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CAV-1 monoclonal antibody, variable region sequence, hybridoma cell strain and application thereof

A monoclonal antibody, canine adenovirus technology, applied in the biological field, can solve the problems of inability to resist virus, complex preparation procedure of high immunity serum, etc. Effect

Active Publication Date: 2019-12-31
LUOYANG PULIKE WANTAI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The prevention and treatment of canine CAV-1 and 2 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 are scattered viruses after immunization of animals and it will take some time to produce antibodies after immunization, and the resistance to the virus cannot be quickly established; while 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 canine polyvalent hyperimmune serum. Chinese Journal of Immunology, 2005,21(7):521-523)

Method used

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  • CAV-1 monoclonal antibody, variable region sequence, hybridoma cell strain and application thereof
  • CAV-1 monoclonal antibody, variable region sequence, hybridoma cell strain and application thereof
  • CAV-1 monoclonal antibody, variable region sequence, hybridoma cell strain and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 Existing product detection clinical situation analysis

[0069] Of the 193 clinical samples of dogs and foxes collected, 95 were positive and 98 were negative by PCR detection method, and 24 were positive and 169 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 follow-up investigations, it was found that dogs and foxes with missed tests and false negatives usually subsequently infect more serious patients, and their excrement or secretions are used as the source of infection to cause mass infections, and severe cases lead to death due to lack of timely treatment. Based on this, the inventors carried out research on canine adenovirus type 1 monoclonal antibody and related products.

[0070] In addition, epidemiological surveys found that the ...

Embodiment 2

[0071] Example 2 Preparation, purification, identification and testing of canine adenovirus type 1 monoclonal antibody

[0072] 2.1 Preparation and purification of canine adenovirus type 1 monoclonal antibody

[0073] Cultivate canine adenovirus type 1, 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-1 Utrecht strain derived from ATCC uses human type O erythrocytes to carry out the hemagglutination test, namely the HA test, according to the "Chinese Veterinary Drug Code", prepare 8 units of antigen according to the results, and then carry out the hemagglutination inhibition test for the samples to be tested respectively That is, HI titer detection, according to "Chinese Veterinary Pharmacopoeia") subcloning screening and culture were carried out to obtain 10...

Embodiment 3

[0100] Embodiment 3 establishment and application of immunohistochemical method

[0101] 3.1 Establishment of immunohistochemical method

[0102] The method of immunohistochemistry includes the following steps:

[0103] 1) Material collection: Tissue samples such as liver, gallbladder, kidney, and intestine of dogs were collected, fixed in formalin quickly, and appropriately trimmed when necessary.

[0104] 2) Preparation of paraffin sections: After the tissue samples are fixed, they are rinsed with running water and then dehydrated, transparent, and soaked in wax with an automatic dehydrator. Oxysilane-treated slides were baked on the slides.

[0105] 3) Inhibition of endogenous enzymes: Dewax the slides to distilled water as usual, add 3% H 2 o 2 Stand to inhibit endogenous enzymes.

[0106] 4) Antigen retrieval: Use antigen heat restoration such as high pressure heat restoration, boiling heat restoration, microwave heat restoration, or enzyme digestion to treat the bak...

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Abstract

The invention provides a variable region sequence of a mouse monoclonal antibody specifically bound with CAV-1 (Canine Adenovirus type I) Fiber protein, and an antibody specifically bound with the CAV-1 Fiber protein, wherein the antibody can be used for preparing a kit and a pharmaceutical composition. The kit provided by the invention overcomes the problem of low detection sensitivity in the prior art, avoids the occurrence of phenomena of missed detection and false negative, can detect various targets of dogs with high sensitivity, and can detect various targets of non-dog animals with highsensitivity; the kit has the advantages of rapidness, simpleness, convenience and accuracy; the pharmaceutical composition of the invention can effectively prevent and treat diseases caused by the CAV-1.

Description

technical field [0001] The invention relates to a canine adenovirus type 1 monoclonal antibody, the variable region sequence of the monoclonal antibody, a hybridoma cell line secreting it, and a pharmaceutical composition, kit and application prepared using the monoclonal antibody, belonging to biotechnology field. Background technique [0002] Canine infectious hepatitis and bear and fox encephalitis are both caused by canine adenovirus type I (CAV-1) and are distributed globally. Canine infectious hepatitis can infect dogs of different breeds, ages and sexes, and can occur in all seasons, with the highest infection rate and mortality rate in puppies (up to 25% to 40% and sudden death within 1-2 days); clinical symptoms Complicated, including vomiting, abdominal pain, diarrhea, elevated body temperature and even sudden death, eye damage, and temporary corneal cloudiness, commonly known as "blue eye disease"; easily associated with other viruses such as canine distemper vir...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/577
CPCG01N33/56983G01N33/577Y02A50/30
Inventor 田克恭邓均华李凡
Owner LUOYANG PULIKE WANTAI BIOTECH
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