Highly neutralizing active anti-sars-cov-2 fully human monoclonal antibody and its application

A monoclonal antibody, sars-cov-2 technology, applied in the fields of microbiology and immunology, can solve the problems of limited plasma donation sources, batch differences, difficult large-scale preparation, etc., and achieve good neutralization protection effect and stability. Good, low immunogenic effect

Active Publication Date: 2022-01-07
ACADEMY OF MILITARY MEDICAL SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages: limited sources of donated plasma, difficulty in large-scale preparation, batch-to-batch differences, etc.; whether there is antibody-dependent enhancement (ADE effect) is still controversial

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Highly neutralizing active anti-sars-cov-2 fully human monoclonal antibody and its application
  • Highly neutralizing active anti-sars-cov-2 fully human monoclonal antibody and its application
  • Highly neutralizing active anti-sars-cov-2 fully human monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Screening and Preparation of Human Anti-SARS-CoV-2 Monoclonal Antibody

[0032] 1.1 Sorting single cells by flow cytometry

[0033] The collected blood samples were separated from PBMC by Ficoll density gradient centrifugation, and the process was as follows:

[0034] 1.1.1 Take fresh anticoagulated whole blood (EDTA anticoagulated) and dilute the whole blood with an equal volume of PBS.

[0035] 1.1.2 Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1.

[0036] 1.1.3 Trim, room temperature, horizontal rotor 700-800g (2000-2500rpm), acceleration 3-4acc, centrifuge for 20-30min.

[0037] 1.1.4 After centrifugation, the bottom of the tube is red blood cells, the middle layer is the separation li...

Embodiment 2

[0093] Example 2 Screening of Binding Activity of Human Anti-SARS-CoV-2 Monoclonal Antibody to S Protein and Analysis of Antibody 4A8 Recognition Epitope

[0094] 2.1 Coating: Dilute the recombinant S antigen, S1 antigen, RBD antigen and S2 antigen with the coating solution to a concentration of 2 μg / mL, coat the microtiter plate, 100 μL per well, and coat overnight at 4 °C.

[0095] 2.2 Blocking: add 300 μL of PBST washing solution to each well, wash 3 times × 3 min each time; tap the liquid in the well, add 2% BSA, 200 μL / well, and block at 37°C for 1 hour.

[0096] 2.3 Sample incubation: add 300 μL PBST washing solution to each well, wash 3 times × 3min / time; clap the liquid in the well, add purified monoclonal antibody diluted in PBS, the first well 9ug / ml, 3-fold serial dilution 100 μL / well, 37 Incubate at ℃ for 1h.

[0097] 2.4 Secondary antibody incubation: wash, same as above; add HRP goat anti-human F C Secondary antibody (diluted 1:20000), 100 μL / well, incubated at...

Embodiment 3

[0104] Example 3 Analysis of Neutralizing Activity on Cell Models During SARS-CoV-2 Infection

[0105] 3.1 After digesting Vero E6 cells with 0.25% trypsin, dilute to 3×10 with culture medium (DMEM+10% FBS)5 The concentration of cells / mL was inoculated into a 96-well cell culture plate with an inoculation volume of 100 μL / well, and cultured overnight in a 5% CO2 incubator at 37°C.

[0106] 3.2 On the day of the experiment, add the purified monoclonal antibody to the 96-well culture plate from the initial concentration (4A8 monoclonal antibody initial concentration 154ug / ml, 3-fold serial dilution, volume 120μL / well; then add 120μL per well COVID-19 virus suspension (dilute virus with DMEM+2%FBS, add 100TCLD 50 / well), mix thoroughly, and incubate for 1 h in a cell culture incubator.

[0107] 3.3 Discard the cell culture supernatant in the 96-well plate, and add 200 μL of co-incubated virus-antibody mixed suspension to each well; set up a survival control (without adding virus...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
Login to view more

Abstract

The invention discloses an anti-SARS-CoV-2 fully human monoclonal antibody, which is obtained through flow sorting-single-cell PCR technology screening and has a unique CDR partition. The invention also discloses the antibody Application in the preparation of 2019‑coronavirus disease therapeutic drugs. The monoclonal antibody disclosed in the present invention has high-efficiency and specific anti-SARS-COV-2 virus activity, and also has the characteristics of high expression, full human origin and good stability, and is suitable for industrial production.

Description

technical field [0001] The invention discloses an antibody and belongs to the fields of microbiology and immunology. Background technique [0002] The causative agent of COVID-19 is SARS-CoV-2, also known as 2019-coronavirus. SARS-CoV-2 belongs to the family Coronaviridae (Coro-naviridae) and belongs to the genus Coronavirus. It is a class of enveloped single-stranded positive-sense RNA viruses. The total length of the genome is 30kb, which consists of 5'-terminal non-coding region, non-structural protein open reading frame (ORF) 1a / b coding region, spike glycoprotein S coding region, envelope protein E coding region, membrane protein M protein Coding region, nucleocapsid protein N protein coding region and 3'-terminal non-coding region. Among them, the polyprotein encoded by the non-structural protein ORF1a / b region can be cleaved to form RNA-dependent RNA polymerase (RdRp), etc., to guide the replication, transcription and translation of the viral genome. Structural pro...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/10C12N15/13C12N15/85C12N5/10A61K39/42A61P31/14
CPCC07K16/10C12N15/85A61P31/14C07K2317/56C07K2317/52C07K2317/92C07K2317/34C07K2317/565C07K2317/76A61K2039/505
Inventor 陈薇李建民迟象阳张军付玲于长明徐俊杰侯利华张冠英范鹏飞郝勐董韵竹宋小红陈旖张金龙房婷刘树玲
Owner ACADEMY OF MILITARY MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap