Monoclonal antibody 20D8 of anti-SARS-CoV-2 epidemic mutant strain

A monoclonal antibody, sars-cov-2 technology, applied in antiviral immunoglobulins, instruments, peptides, etc., can solve the effect of monoclonal antibody neutralization activity, affect the transmission speed of mutant strains, pathogenicity, virus immunity Escape and other issues

Active Publication Date: 2021-09-07
WUHAN INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Emerging mutant strains such as P.1 strain (containing mutations such as K417T, E484K and N501Y), B.1.351 (also known as 501Y.V2, containing mutations such as K417N, E484K and N501Y) and B.1.1.7 (containing mutations such as E484K Multiple point mutations in the amino acid sequence of the spike protein (including RBD and NTD regions) will not only affect the transmission speed and pathogenicity of mutant strains, but also have a greater effect on the neutralizing activity of monoclonal antibodies The impact of the virus may lead to the occurrence of immune escape of the virus

Method used

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  • Monoclonal antibody 20D8 of anti-SARS-CoV-2 epidemic mutant strain
  • Monoclonal antibody 20D8 of anti-SARS-CoV-2 epidemic mutant strain
  • Monoclonal antibody 20D8 of anti-SARS-CoV-2 epidemic mutant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, Preparation and Purification of Monoclonal Antibody 20D8

[0037] 1. Preparation: After injecting Freund's incomplete adjuvant 7 days in advance, monoclonal hybridoma cell lines (0.5mL, 3×10 6 cells / mL) were injected into the peritoneal cavity of mice, and cultured for 7-10 days.

[0038] 2. Purification: Harvest the ascites, let it stand at 37°C for 2 hours, centrifuge at 5000rpm for 30 minutes, collect the supernatant in the middle layer and filter it, and then purify it with a Protein G affinity chromatography column. The purification steps are briefly described as:

[0039] Equilibrate with 0.1M Tris buffer at pH 7.0;

[0040] After loading, rinse with 0.1M Tris buffer at pH 7.0;

[0041] Subsequent elution was performed with 1.0 M Tris buffer at pH 8.0.

[0042] The eluate was collected and further dialyzed in PBS buffer. The purified antibody was taken for SDS-PAGE and HPLC-SEC detection and analysis.

[0043] 3. Result analysis: SDS-PAGE results ...

Embodiment 2

[0056] Example 2, Functional Analysis of Antibodies

[0057] 1. Detection of binding activity of anti-SARS-CoV-2 monoclonal antibody 20D8 to antigen

[0058] The ELISA method was used to determine the binding ability of the 20D8 antibody to the spike protein S1 of the SARS-CoV-2 virus. The steps are briefly described as:

[0059] (1) Using SARS-CoV-2 spike S1-His recombinant protein (Sino company, product number: 40591-V08H) as the coating antigen, coat 2.0 μg / mL antigen on the microtiter plate with carbonate buffer, Incubate at 37°C for 2 hours;

[0060] (2) Block with casein buffer at 37°C for 2h; add serially diluted antibody to be tested, and incubate at 37°C for 1h;

[0061] (3) Add 1:10 000 diluted goat anti-mouse IgG-HRP (Biodragon Company, product number: BF03001X), and incubate at 37°C for 1 hour;

[0062] (4) After developing the color with the chromogenic solution, 2M HCl terminates the reaction; the absorbance A450nm value is detected by a microplate reader.

...

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Abstract

The invention relates to a monoclonal antibody 20D8 of an anti-SARS-CoV-2 epidemic mutant strain. The antibody can neutralize wild type SARS-CoV-2 and various SARS-CoV-2 mutant strains, including a P.1 strain, a B.1.351 strain and a B.1.1.7 strain.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a monoclonal antibody 20D8 against the epidemic mutant strain of SARS-CoV-2. Background technique [0002] SARS-CoV-2 is a novel coronavirus with high infection rate and fatality rate. Since its outbreak, it has caused a global pandemic and seriously threatened global public health. Vaccines can effectively prevent infectious diseases, and some new crown vaccines have been approved for use, but they are mainly aimed at healthy people. Neutralizing antibodies against SARS-CoV-2 can effectively block the combination of virus and cells, and have shown positive effects in clinical trials. [0003] Currently, some monoclonal antibodies against SARS-CoV-2 have been approved for emergency use, and a large number are in various stages of development. However, most of the existing antibodies are mainly against the unmutated SARS-CoV-2 wild strain (Wild-type, WT). Emergi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/569
CPCC07K16/10G01N33/56983C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/76G01N2333/165Y02A50/30
Inventor 王文辉郭靖万鑫卢佳杨东升林凤杰王泽鋆
Owner WUHAN INST OF BIOLOGICAL PROD CO LTD
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