Anti-type a foot-and-mouth disease virus neutralizing monoclonal antibody and its application

A technology of foot-and-mouth disease virus and monoclonal antibody, which is applied to the preparation and application of type A foot-and-mouth disease virus monoclonal antibody, and the application in the prevention or treatment of type A foot-and-mouth disease. In the field of diagnosis, monoclonal antibodies can solve antigenic differences and diagnosis of foot-and-mouth disease and the difficulty of controlling

Active Publication Date: 2019-07-23
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different subtypes within the same serotype have different degrees of antigenicity, and the degree of serological cross-reactivity also varies, making the diagnosis and control of foot-and-mouth disease very difficult

Method used

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  • Anti-type a foot-and-mouth disease virus neutralizing monoclonal antibody and its application
  • Anti-type a foot-and-mouth disease virus neutralizing monoclonal antibody and its application
  • Anti-type a foot-and-mouth disease virus neutralizing monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1A

[0040] Preparation of experimental example 1A type foot-and-mouth disease virus neutralizing monoclonal antibody

[0041] 1. Test method

[0042] 1.1 Mouse immunization and preparation of monoclonal antibody

[0043] (1) Type A FMDV is inoculated on the overgrown BHK-21 cell monolayer, and the virus is harvested after more than 75% of the cells have pathological changes. The harvested cell culture medium was repeatedly frozen and thawed three times to initially lyse the cells and release the virus. The frozen-thawed virus solution was lysed and inactivated by adding 1:1000 TritonX-100 and 4:10000 formaldehyde for more than 48 hours, and 1 mL of the inactivated virus solution was taken to BHK-21 cells for three generations to test whether the inactivation was complete. Centrifuge the freeze-thawed culture medium at 9000rpm (Beckman high-speed centrifuge, JA-10 rotor) at 4°C for 90min, recover the supernatant of the virus solution, and discard the cell pellet. Add 80g / L PEG60...

experiment example 2A

[0065] Experimental example 2 Establishment of detection method for type A foot-and-mouth disease virus antibody

[0066] 1. Test method

[0067] 1.1 Purification of monoclonal antibodies

[0068] The prepared ascites was purified by octanoic acid-saturated ammonium sulfate method, and the operation steps were briefly described as follows:

[0069] (1) Take 3ml of pretreated ascitic fluid and add 6ml of 0.06mol / L pH4.8 acetate buffer solution; add 99ul octanoic acid to the ascitic fluid, stir at room temperature for 30 minutes, and let it stand at 4°C for more than 2 hours; take it out and centrifuge at 11000rpm for 30 minutes, and discard the precipitate; The supernatant was adjusted to pH 7.4 with 2mol / L NaOH.

[0070] (2) Add saturated ammonium sulfate to 50% saturation at 4°C, stir in an ice bath for 30 minutes, and let stand at 4°C for more than 2 hours; centrifuge at 11,000 rpm for 30 minutes, discard the supernatant; dissolve the precipitate in 5ml of 0.1M In PBS of ...

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Abstract

The invention discloses preparation and application of a monoclonal antibody of an A-type foot and mouth disease virus (FMDV) and belongs to the field of prevention and treatment of foot and mouth diseases. According to the preparation and the application, a hybridoma cell line secreting the neutralizing monoclonal antibody of the A-type FMDV is constructed, the microbiological collection number of the hybridoma cell line is CGMCC 12049, and the secreted monoclonal antibody is an A-type FMDV serotype specific monoclonal antibody and has high-strength neutralizing ability; and a detection method for the A-type FMDV antibody is created by using the monoclonal antibody. The monoclonal antibody prepared by the invention shows good competitive ability with strongly-positive, weakly-positive and suspected serum of the A-type FMDV in the detection method for the A-type FMDV antibody and is free of competitiveness with strongly-positive serum of an O-type FMDV, strongly-positive serum of an Asia1-type FMDV and negative serum of an FMDV, so that the monoclonal antibody can be used for specifically discriminating the negative and positive serum of the A-type FMDV and has an important application prospect in aspects such as the diagnosis, prevention and treatment of the A-type foot and mouth diseases.

Description

technical field [0001] The present invention relates to a monoclonal antibody, in particular to a neutralizing monoclonal antibody against type A foot-and-mouth disease virus and a hybridoma cell line secreting the neutralizing monoclonal antibody. The present invention further relates to the use of the monoclonal antibody in diagnosis and prevention Or the application in treating type A foot-and-mouth disease, belonging to the field of preparation and application of type A foot-and-mouth disease virus monoclonal antibody. Background technique [0002] Foot and Mouth Disease Virus (Foot and Mouth Disease Virus, FMDV) belongs to Picornaviridae, Foot and Mouth Disease Virus genus, FMDV genome RNA length is about 8.5kb. Foot and mouth disease (FMD) is caused by FMD virus (FMDV), and is a highly contagious infectious disease that mainly affects cattle, pigs, sheep and other domestic animals and a variety of wild cloven-hoofed animals. The outbreak and prevalence of this disease...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/569G01N33/577A61K39/42A61P31/14
CPCC07K16/1009C07K2317/10
Inventor 于力周国辉王海伟杨德成
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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