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Monoclonal antibody of anti lymphocyst virus of and preparation method

A technology of lymphocyst virus and monoclonal antibody, which is applied in the cross field of molecular immunology and virology, to achieve the effect of good adhesion, uniform cell shape and strong refraction

Active Publication Date: 2005-08-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report on the development of monoclonal antibody against flounder lymphocyst virus

Method used

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  • Monoclonal antibody of anti lymphocyst virus of and preparation method
  • Monoclonal antibody of anti lymphocyst virus of and preparation method
  • Monoclonal antibody of anti lymphocyst virus of and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Development of monoclonal antibodies against flounder lymphocyst virus:

[0038] 1. Purification of flounder lymphocyst virus:

[0039] (1) Take the diseased flounder, first use 70% alcohol cotton ball to disinfect the affected part, then cut off the cyst with a sterilized scalpel, add an appropriate amount of quartz sand and 10 times the volume of TNE buffer (100mM NaCl, 1mM EDTA, 50mM Tris , pH 7.4), homogenate;

[0040] (2) The homogenate was centrifuged at 4°C, 500g, for 30min, and the supernatant was taken;

[0041] (3) The supernatant was centrifuged at 4°C, 1800g, for 30min, and the supernatant was taken again;

[0042] (4) Prepare 30% (W / V) sucrose solution with sucrose and supernatant, centrifuge at 78500g for 120min at 4°C, discard supernatant;

[0043] (5) Add TNE to the precipitate to 1ml, place it lightly above the sucrose gradient (37%, 40%, 47%, 52%, 57%, 62%) (W / V), centrifuge at 78500g for 120min at 4°C ;

[0044] (6) Carefully suck out the virus b...

Embodiment 2

[0081] Identification of flounder lymphocyst virus monoclonal antibody of the present invention:

[0082] 1. Identification by indirect immunofluorescence antibody method:

[0083] First, the production of frozen sections:

[0084] Fresh cysts were taken, cut into small pieces about 3mm square, washed with normal saline, blotted dry, embedded with frozen embedding agent, placed at -20°C for 30min, and sliced ​​at -20°C with a cryostat, with a thickness of 5 μm. The slices were fixed with acetone for 20 min, dried at room temperature, and frozen at -20°C for later use.

[0085] Second, fluorescent antibody staining:

[0086] (1) The culture supernatant of the hybridoma cells screened and cloned above was used as the primary antibody, and added to the slice.

[0087] (2) Incubate in a humid box at 37°C for 45 minutes.

[0088] (3) Take out the glass slide and wash it 3 times with 0.01M phosphate buffered saline, 5 min each time.

[0089] (4) Goat anti-mouse IgG antibody lab...

Embodiment 3

[0110] Using the developed monoclonal antibody against flounder lymphocyst virus to identify Sebastesschlegeli lymphocyst virus;

[0111] First, the production of frozen sections:

[0112] Take the diseased flat scorpionfish cyst tissue, cut into small pieces about 3 mm square, wash with normal saline, blot dry, embed with frozen embedding agent, place at -20°C for 30min, and slice at -20°C with a frozen microtome. The slice thickness is 5 μm. The slices were fixed with acetone for 20 min, dried at room temperature, and frozen at -20°C for later use;

[0113] Second, fluorescent antibody staining:

[0114] (1) Aspirate 15 μl of monoclonal antibody against flounder lymphocyst virus and add it to the slice.

[0115] (2) Incubate in a humid box at 37°C for 45 minutes;

[0116] (3) Take out the glass slide and wash it 3 times with 0.01M phosphate buffered saline, 5 min each time.

[0117] (4) Goat anti-mouse IgG antibody labeled with fluorescein isothiocyanate, added to the s...

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Abstract

Two monoclonal antibodies B and C for the lymphocystics virus of bastard halibut are prepared from two hybrid tumor cells (CCTCC-C200419 and CCTCC-C200420) through conventional culture for 2-3 days to secrete a lot of said monoclonal antibodies B and C.

Description

technical field [0001] The present invention relates to the improvement of antibody application technology, specifically a monoclonal antibody against flounder (Paralichthys olivaceus) lymphocystis virus (Lymphocystis virus) secreted by hybridoma cells and its preparation method, belonging to the intersection of molecular immunology and virology technology field. Background technique [0002] Lymphocystic disease is a chronic viral disease. Many cysts appear on the epidermis, fins and tail of diseased fish. The pathogen of lymphocystis disease is Lymphocystis virus (Lymphocystis virus), which belongs to the family Iridoviridae. Lymphocyst disease is endemic in a wide area and is distributed worldwide. It is currently known that at least 42 families and more than 125 species of fish can be infected by lymphocyst virus. In recent years, the disease has occurred in perch, grouper, red sea bream, turbot and American redfish in Japan and my country's Guangdong, Fujian, Zhejiang...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C07K16/28C12P21/08G01N33/577
Inventor 战文斌程顺峰
Owner OCEAN UNIV OF CHINA
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