Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, applied in the biological field, can solve problems such as hybridoma cell lines that cannot secrete anti-amantadine monoclonal antibody, and achieve accurate immune detection and immune analysis, immune titer High, specific effect

Active Publication Date: 2015-04-01
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The core of hybridoma technology to prepare monoclonal antibodies is to obtain ideal hybridoma cell lines, but there is no hybridoma cell line that can secrete anti-amantadine monoclonal antibodies in the prior art

Method used

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  • Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain
  • Hybridoma cell strain secreting anti-amantadine monoclonal antibody and application of hybridoma cell strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation of embodiment 1 hybridoma cell line

[0030] (1) Immunization of mice

[0031] Select 8-week-old BALB / c female mice to mix 10 μg of amantadine artificial antigen and quick antibody-5w adjuvant in equal volumes to form 100 μl, and inject the rear calf muscle of the mouse; the rear calf muscle part is exposed to the mouse leg Where there is a large muscle on the outside, and most of the mouse's thighs are covered by the abdominal skin, the knee joints can also be found to distinguish the calf from the thigh; two weeks later, the second injection was injected in the same way; two weeks later, intraperitoneal injection was performed with 10 μg of antigen injection.

[0032] The molecular structural formula of amantadine artificial antigen is shown in formula (I):

[0033]

[0034] In formula (I), BGG is bovine gamma globulin.

[0035] (2) Preparation of lymphocytes

[0036] Get the spleen of the BALB / c female mouse treated through step (1), grind the ...

Embodiment 2

[0064] Example 2 Reactivity test of anti-amantadine monoclonal antibody

[0065] The reactivity of the anti-amantadine monoclonal antibody prepared in Example 1 was detected by an indirect ELISA method, and the detection steps included: ① Dilute the amantadine artificial antigen to 500 ng / mL with a carbonate buffer solution with a pH of 9.6 and a concentration of 0.05M , then add 100 μl / well diluted amantadine artificial antigen to the 96-well ELISA plate, and coat overnight at 4°C;

[0066] ②Wash the plate 3 times with PBS buffer containing 0.05% tween20; then add 100 μl anti-amantadine monoclonal antibody solution with a concentration of 0.3 μg / ml, make a 1:3 dilution, and set up a negative control at the same time, react at 37 ° C for 60 minutes Afterwards, wash the plate once with PBS buffer containing 0.05% tween20;

[0067] ③ Add PBS buffer to goat anti-mouse IgG-HRP and dilute to 5000 times the volume, then add 100 μl / well diluted goat anti-mouse IgG-HRP to a 96-well c...

Embodiment 3

[0072] Such as figure 1 As shown, a kind of amantadine colloidal gold detection test strip of the present embodiment comprises sample pad, colloidal gold pad, nitrocellulose membrane and water absorption pad; Sample pad, colloidal gold pad, nitrocellulose membrane and water absorption pad are sequentially head to tail Lap on the base plate. Wherein, the colloidal gold pad is coated with the anti-amantadine monoclonal antibody prepared in Example 1 labeled with colloidal gold, and the detection line (T) of the nitrocellulose membrane is coated with the amantadine-bovine serum albumin complex, The quality control line (C) is coated with goat anti-mouse IgG. The coating amount of amantadine-bovine serum albumin complex is the detection limit of amantadine by the anti-amantadine monoclonal antibody (take 20 μg / kg as an example). The preparation method of the test strip is a conventional method in the art.

[0073] When testing, insert one end of the test strip sample pad into t...

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Abstract

The invention discloses a hybridoma cell strain secreting an anti-amantadine monoclonal antibody and application of the hybridoma cell strain. The name of the hybridoma cell strain is hybridoma cell strain 7G4.3, and the collection number is CCTCC No. C2014169. The hybridoma cell strain is obtained by immunizing a mouse with a self-designed amantadine artificial antigen and using spleen cells of the immunized mouse, the anti-amantadine monoclonal antibody secreted by the hybridoma cell strain has high titer and strong specificity, and can be used for fast and accurate immunodetection and immunoassay of amantadine.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell strain secreting anti-amantadine monoclonal antibody and application thereof. Background technique [0002] Amantadine can inhibit virus reproduction and is clinically used to prevent and treat various type A influenza virus infections. However, due to economic interests and the pressure of disease prevention and control, amantadine is abused in poultry breeding in my country. The residual accumulation of amantadine will cause drowsiness, insomnia, dizziness, depression, nausea and other symptoms to consumers; long-term use will also make viruses such as avian influenza resistant to drugs, and induce new drug-resistant viruses. [0003] However, in November 2012, the media revealed that domestically raised "fast-growing chickens" only take 45 days from hatching to being served on the dining table. In order to ensure their survival, unscrupulous traders f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/68C12R1/91
Inventor 钱鸣蓉苏娟杨华张巧艳汪建妹邵越水肖英平郑孝君陈东
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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