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Anti-SARS-CoV-2 neutralizing antibody and application thereof

A sars-cov-2, antibody technology, applied in the direction of antibodies, applications, antiviral agents, etc., to achieve the effect of high-efficiency neutralization activity

Active Publication Date: 2022-05-31
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the spread and continuation of the global pandemic of SARS-CoV-2, the emergence of new mutant strains poses great challenges to the protective effect of neutralizing antibodies.

Method used

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  • Anti-SARS-CoV-2 neutralizing antibody and application thereof
  • Anti-SARS-CoV-2 neutralizing antibody and application thereof
  • Anti-SARS-CoV-2 neutralizing antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Screening of anti-SARS-CoV-2 neutralizing antibodies

[0091] (1) Plasma neutralization activity detection

[0092] 1. Sample: Plasma of volunteers immunized twice with the novel coronavirus inactivated vaccine (BBIBP-CorV).

[0093] 2. Neutralization activity detection: Detect the neutralization of 28 samples against SARS-CoV-2 wild type and mutant strains (Alpha, Betav1, Beta v2, Beta v3, Gamma, Delta, Lambda, Kappa, Epsilon, Zeta) pseudoviruses Titer. Methods as below:

[0094] (1) Plasma samples (named plasma No. 1-No. 28) were heat-inactivated at 56°C for 30 min, and centrifuged briefly to remove insoluble matter.

[0095] (2) In a 96-well cell culture plate, add 150 µl GM (DEME+10%FBS+1% double antibody) to the cell control (column 1), add 100 µl GM to the remaining wells, and add another 30 µl GM to 3A- 3H and 8A-8H (1-12 is column 1-12, A-H is row A-H, for example, 3A is the treatment well of column 3 and row A in a 96-well plate).

[0096] (3) T...

Embodiment 2

[0181] Example 2. Preparation of neutralizing antibodies

[0182] (1) Primary screening of antibody activity

[0183] 1. Antibody expression in 293T cell system

[0184] Transiently transfect the successfully paired antibody heavy chain and light chain gene expression vectors into 293T cells:

[0185] (1) 24 hours before transfection in a 12-well plate, each well was plated with 5×10 5 293T cells.

[0186] (2) On the day of transfection, observe the confluence of the cells, preferably 70%-80%.

[0187] (3) Take 1 μg of the plasmid DNA extracted by the plasmid mini-extraction kit (Tiangen, DP103) and dilute it with 60 μl of Opti-MEM medium.

[0188] (4) Dilute 4 μl Lipofectamine® 2000 Reagent with 60 μl Opti-MEM medium.

[0189] (5) Mix the above-mentioned diluted DNA and Lipofectamine® 2000 at a volume of 1:1 to obtain a mixture, and incubate at room temperature for 5 min.

[0190] (6) Take 120 μl of the mixture and gently add it to 293T cells in 5% CO 2 Incubate at 37...

Embodiment 3

[0235] Example 3. Antibody 6-2C affinity and neutralizing activity analysis

[0236] The neutralizing antibody 6-2C prepared in Example 2 was used to carry out the following experiments.

[0237] (1) Affinity analysis of neutralizing antibodies

[0238] 1. BLI detection of the binding ability of neutralizing antibody 6-2C to SARS-CoV-2 RBD

[0239] (1) Ligand coupling: In the high-throughput molecular interaction instrument Octet RED 384, biotin-labeled 10 µg / ml RBD protein (Acrobiosystems, SPD-C82E9) was coupled to the SA sensor (SARTORIUS, 18-0009) superior.

[0240] (2) Antibody 6-2C was serially diluted 2-fold with PBS starting from 320 nM for 6 gradients.

[0241] (3) According to the table 4 below, set the injection analysis from low concentration to high concentration.

[0242]

[0243] (4) Data analysis: set the following table 5 in the analysis software, and perform curve fitting.

[0244]

[0245] 2. Analysis of the results: the affinity Kd between 6-2C an...

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Abstract

The invention relates to a neutralizing antibody for resisting SARS-CoV-2 and application of the neutralizing antibody. The invention provides an anti-SARS-CoV-2 neutralizing antibody or an antigen binding fragment thereof, the anti-SARS-CoV-2 neutralizing antibody has a heavy chain variable region containing VHCDR1, VHCDR2 and VHCDR3 and a light chain variable region containing VLCDR1, VLCDR2 and VLCDR3, the VHCDR1, VHCDR2 and VHCDR3 respectively comprise amino acid sequences at positions 26-33, 51-58 and 97-111 in SEQ ID No. 3, and the VLCDR1, VLCDR2 and VLCDR3 respectively comprise amino acid sequences at positions 27-32, 50-52 and 89-97 in SEQ ID No. 4. The neutralizing antibody disclosed by the invention has efficient neutralizing activity and can be used for neutralizing a mutant strain of SARS-CoV-2 in a broad spectrum.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to neutralizing antibodies against novel coronaviruses and applications thereof. Background technique [0002] Since the novel coronavirus (SARS-CoV-2) was first reported at the end of 2019, it has posed a huge challenge and impact on public health and social life. Existing vaccines are effective in preventing infection with wild-type strains, but as the global pandemic of SARS-CoV-2 continues, emerging mutant strains are causing breakthrough infections. In particular, a large number of mutations in important target sites of the Omicron mutant strain (B.1.1.529) greatly reduced the neutralizing activity of the immune serum, posing a threat to the protective effect of the vaccine. Therefore, efficient and broad-spectrum prevention and treatment methods are urgently needed to control the prevalence of SARS-CoV-2. The broad-spectrum neutralizing antibody against SARS-CoV-2 can block the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12P21/02A61K39/42A61P31/14
CPCC07K16/10A61P31/14C07K2317/56C07K2317/565C07K2317/76C07K2317/92A61K2039/505
Inventor 程功刘玉斌太万博邹艳盛洁
Owner TSINGHUA UNIV