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Monoclonal antibody for resisting foot and mouth disease virus type A and application thereof

A monoclonal antibody, foot-and-mouth disease virus technology, applied in the direction of anti-viral immunoglobulin, immunoglobulin, instruments, etc., can solve the problems of biological safety risks, complicated ELISA kit operation steps, unstable test results and other problems

Active Publication Date: 2021-06-01
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus neutralization test is the gold standard for antibody determination, but this method requires the use of live viruses and can only be operated in a specific laboratory. The operation is relatively complicated and there are biosafety risks
The operation steps of the liquid phase blocking ELISA kit are cumbersome, and the test results are unstable between batches of the kit
[0004] In recent years, after Type O FMD, Type A FMD has become prevalent and broke out. In the domestic market, the liquid phase blocking ELISA kit for antibody against FMD A virus is widely used in the domestic market, but the kit captures Antibodies and enzyme-labeled antibodies are all polyantibodies, and polyantibody serum often has uneven quality, which affects the test results
The monoclonal antibody has a single biological activity, is easy to produce and standardize, so the monoclonal antibody of suitable A-type foot-and-mouth disease virus is screened to prepare an ELISA kit, which solves the problem of poor stability and improves sensitivity and specificity. prevention is important

Method used

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  • Monoclonal antibody for resisting foot and mouth disease virus type A and application thereof
  • Monoclonal antibody for resisting foot and mouth disease virus type A and application thereof
  • Monoclonal antibody for resisting foot and mouth disease virus type A and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The acquisition of embodiment 1 hybridoma cell

[0032] 1 mouse immunization

[0033] 5-6 weeks old Balb / c mice were immunized with foot-and-mouth disease type A virus-like granule protein as the immunogen, and the immunization method was multi-point subcutaneous injection on the back, and the injection volume of each mouse was 150 μg. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. The time interval between each immunization was two weeks, and the mouse serum was collected one week after the third immunization to detect the antibody titer (Lan animal research liquid phase resistance ELISA kit), when the antibody titer was not lower than 1:1440, shock immunization was performed, that is, the antigen was injected into the tail vein, and three days after the shock immunization, the spleen of the mouse was taken for fusion with myeloma cells.

[0034] 2 cell fusion

[0035] Ob...

Embodiment 2

[0038] Preparation and identification of embodiment 2 monoclonal antibody

[0039] 1 Preparation of monoclonal antibody ascites

[0040] Sensitize 7-8 weeks old Balb / c mice with sterilized liquid paraffin, 0.5ml / mouse, inject 0.5ml (about 2.0×10 6 cells / ml) of hybridoma cells. After the injection, the abdomen of the mice was rubbed lightly every day, and the ascites was extracted when the abdomen of the mice was obviously enlarged laterally. Centrifuge the ascitic fluid at 10,000 rpm for 10 min, take the supernatant, aliquot and store at -70°C.

[0041] 2 Ascites potency determination of hybridoma cells

[0042] According to the instructions of the Lanshouyan Foot-and-Mouth Disease Virus Type A Antibody Liquid Phase Blocking ELISA Detection Kit, the antibody titers of the prepared monoclonal antibodies 1 to 8 were measured respectively, which were 1:5760, 1:5760, 1:8192, and 1: 2880, 1:4096, 1:5760, 1:4096, and 1:11520. It is proved that the monoclonal antibodies 1-8 have...

Embodiment 3

[0043] Purification and content determination of embodiment 3 monoclonal antibody

[0044] Use affinity chromatography to purify hybridoma mouse ascites, the specific operation is as follows: thaw the frozen ascites sample, centrifuge at 10000rpm 4°C for 10min, absorb the clear liquid, and use 3 times the column volume of sample buffer (Bindingbuffer formula : 20mM Na 2 HPO 4 , 0.15M NaCl, pH8.0) after dilution, carry out Protein G affinity chromatography column purification, column chromatography eluent is pH 2.5 0.1M glycine buffer, the eluted antibody needs to be immediately neutralized with buffer (1MTris-HCl, pH8.5) adjusted the pH value to neutral, and carried out SDS-PAGE gel analysis and protein content determination, the results showed that the purity of purified monoclonal antibodies 1 to 8 was greater than 90%, and the protein content was respectively 3.3mg / ml, 3.5mg / ml, 3.6mg / ml, 3.2mg / ml, 3.3mg / ml, 3.5mg / ml, 3.3mg / ml and 3.9mg / ml can meet the needs of clinical a...

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Abstract

The invention relates to monoclonal antibodies 2E11 and 3B4 of a foot and mouth disease virus type A. The kit comprises an effective amount of the monoclonal antibody 2E11, an effective amount of a foot and mouth disease virus type A antigen, an effective amount of an enzyme-labeled monoclonal antibody 3B4, a detection reagent for detecting the antigen-antibody reaction of the foot and mouth disease virus type A, and a support medium, wherein the monoclonal antibody 2E11 is coated on a support medium. The solid-phase competitive ELISA detection kit provided by the invention solves the problems of large batch-to-batch difference and poor stability of the existing liquid-phase blocking ELISA kit, the detection titer reaches 1: 128, and the sensitivity is high.

Description

technical field [0001] The present invention relates to a hybridoma cell strain, a monoclonal antibody secreted by it, the variable region sequence of the monoclonal antibody, a hybridoma cell strain secreting it, and a kit and application prepared using the monoclonal antibody, belonging to Field of pharmaceutical formulations containing antibodies and immunoassays. Background technique [0002] Foot-and-mouth disease is a febrile, acute, and highly contagious infectious disease caused by foot-and-mouth disease virus infection in cloven-hoofed animals. The disease spreads rapidly and has a high incidence rate. The outbreak and prevalence of the disease have brought huge economic losses to the global aquaculture industry. It is listed as a class A severe infection by the World Organization for Animal Health (OIE) and the Food and Agriculture Organization of the United Nations (FAO). It is listed as the first class of infectious diseases in my country. At present, the preven...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N5/20G01N33/569G01N33/543G01N33/577
CPCC07K16/1009G01N33/56983G01N33/543G01N33/577C07K2317/56G01N2333/09
Inventor 田克恭燕贺
Owner LUOYANG PULIKE WANTAI BIOTECH
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