Anti-SARS-CoV-2 S1-RBD monoclonal antibody and application thereof

A monoclonal antibody, sars-cov-2s1-rbd technology, applied in antiviral immunoglobulins, instruments, peptides, etc., can solve the problems of false negative test results, cumbersome operation, large human error, etc., and achieve subjective factor error. The effect of small size, low color rendering background, and short detection period

Active Publication Date: 2022-01-14
国际遗传工程和生物技术中心泰州区域研究中心 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, virus isolation, IFA and IMPA methods are cumbersome and time-consuming, require high equipment, large human errors, and high technical requirements for operators; improper operation of RT-PCR method personnel, failure of nucleic acid extraction, poor method performance, etc. will cause detection The result is false-negative or false-positive, and it needs to rely on expensive instruments with high cost; gene sequencing takes a long time and requires high equipment, which is not suitable for clinical rapid and large-scale diagnosis; the sensitivity and specificity of immunocolloidal gold method are relatively low. Low, high probability of false positive rate and false negative rate

Method used

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  • Anti-SARS-CoV-2 S1-RBD monoclonal antibody and application thereof
  • Anti-SARS-CoV-2 S1-RBD monoclonal antibody and application thereof
  • Anti-SARS-CoV-2 S1-RBD monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The preparation of embodiment 1 mouse anti-SARS-CoV-2 S1 protein monoclonal antibody

[0043] The monoclonal antibody of the present invention uses SARS-CoV-2 S1 recombinant protein as the immunogen and is secreted from the hybridoma cell line obtained from immunized mice, and is hereinafter referred to as 28D9 and 6B11. Wherein, the amino acid sequence of the heavy chain variable region of monoclonal antibody 28D9 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of monoclonal antibody 6B11 is shown in SEQ ID NO.2. The amino acid sequence is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4. Specific steps are as follows:

[0044] (1) Five 8-week-old and healthy female purebred Balb / c mice were selected for antigen immunization with SARS-CoV-2 S1 recombinant protein (purchased from Beijing ...

Embodiment 2

[0056] Example 2 Double Antibody Sandwich ELISA Detection

[0057] In order to obtain antibody pairs suitable for quantitative detection of SARS-CoV-2 virus or its S1 protein, the following double-antibody sandwich ELISA crossover experiment was performed to obtain purified monoclonal antibodies 26D8, 28D9, 30G10, and 6B11, and these 4 antibodies were labeled separately HRP (Baiying Biotechnology Co., Ltd. provides labeling HRP services), and obtained 4 kinds of enzyme-labeled monoclonal antibodies HRP-26D8, HRP-28D9, HRP-30G10, and HRP-6B11. Monoclonal antibodies 26D8, 28D9, 30G10, and 6B11 were coated on 96-well ELISA plates, and incubated overnight at 4°C for 20h; after washing, the blocking solution was blocked at 37°C for 2h. After washing, add SARS-CoV-2 S1 recombinant protein to be detected (diluted to 20000pg / ml in blocking solution) or SARS-CoV-2 inactivated virus vaccine (gifted by Institute of Medical Biology, Chinese Academy of Medical Sciences, diluted 20 times in...

Embodiment 3

[0064] One of the purposes of the present invention is to apply the best antibody obtained from the antibody provided by the present invention to double-antibody sandwich ELISA, which can quantitatively detect SARS-CoV-2 virus or its S1 protein. In order to achieve the high sensitivity of the present invention, A series of experiments were optimized, and the technical scheme is as follows:

[0065] (1) Determination of optimal antibody dilution concentration

[0066] Fix the concentration of the test substance (when the test sample is S1 protein, the concentration is 25000pg / ml; when the test sample is an inactivated SARS-CoV-2 virus vaccine, the dilution factor is 30 times), the coated antibody (when When the sample to be tested is S1 protein, the coating antibody is 6B11; when the sample to be tested is an inactivated SARS-CoV-2 virus vaccine, the coating antibody is 28D9) and the enzyme-labeled antibody (HRP-28D9) are set at different concentrations ( 1, 2, 3, 4, 5, 6, 7, ...

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Abstract

The invention relates to an anti-SARS-CoV-2S1-RBD monoclonal antibody 28D9. A heavy chain of the monoclonal antibody 28D9 is IgG1 type, a light chain of the monoclonal antibody 28D9 is Kappa type, an amino acid sequence of a heavy chain variable region is as shown in SEQ ID NO.1, and an amino acid sequence of a light chain variable region is as shown in SEQ ID NO.2. The antibody has native conformation epitopes, has good reactogenicity with SARS-CoV-2S1 protein and SARS-CoV-2S1-RBD, and has the characteristics of high specificity, high sensitivity, high titer and the like. A double-antibody sandwich ELISA detection method is established on the basis of the SARS-CoV-2S1 protein epitope, can be used for quantitatively detecting the SARS-CoV-2 inactivated virus and the SARS-CoV-2S1 protein, and is good in repeatability, high in precision, high in sensitivity, low in color development background and good in linearity.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-SARS-CoV-2 S1-RBD monoclonal antibody and its application. Background technique [0002] SARS-CoV-2 is a new strain of coronavirus that has never been found in humans before. SARS-CoV-2 and two other closely related highly pathogenic viruses, SARS-CoV and MERS-CoV, belong to the Coronaviridae βcoronavirus Viruses. SARS-CoV-2 can infect humans across species barriers, and can be transmitted through close contact, respiratory droplets, and high-concentration aerosols, causing infectious diseases mainly involving lung lesions, and can also induce diseases including nervous system and digestive system. Systemic damage, severe cases can lead to death. At present, there is no specific treatment for patients infected with SARS-CoV-2. Early diagnosis and timely control are the keys to preventing the further spread of the epidemic and controlling new infection clues. Therefore, the de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/543G01N33/535
CPCC07K16/10G01N33/54306G01N33/535C07K2317/56G01N2333/165
Inventor 陶诗怡杨义力吴宏斌邓大伟孔秀芹陈京丁敏
Owner 国际遗传工程和生物技术中心泰州区域研究中心
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