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Anti-koi herpes virus monoclonal antibody and its cell line and application

A koi herpes virus and monoclonal antibody technology, applied in the direction of anti-viral immunoglobulin, chemical instruments and methods, biochemical equipment and methods, etc., can solve false positives, PCR detection missed detection, unfavorable virus amplification virus antibody Preparation and other issues, to achieve a highly specific effect

Active Publication Date: 2020-03-31
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Because the separation of KHV cells is difficult, it is not conducive to the large-scale amplification of the virus and the preparation of virus antibodies, which makes it difficult to carry out serological detection methods. At present, the detection of KHV is mainly based on the PCR method, but there are problems of missed detection and false positives in PCR detection. And mostly only as a qualitative detection of the virus
So far, there have been no reports of monoclonal antibodies with strong specificity against koi herpes virus

Method used

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  • Anti-koi herpes virus monoclonal antibody and its cell line and application
  • Anti-koi herpes virus monoclonal antibody and its cell line and application
  • Anti-koi herpes virus monoclonal antibody and its cell line and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of the monoclonal antibody of embodiment 1 anti-koi herpes virus

[0050] 1. Antigen Preparation

[0051] CCB (common carp brain cells) is a koi herpes virus sensitive cell line. When 80% of CCB cells are covered with cell culture flasks, the virus titer is 10 6 TCID 50 / ml of KHV-GZ1301 virus strain was inoculated in CCB, and the infected cells were placed in a 22°C incubator, and observed every day. The cells were repeatedly frozen and thawed at -80°C, the cells were lysed to release the virus particles, the virus suspension was collected, and the virus suspension was purified by 20% to 66% sucrose density gradient ultracentrifugation, and the Koi herpes virus was confirmed by electron microscope observation The samples were then stored at -80°C.

[0052] 2. Immunization of Mice

[0053] The mice used for immunization were 5-week-old female BALB / c mice of SPF grade. Each mouse was subcutaneously injected with the above-mentioned purified koi herpe...

Embodiment 2

[0062] Example 2: Characterization of Monoclonal Antibodies Against Koi Herpes Virus

[0063] 1. Ascites titer identification of monoclonal antibodies

[0064] Methods: The titer of monoclonal antibody in ascites was identified by indirect ELISA.

[0065] Result: the monoclonal antibody ascites titer of the present invention is 1:1.28×10 5 , indicating that the hybridoma cell line has the ability to secrete high-titer antibody.

[0066] 2. Isotype Identification of Monoclonal Antibody

[0067] Method: The typing kit (SBA Clonotyping TM System / HRP), and identify the Ig subtype of the monoclonal antibody of the present invention according to its instructions.

[0068] Results: The subtype of the monoclonal antibody of the present invention is IgG2b.

[0069] 3. Specificity analysis of monoclonal antibodies

[0070] Western blotting: The purified koi herpes virus was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins on the ...

Embodiment 3

[0074] Embodiment 3 detects the ELISA kit of koi herpes virus

[0075] The ELISA kit that detects koi herpes virus consists of: the monoclonal antibody prepared in Example 1; the solid-phase carrier coated with the polyclonal antibody against koi herpes virus; horseradish peroxidase-labeled goat anti-mouse antibody ( purchased from sigma company); enzyme substrate reaction solution; positive control; negative control; washing solution; reaction termination solution.

[0076] Among them, preparation of rabbit polyclonal antibody against koi herpes virus and embedding ELISA strip plate: use the purified recombinant expression protein of koi herpes virus as antigen, immunize rabbits by multi-point injection, and immunize 4 times in total; Polyclonal antibodies against koi herpesvirus. Dilute the purified rabbit anti-koi herpes virus polyclonal antibody to a concentration of 2 μg / ml with a coating buffer (0.05mol / L carbonate buffer, pH 8.2), and coat a 96-well ELISA plate (USA C...

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Abstract

The invention discloses a preparation method and application of a monoclonal antibody against koi herpesvirus (KHV). The prepared monoclonal antibody against KHV is secreted by a mouse hybridoma cellstrain of which the preservation number is CCTCC NO: C201887, and the titer of the monoclonal antibody is 1:(1.28 is multiplied by 105); and the hybridoma cell strain has high activity, can recognizethe structural protein of the KHV, and has strong specificity. On the basis, a sandwich ELISA detection method for detecting the KHV is established, and the minimum detection concentration is 3.923ng / mL. The monoclonal antibody is utilized for establishing an indirect sandwich ELISA kit for detecting the KHV, and the kit can be used for large-scale detection of the KHV and has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody against koi herpes virus, a cell line and application thereof. Background technique [0002] Carp herpesvirus type 3, also known as Koi herpesvirus (Koi herpesvirus, KHV), is the pathogen that causes carp, Koi and its variants to occur Koi herpesvirus disease (KHVD), with a mortality rate of up to 80%. More than, the carp of many countries and regions all over the world and koi breeding industry have caused huge economic loss. Koi herpes virus disease is a fish disease identified at the end of the 20th century. It is a disease that seriously threatens the safety of carp and koi farming. The World Organization for Animal Health (OIE) lists it as a disease that must be declared. Classified as a second-class disease. Since koi herpes virus disease was first discovered in the UK in 1996, the disease has broken out in Germany, Belgium, France, Poland, J...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/08G01N33/577G01N33/535
CPCC07K16/085G01N33/535G01N33/577G01N2333/03
Inventor 李莹莹王庆曾伟伟王英英石存斌尹纪元任燕
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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