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Novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof

A sars-cov-2 and single-domain antibody technology, applied in the field of biomedicine, can solve problems such as changes in pathogenicity, failure of monoclonal antibody drugs or reduction of neutralization ability, and decline in vaccine protection

Active Publication Date: 2021-12-31
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More and more mutant strains have various consequences, including accelerated transmission and changes in pathogenicity. Another direct consequence is the decline in the protection of vaccines, and the failure or neutralization of some monoclonal antibody drugs developed in the early stage. Greatly reduced

Method used

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  • Novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof
  • Novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof
  • Novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Screening and preparation of single-domain antibodies that specifically bind to the RBD region of the SARS-CoV-2 spike protein

[0066] 1. Phage library screening

[0067] The basic principle and basic operation process of phage library screening refer to "Biological Library Technology-Phage Display and SELEX Technology" written by Shao Ningsheng et al. The screening steps are briefly described as follows: First, 50 μg of SARS-CoV- 2 Recombinant protein in the RBD region of the spike protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., catalog number: 40592-V08H86, 40592-V08H88, referred to as RBD protein) antigen using the commercial kit EZ-Link of Thermo Fisher Scientific TM Sulfo NHS-LC-LC-Biotin was labeled with biotin and desalted and purified (the desalting column was purchased from Thermo Fisher Scientific). The first round of screening was liquid-phase selection, firstly, 12 μg of biotin-labeled RBD protein was mixed with 1 mL (10 12 -10 13 ) pha...

Embodiment 2

[0091] Example 2 Affinity Determination of Anti-RBD Single Domain Antibody

[0092] Antibodies to be tested: two RBD single domain antibodies B3A3 and I3A10 purified in Example 1.

[0093] BIAcore T-200 biomolecular interaction instrument (GE Life Sciences) was used to measure the affinity of antibodies. This technology was developed based on surface plasmon resonance (SPR) technology, which can be used for label-free and rapid detection of interactions between biomacromolecules. , Real-time, automatic operation and detection.

[0094] When measuring the interaction between the recombinant RBD protein and its corresponding single domain antibody, first coat the antigen RBD protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., Gamma strain RBD product number: 40592-V08H86, Kappa strain RBD product number: 40592-V08H88) on On the sensor chip, the association constant, dissociation constant and affinity constant were determined using the single domain antibody as the mobile pha...

Embodiment 3

[0101] SARS-CoV-2 pseudotyped virus neutralizing activity assay of embodiment 3 anti-RBD single domain antibody

[0102] Antibodies to be tested: two single domain antibodies B3A3 and I3A10 purified in Example 1.

[0103] SARS-CoV-2 pseudotyped virus is a new type of virus particle formed by assembling the replication core element of retrovirus and the envelope spike glycoprotein (ie, S protein) of SARS-CoV-2 virus. Compared with true viruses, pseudotyped viruses can only infect cells at one time, have a wide host range, high titers, and are not easily inactivated by serum complement, so they can replace true viruses for neutralization detection. The ability of pseudotyped virus to infect cells depends on the type and characteristics of the glycoprotein wrapped outside it, which is an ideal tool for studying the inhibition efficiency of neutralizing antibodies, receptor utilization and invasion infection mechanism of SARS-CoV-2.

[0104] 1. Packaging preparation of SARS-CoV-2...

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Abstract

The invention discloses a novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof, and particularly discloses a single-domain antibody capable of effectively inhibiting various novel coronavirus SARS-CoV-2 mutant strains. By means of a phage antibody library technology, broad-spectrum neutralizing single-domain antibodies B3A3 and I3A10 specifically combined with SARS-CoV-2 spike protein RBD are successfully obtained. The single-domain antibody is high in antigen affinity, has an obvious inhibiting effect on SARS-CoV-2 main epidemic strains, is a neutralizing antibody with a broad-spectrum effect, and is good in broad spectrum and neutralizing activity. The single-domain antibody can be prepared into specific antibody medicine, SARS-CoV-2 diagnostic reagents or kits and the like clinically used for preventing and treating novel coronavirus pneumonia (COVID-19), and has very wide prospects and important significance in the fields of medicine application, clinical diagnosis and the like.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a novel coronavirus SARS-CoV-2 broad-spectrum neutralizing antibody and application thereof. In particular, it relates to a single-domain antibody that effectively inhibits a variety of novel coronavirus SARS-CoV-2 mutant strains and applications thereof. Background technique [0002] Antibody (Ab) is an effector immune molecule produced by B cells that proliferate and differentiate into plasma cells after being specifically stimulated by B cell antigen epitopes. It mediates humoral immunity and is a class of immunoglobulins that can specifically bind to antigens ( immunoglobulin, Ig). Ig specifically recognizes and binds to the corresponding antigen epitope in a lock-key complementary relationship through the antigen-binding groove composed of its V region CDRs. When the antibody binds to the surface of the pathogen or the key epitope of the bacterial toxin, the virulence structure of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/70C12N1/21G01N33/569G01N33/68A61K39/42A61P31/14A61P11/00A61P1/00C12R1/19
CPCC07K16/10G01N33/56983A61P31/14G01N2333/165C07K2317/565A61K2039/505C07K2317/569C07K2317/56Y02A50/30
Inventor 杨威迟晓静刘秀英张心慧潘胜男段花蕊
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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