Anti-foot-and-mouth disease (FMD) O-type virus monoclonal antibodies and application thereof

A technology of monoclonal antibody and foot-and-mouth disease virus, which is applied in antiviral immunoglobulin, immunoglobulin, instruments, etc., can solve the problems of large batch-to-batch variation, monoclonal antibody biological activity, and poor stability, and achieve high sensitivity Detection effect

Active Publication Date: 2021-03-19
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The common antibody level detection kit in the market is the liquid-phase blocking ELISA kit, which uses polyclonal antibodies, which have problems of large batch-to-batch variance and poor stability.
Monoclonal antibodies have a single biological activity, are easy to produce and standardize, and O-type foot-and-mouth disease is very prevalent in my country and belong to different genetic topologies. ELISA kit, so screening the appropriate monoclonal antibody is of great significance for the diagnosis and prevention of the disease

Method used

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  • Anti-foot-and-mouth disease (FMD) O-type virus monoclonal antibodies and application thereof
  • Anti-foot-and-mouth disease (FMD) O-type virus monoclonal antibodies and application thereof
  • Anti-foot-and-mouth disease (FMD) O-type virus monoclonal antibodies and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The acquisition of embodiment 1 hybridoma cell

[0034] 1. Mice Immunization

[0035] 5-6 weeks old Balb / c mice were immunized with foot-and-mouth disease type O virus-like particles as the immunogen, and the immunization method was multi-point subcutaneous injection on the back, and the injection volume of each mouse was 200 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.

[0036] 2. Cell Fusion

[0037] Obtain ...

Embodiment 2

[0040] Preparation and identification of embodiment 2 monoclonal antibody

[0041] 1. Preparation of Monoclonal Antibody from Ascites

[0042] Sensitize 7-8 weeks old Balb / c mice with sterilized liquid paraffin, 0.5ml / mouse, inject 0.5mL (about 3.0×10 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.

[0043] 2. Ascites titer determination of hybridoma cells

[0044] According to the instructions of Lanshouyan Foot-and-Mouth Disease Virus Type O Antibody Liquid Phase Blocking ELISA Detection Kit, the prepared monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, monoclonal antibody 4, monoclonal antibody 5, and monoclonal antibody were measured respectively. The antibody titers of antibody 6, mAb 7, mAb 8, m...

Embodiment 3

[0045] Purification and content determination of embodiment 3 monoclonal antibody

[0046] 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, the eluent of column chromatography is the glycine buffer solution of pH 2.5 0.1M, the eluted antibody needs to be neutralized immediately with the buffer solution ( 1M Tris-HCl, pH8.5) adjusted the pH value to neutral, and carried out SDS-PAGE gel analysis and protein content determination, the results showed: the purified monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, monoclonal antibody Antibody 4, Monoclonal Antibody 5, Monoclonal Antibody 6, Monoclonal Antibody 7, Monoclonal A...

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Abstract

The invention provides monoclonal antibodies 6G6 and 7D7 which can specifically bind with different genetic topology O-type foot-and-mouth disease (FMD) viruses, and an ELISA detection kit prepared from the monoclonal antibodies 6G6 and 7D7 can be used for carrying out high-sensitivity detection on the different genetic topology O-type FMD viruses.

Description

technical field [0001] The invention relates to a monoclonal antibody capable of reacting with foot-and-mouth disease type O virus, the variable region sequence of the monoclonal antibody, a hybridoma cell line secreting it, and a kit and application prepared using the monoclonal antibody, which belong to the antibody-containing Pharmaceutical preparations and the field of immunoassays. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, highly contagious infectious disease caused by foot-and-mouth disease virus, and artiodactyls are the most susceptible. The outbreak and prevalence of the disease have brought huge economic losses to the global breeding industry. The World Organization for Animal Health lists it as a A type of infectious disease. FMD virus is divided into seven serotypes, namely A, O, Asia I, C, SAT1, SAT2, and SAT3, among which O-type FMD virus is the most prevalent. According to genetic classification, there are currently three types ...

Claims

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Application Information

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