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Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell

A canine distemper virus and monoclonal antibody technology, which is used in the production of therapeutic canine distemper virus monoclonal antibodies and hybridoma cells. , to achieve the effect of small difference between batches, low cost and stable production process

Active Publication Date: 2013-12-11
北京世纪元亨动物防疫技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Generally speaking, various miscellaneous proteins (including Ig) of mice are often mixed in this type of ascites, so it can only be used after purification; in addition, this method has low yield, long production cycle, and large batch-to-batch variation, so it is not suitable for Mass production of therapeutic monoclonal antibodies

Method used

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  • Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell
  • Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell
  • Method for producing therapeutic monoclonal antibody of canine distemper virus, product thereof and hybridoma cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Obtaining of Hybridoma Cell Line Secreting Anti-CDV Monoclonal Antibody

[0060] 1.1 Use canine distemper virus CDV (CDV MD-77 strain, HA titer 1:5120, from the Veterinary Diagnostic Center of the Ministry of Agriculture) to immunize Balb / c mice. Equal volumes of the virus solution and complete Freund's adjuvant (CFA) were mixed and emulsified, and injected into the muscles of the limbs at multiple points, each with 300ul per injection. 15d and 30d after the first immunization, booster immunization was carried out with the same dose of virus solution plus incomplete Freund's adjuvant (IFA). Blood was collected after the second booster, and ELISA titer and immunoenzyme titer were tested simultaneously on the serum.

[0061] 1.2 Preparation of hybridoma:

[0062] When the titer of ELISA reaches 1:20000 and the titer of immune enzyme reaches 1:640, the mouse spleen is taken for fusion. The immunization was boosted again 72 hours before the fusion, and the viru...

Embodiment 210L

[0067] Example 210L (Bereng) bioreactor cultivation

[0068] (1) The CDV1H2 cell line in Example 1 was pipetted with cell culture medium, dispersed and passaged, and continued to be cultured in the cell culture medium at 37°C. When a good monolayer was formed, it was used for continued passage; the total number of cells reached 10 9 / 500ml or more.

[0069] Identification of seed cells: when the number of cells reaches 5×10 5 When the number / ml is more than 98017 strains of canine distemper virus, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), and reovirus type 3 (Reo-3) cell culture fly slices, carry out Immunoenzyme test. Canine distemper virus monoclonal antibody is positive for canine distemper virus cell culture flyer, and other virus flyer is negative; secreted monoclonal antibody is IgG1 type;

[0070] (2) Cultivation and production of therapeutic monoclonal antibodies: The hybridoma cells secreting therapeutic monoclonal antibodies were ta...

Embodiment 330L

[0079] Example 330L (Bereng) bioreactor cultivation

[0080] (1) The CDV1H2 cell line in Example 1 was pipetted with cell culture medium, dispersed and passaged, and continued to culture in the cell culture medium at 37°C. When a good monolayer was formed, it was used for continued passage; the total number of cells reached 3×10 9 / 500ml or more.

[0081] Identification of seed cells: when the number of cells reaches 5×10 5 When the number / ml is more than 98017 strains of canine distemper virus, canine parvovirus AMS-1 strain, canine infectious hepatitis virus (ICHV), and reovirus type 3 (Reo-3) cell culture fly slices, carry out Immunoenzyme test. Canine distemper virus monoclonal antibody is positive for canine distemper virus cell culture flyer, and other virus flyer is negative; secreted monoclonal antibody is IgG1 type;

[0082] (2) Cultivation and production of therapeutic monoclonal antibodies: The hybridoma cells secreting therapeutic monoclonal antibodies were take...

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Abstract

The invention provides a method for producing a therapeutic monoclonal antibody of a canine distemper virus, a product thereof and a hybridoma cell. The method provided by the invention comprises the following steps: (1) carrying out amplification culture on the hybridoma cell which secretes and produces the therapeutic monoclonal antibody of the canine distemper virus in a cell culture solution; (2) then, inoculating the hybridoma cell into a cell culture solution in a bioreactor and culturing; (3) harvesting the therapeutic monoclonal antibody, produced by the hybridoma cell, of the canine distemper virus. The method provided by the invention has the advantages that the produced therapeutic monoclonal antibody of the canine distemper virus is high in purity, little in batch difference and easy in quality control, and the quality of the therapeutic monoclonal antibody can be improved remarkably.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibody production, in particular to a method for producing therapeutic canine distemper virus monoclonal antibody, its product and hybridoma cells. Background technique [0002] In 1975, Koehler and Milstein (Nature Vol.256, pp495-497) used cell hybridization technology to fuse mouse spleen cells immunized with sheep red blood cells (SRBC) with mouse myeloma cells, and successfully obtained an anti-SRBC monoclonal Antibody. Created the epoch-making hybridoma monoclonal antibody technology, which opened a new field for biotechnology. At present, the application of this technology has involved many fields such as medicine, agriculture, food, environment, etc., and is widely used in basic research, disease diagnosis, environment and food detection, treatment, prevention, protein purification, etc. With the application of monoclonal antibodies in in vivo diagnosis and treatment, higher requirem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/08C07K16/10C12N5/20
Inventor 孙明陈西钊田克恭曹振张丽
Owner 北京世纪元亨动物防疫技术有限公司
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