Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application

A monoclonal antibody and hybridoma cell technology, applied in the direction of antibodies, antibody medical components, and methods based on microorganisms, can solve economic losses, sequelae of sick animals, affect the health of dogs and other susceptible animals, etc., and achieve improvement The effect of the detection rate

Active Publication Date: 2016-06-22
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mortality rate of infected dogs is as high as 30%-80%, which seriously affects the health of dogs and other susceptible animals, causes a large number of dogs to die, and can cause serious sequelae of sick animals, resulting in serious economic losses
[0004] At present, the detection methods of canine distemper virus at home and abroad include electron microscope observation, avidin-biotin-complex method (ABC), immunofluorescence method (IFA), polymerase chain reaction method (PCR), and detection requires expensive Equipment and professional technicians limit its application in large-scale screening, making these methods biased towards laboratory diagnosis and not suitable for grassroots or on-site rapid detection

Method used

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  • Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application
  • Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application
  • Mouse bone marrow hybridoma cell strains, monoclonal antibody generated by same and application

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Embodiment approach

[0026] As an embodiment of the present invention, the monoclonal antibody is monoclonal antibody 6E11, which is secreted and produced by the mouse bone marrow hybridoma cell line 6E11.

[0027] The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6E11 is the amino acid sequence shown in SEQ ID No.2; the amino acid sequence of the light chain variable region of the monoclonal antibody 6E11 is the amino acid sequence shown in SEQ ID No.4.

[0028] The relative affinity constant of the monoclonal antibody 6E11 of the present invention is 1:5120, that is to say, its binding strength with the epitope of the antigen is very high; the monoclonal antibody 6E11IPMA (immunoperoxidase monolayer cell assay) The titer is 1:12800, and it has good reactivity with canine distemper virus.

[0029] The present invention relates to the mouse bone marrow hybridoma cell 1G5 strain (Hybridoma-Balb / cmousespleencellsandSp2 / 0, strain1G5) preserved in the China Center f...

Embodiment 1

[0074] Embodiment 1 Preparation, purification, identification and inspection of anti-canine distemper virus monoclonal antibody

[0075] 1.1 Preparation and purification of anti-canine distemper virus monoclonal antibody

[0076] Canine distemper virus CVCCAV299 strain (see http: / / cutke.com / index.php / Good / index / model / 0 / id / 92311.html) was inoculated into Vero cells, and harvested when the lesion was 85% Viruses were quickly frozen and thawed once at -40°C, centrifuged at 3000r / min for 30min, and cell debris was removed and then aliquoted. According to the operation method of HarlowE et al. (HarlowE, LaneD. Antibodies: laboratorymanual. NewYork: Cold Spring Harbor Laboratory Press, 1998, 139-312), the mouse bone marrow hybridoma cell 6E11 strain and the mouse bone marrow hybridoma cell 1G5 strain were prepared and submitted for preservation. Further using the above two cell lines, anti-canine distemper virus monoclonal antibody 6E11 and anti-canine distemper virus monoclonal an...

Embodiment 2

[0124] Example 2 Preparation and detection method, quality research and application of canine distemper virus antigen detection kit

[0125] 2.1 Preparation and detection method of antigen detection kit

[0126] Kit 1 is the preparation of enzyme immunochromatography detection test strips: prepare enzyme immunochromatography detection test strips according to CN104062430A (such as figure 1 shown), the monoclonal antibody 1G5 prepared in Example 1 was enzyme-labeled, and the monoclonal antibody 6E11 prepared in Example 1 and goat anti-mouse IgG (purchased from Sigma Company) were sprayed on the nitrocellulose membrane as a detection line and the quality control line, the nitrocellulose membrane, the enzyme label pad and the substrate pad are packed in a plastic casing to make an enzyme immunoassay test strip. And prepare phosphate buffered saline as sample treatment solution. When testing, mix the sample with the sample treatment solution, add 2 to 3 drops (about 80 μl) of th...

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Abstract

The invention provides two mouse bone marrow hybridoma cell strains, a monoclonal antibody generated by the same and application, canine distemper virus resistant monoclonal antibodies 6E11 and 1G5 respectively secrete by the mouse bone marrow hybridoma cell strains, a prepared detecting kit and pharmaceutical composition containing the monoclonal antibodies 6E11 and 1G5, and application of the detecting kit and pharmaceutical composition, and belongs to the technical field of biology. The prepared canine distemper virus antigen detection kit containing the monoclonal antibodies 6E11 and 1G5 is high in sensitivity, and is easy and convenient to operate, quick and time-saving, or the detection rate of CDV infection is improved, application in the aspect of large-scale screening is facilitated, and the prepared canine distemper virus antigen detection kit containing the monoclonal antibodies 6E11 and 1G5 is suitable for quick detection in grassroots or in site.

Description

technical field [0001] The invention relates to a canine distemper virus mouse bone marrow hybridoma cell line, a monoclonal antibody produced by it, a detection kit containing the monoclonal antibody and an application thereof, belonging to the field of biotechnology. Background technique [0002] Canine distemper virus (CDV) belongs to the Morbillivirus genus of the Paramyxoviridae family. The viral nucleic acid is single-stranded RNA, and the virus particles are round or shapeless, sometimes filamentous. CDV is sensitive to heat and dryness, and can be inactivated at a temperature of 50-60°C for 30 minutes. CDV cannot survive in dogs for a long time in hot seasons. At cooler temperatures, CDV can survive for longer periods of time, several weeks at 2-4°C and more than 7 years at -60°C. [0003] Canine distemper is caused by canine distemper virus. It is an acute and highly contagious infectious disease of Canidae, Procyonidae and Mustelidae. It is characterized by biphas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10A61K39/42A61P31/14G01N33/577G01N33/569G01N33/558C12R1/91
CPCA61K2039/505A61K2039/54A61K2039/545C07K16/1027C07K2317/56G01N33/558G01N33/56983G01N33/577
Inventor 田克恭郝丽影
Owner LUOYANG PULIKE WANTAI BIOTECH
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