Canine adenovirus type 2 monoclonal antibody, variable region sequence, hybridoma cell and application thereof

A monoclonal antibody, canine adenovirus technology, applied in the direction of antibodies, antiviral agents, antiviral immunoglobulins, etc., can solve the problems of scattered toxin, inability to resist virus, complicated preparation procedures of hyperimmune serum, etc. toxicity, reducing morbidity, and reducing mortality

Active Publication Date: 2019-12-31
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The prevention and treatment of dogs are currently mainly immunized with vaccines containing canine adenovirus type 2 and hyperimmune serum, but the canine adenovirus type 2 strains contained in the vaccine are all attenuated strains, and there is a risk of shedding the virus after immunization of animals, and the immune It still takes a while to produce antibodies afterward, and the resistance to the virus cannot be quickly established; and the preparation procedure of hyperimmune serum is complicated, not only need to screen qualified experimental animals, but also to immunize with attenuated and strong viruses for many times (Li Zhimin et al. Development and application of high-valent immune serum. Chinese Journal of Immunology, 2005,21(7):521-523)

Method used

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  • Canine adenovirus type 2 monoclonal antibody, variable region sequence, hybridoma cell and application thereof
  • Canine adenovirus type 2 monoclonal antibody, variable region sequence, hybridoma cell and application thereof
  • Canine adenovirus type 2 monoclonal antibody, variable region sequence, hybridoma cell and application thereof

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Experimental program
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Effect test

Embodiment 1

[0058] Embodiment 1 Existing product detection clinical situation analysis

[0059] 300 clinical canine disease materials were collected, 180 were positive and 120 were negative by CAV-2 PCR method, 82 were positive and 218 were negative by commercial test strips. The coincidence rate between commercial test strips and PCR methods is low, and there are serious missed detections, which mislead doctors or farmers to relax their vigilance and prevent early prevention and treatment. Through the follow-up investigation, it was found that the missed detection and false-negative dogs were subsequently infected more seriously, and the secretions of the sick dogs were used as the source of infection to cause mass infections, and severe cases resulted in death due to lack of timely treatment. Based on this, the inventors carried out research on canine adenovirus type 2 monoclonal antibody and related products.

[0060] In addition, the epidemiological survey found that the existing com...

Embodiment 2

[0061] Example 2 Preparation, Purification, Identification and Testing of Canine Adenovirus Type 2 Monoclonal Antibody

[0062] 2.1 Preparation and purification of canine adenovirus type 2 monoclonal antibody

[0063] Cultivate canine adenovirus type 2, harvest the cell culture, centrifuge at 3000 rpm for 30 minutes after freezing and thawing, take the supernatant as the antigen and emulsify it with Freund's adjuvant, and immunize mice with a final content of 200 μg / ml. mice for cell fusion. Use the HI method (the CAV-2 Toronto A26 / 61 strain derived from ATCC uses human type O erythrocytes to conduct the erythrocyte agglutination test, that is, the HA test according to the "Chinese Veterinary Pharmacopoeia", prepare 8 units of antigen according to the results, and then conduct the erythrocyte agglutination test on the samples to be tested respectively. The agglutination inhibition test (HI titer detection, carried out according to "Chinese Veterinary Pharmacopoeia") was carri...

Embodiment 3

[0095] Preparation and application of embodiment 3 test strips

[0096] 3.1 Preparation and detection of colloidal gold test strips

[0097] 3.1.1 Preparation and testing of test strips 1-4

[0098] Heat and boil 0.01% chloroauric acid solution, then add 1% trisodium citrate solution to prepare colloidal gold, and restore to the original volume with distilled water after cooling to room temperature.

[0099] With 0.2mol / L K 2 CO 3 Adjust the pH of the colloidal gold solution to 7.5. After stirring at a constant speed, add the monoclonal antibody 4H12 solution (working concentration: 10-60 μg / ml) to the colloidal gold solution for labeling. After stirring, add an appropriate amount of 10% BSA drop by drop, and stir at a constant speed. 30min. After standing still at 2-8°C for 2 hours, centrifuge at 2000r / min at 4°C for 30min, and dissolve the precipitate with 1 / 10 volume of PBS buffer containing 1% BSA to obtain the gold-labeled monoclonal antibody 4H12. The standard monoc...

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Abstract

The invention provides a variable region sequence of a mouse monoclonal antibody specifically binding to canine adenovirus type 2 fiber protein, and an antibody specifically binding to canine adenovirus type 2 fiber protein conformational epitope. The antibody can be used for preparing kits and pharmaceutical compositions. The kit provided by the invention overcomes the problem of low detection sensitivity in the prior art, avoids missing detection and false negative phenomena, can detect various targets, and has the advantages of rapidness, simplicity, convenience and accuracy; and the pharmaceutical composition disclosed by the invention can be used for effectively preventing and treating diseases caused by CAV-2.

Description

technical field [0001] The invention relates to a canine adenovirus type 2 monoclonal antibody, a variable region sequence, a hybridoma cell secreting the monoclonal antibody, a pharmaceutical composition prepared by using the monoclonal antibody, a kit and application thereof, belonging to the field of biotechnology. Background technique [0002] Both canine infectious laryngotracheitis and enteritis are caused by canine adenovirus type II (CAV-2) and have a global distribution. Canine infectious laryngotracheitis can infect dogs of different breeds, ages and sexes, and can occur in all seasons, with the highest infection rate and mortality in puppies; the clinical symptoms are complex, including persistent high fever, cough, serous to mucous nasal Lesions, tonsillitis, laryngotracheitis, pneumonia and other respiratory symptoms; easy to mix infection or secondary infection with other viruses such as canine distemper virus, canine parvovirus, canine parainfluenza virus, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N5/20G01N33/569A61K39/395A61P31/20
CPCC07K16/081G01N33/56983A61P31/20C07K2317/56G01N2333/075A61K2039/505
Inventor 田克恭王莹
Owner LUOYANG PULIKE WANTAI BIOTECH
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