand the antibody levels of H9 subtype AIV in chickens and humans, a large-scale, accurate, sensitive, safe and convenient method should be established. Antibody detection methods are necessary

Method used

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  • Anti-H9 subtype avian influenza virus monoclonal antibody, hybridoma cell strain and application of monoclonal antibody
  • Anti-H9 subtype avian influenza virus monoclonal antibody, hybridoma cell strain and application of monoclonal antibody
  • Anti-H9 subtype avian influenza virus monoclonal antibody, hybridoma cell strain and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Preparation and Identification of H9N2 Subtype Avian Influenza Virus (H9N2 AIV) SH441 Strain Monoclonal Antibody

[0029] 1. Materials and Methods

[0030] 1.1 Viruses, cells and experimental animals

[0031] H9N2 subtype avian influenza virus SH441 strain (A / chicken / Shanghai / 441 / 2009) was isolated and preserved by our laboratory; MDCK cells, 293T cells and SP2 / 0 cells were preserved by our laboratory; SPF chicken embryos were purchased from Beijing Merial Weitong Experimental Technology Co., Ltd.; clean grade BALB / c mice were purchased from Shanghai Slack Experimental Animal Co., Ltd.; 4-5 week-old test chickens were hatched from SPF chicken embryos and raised in a negative pressure isolator. The pCAGGS-H9-HA recombinant eukaryotic plasmid was constructed and preserved in our laboratory.

[0032] 1.2 Main materials and test serum

[0033]DMEM and Opti-MEM medium were purchased from GBICO; 8-azaguanine, PEG1450, HAT, HT, Freund's complete adjuvant, Freund's...

Embodiment 2

[0050] Example 2 Establishment of H9N2 AIV antibody blocking ELISA method (blocking ELISA, bELISA)

[0051] 1. H9N2 AIV antibody blocking ELISA operation method

[0052] (1) Coat the purified SH441 strain inactivated virus antigen with pH9.6 carbonate buffer solution, 100ng / well per well, overnight at 4°C. After taking it out the next day, wash it three times with 0.05% Tween-20PBS (PBST), 3 minutes each time;

[0053] (2) Block the microtiter plate with PBS of 5% skim milk, 37°C for 2 hours, wash 3 times, the method is the same as above;

[0054] (3) Add the serum to be tested, negative and positive controls diluted 10 times with antibody diluent, incubate at 37°C for 1 hour at 100 μL per well, and wash as above;

[0055] (4) 100 μL S1B1 monoclonal antibody ascites fluid (1000×) per well, incubate at 37°C for 1 hour, wash 3 times;

[0056] (5) Add 100 microliters of horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (5000×) diluted in antibody diluent ...

Embodiment 3

[0078] Embodiment 3 detects the composition and application of the blocking ELISA (B-ELISA) kit of H9 subtype avian influenza virus specific antibody

[0079] 1. The composition of the kit

[0080] According to the contents of the kits listed in Table 4, assemble into kits, and store under corresponding conditions after assembly.

[0081] Table 4 Contents of H9 Subtype Avian Influenza Virus Antibody Blocking ELISA Detection Kit

[0082] Numbering content quantity Remark 1 Removable microtiter plate to be coated 1 block 8×12 format, store at 4°C 2 coat antigen a tube 100μL / tube, store at -20℃ 3 Coating buffer (1×) a bottle 10mL / bottle, store at 4°C 4 blocking solution a bottle 20mL / bottle, store at 4°C 5 positive control serum 1 tube 50μL / tube, store at -20℃ 6 negative control serum 1 tube 50μL / tube, store at -20℃ 7 Monoclonal antibodies 1 tube 100μL / tube, store at -20℃ 8 HRP-anti-mouse ...

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Abstract

The invention discloses an anti-H9 subtype avian influenza virus monoclonal antibody which is bound with HA protein of an H9 subtype avian influenza virus and has hemagglutination inhibition activity. The invention further discloses a reagent kit for detecting the H9 subtype avian influenza virus antibody, and an application of the monoclonal antibody to preparation of an avian influenza diagnosis product. The reagent kit for detecting the H9 subtype avian influenza virus antibody has good specificity and sensitivity, is applicable to antibody detection and screening of massive different host serums, and has good application prospects in diagnosis and the antibody detection of H9 subtype avian influenza virus diseases.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a monoclonal antibody against H9 subtype avian influenza virus, a hybridoma cell line, and a blocking ELISA reagent for detecting H9 subtype avian influenza virus-specific antibodies using the monoclonal antibody box. Background technique [0002] Avian influenza (AI) is an acute infectious disease caused by type A influenza virus that seriously endangers the health of poultry. Since the first outbreak of the disease in chickens in Italy in 1878, AI outbreaks have been caused by various subtypes of avian influenza viruses all over the world. Among them, the H9 subtype of avian influenza was first isolated from a turkey farm in Wisconsin, USA in 1966. Since the 1990s, it has spread more and more widely, and has become endemic in many countries in Asia, the Middle East and North Africa. , Bring heavy blow to poultry industry, also cause immeasurable economic loss. In my ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983G01N33/577
Inventor 李泽君杨健美滕巧泱李雪松戴晓光
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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