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Hybridoma cell capable of secreting an anti-novel coronavirus N protein monoclonal antibody, onoclonal antibody and application

A monoclonal antibody, hybridoma cell line technology, applied in the field of immune detection, can solve the problem of long detection time, and achieve the effects of rapid monitoring and prevention, strong specificity and high sensitivity

Active Publication Date: 2020-10-02
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current commonly used RT-PCR method takes a long time to detect, and requires expensive instruments and equipment and professionals to perform the test in a specialized PCR laboratory, and serological tests usually require 7 days after infection to detect IgM and / or IgG antibodies , the development of new technologies and methods for rapid, real-time, low-cost, high-sensitivity and specificity antigen detection is necessary and important for early diagnosis of new coronaviruses and prevention of their spread and spread

Method used

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  • Hybridoma cell capable of secreting an anti-novel coronavirus N protein monoclonal antibody, onoclonal antibody and application
  • Hybridoma cell capable of secreting an anti-novel coronavirus N protein monoclonal antibody, onoclonal antibody and application
  • Hybridoma cell capable of secreting an anti-novel coronavirus N protein monoclonal antibody, onoclonal antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the acquisition of the hybridoma cell line that can secrete the monoclonal antibody of the N protein of anti-SARS-COV-2

[0045] The N protein of SARS-COV-2 was diluted with normal saline and mixed with an equal volume of Quick Anti-body-Mouse 3W adjuvant. Two Balb / c mice were immunized by intramuscular injection in the calf, and the immunization was boosted in the same way on the 14th day. On the 21st day, blood was collected from the tail of the mice, and the serum titer was detected by indirect ELISA after the serum was separated, and the titer reached the fusion requirement. Three days before cell fusion, a spleen boost was performed with 100 μg of protein.

[0046] Balb / c mouse spleen cells were mixed with NS1 myeloma cells in logarithmic growth phase at a ratio of 1:9, and then fused under the action of 50% PEG1500. After fusion, the cells were cultured in HAT semi-solid medium containing 10% Clone Easy medium for 8-10 days, and cell clones were sel...

Embodiment 2

[0048] Example 2, Preparation, purification and titer of monoclonal antibody N-3G3

[0049] 1. Preparation of monoclonal antibody N-3G3

[0050] 1. Incremental cultivation method

[0051] The preparation method of the cell culture medium (7.4): add fetal bovine serum to RPMI-1640 medium, and the final concentration of fetal bovine serum is 20% (mass percentage).

[0052] The hybridoma cell line N-3G3 was placed in the cell culture medium and cultured at 37°C for 3-4 days, and the cell culture supernatant was purified by Protein A affinity chromatography to obtain a monoclonal antibody (stored at -20°C).

[0053] 2. Ascites preparation

[0054] Balb / c mice were intraperitoneally injected with sterilized paraffin oil (0.5mL / only), and 10 days later, intraperitoneally injected the hybridoma cell line N-3G3 (5×10 5 cells / only), 8-10 days later, ascitic fluid was collected, purified by Protein A affinity chromatography, and then transferred to a dialysis bag for dialysis in pH7.4,...

Embodiment 3

[0067] Embodiment 3, the preparation of colloidal gold rapid detection test paper

[0068] 1. Preparation of colloidal gold-labeled antibody

[0069] 1. Preparation of colloidal gold by trisodium citrate reduction method

[0070] Measure 495ml of deionized water with a measuring cylinder, pour it into a glass two-necked bottle, add 5ml of 1g / 100mL chloroauric acid aqueous solution; put a magnetic stirrer in the glass two-necked bottle, connect a spherical condenser, and use a temperature-controllable electromagnetic Heat the stirrer until boiling; add 5ml of 1g / 100mL trisodium citrate aqueous solution while stirring, the golden chloroauric acid solution turns purple within 2 minutes, continue to boil for 5 minutes, and then cool naturally, which is colloidal gold solution.

[0071] 2. Preparation of colloidal gold-labeled N-3G3 antibody

[0072] Get 20 parts by volume of colloidal gold solution, adjust the pH to 7.5 with 0.1M potassium carbonate aqueous solution, then add m...

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Abstract

The invention discloses a hybridoma cell capable of secreting an anti-novel coronavirus (SARS-COV-2) N protein monoclonal antibody, a monoclonal antibody and anapplication. The invention provides a hybridoma cell strain N-3G3, wherein the preservation number of strain is CCTCC NO: C202075. The invention also protects the monoclonal antibody secreted by the hybridoma cell strain N-3G3. The antibodywith high sensitivity and high specificity is the key to the development and implementation of an antigen detection technology. The specific antibody of the N protein of the SARS-CoV-2 is obtained onthe basis of a monoclonal antibody technology, and an SARS-COV-2 detection test strip is prepared from the N protein of the SARS-CoV-2. When the test strip provided by the invention is used for detecting the SARS-CoV-2, the operation is simple, the sensitivity is high, the specificity is strong, and the rapid monitoring and prevention of the SARS-COV-2 can be realized.

Description

technical field [0001] The invention belongs to the field of immune detection, and in particular relates to a hybridoma cell capable of secreting a monoclonal antibody against the N protein of a novel coronavirus (SARS-COV-2), a monoclonal antibody and an application thereof. Background technique [0002] The novel coronavirus (SARS-CoV-2) is a beta coronavirus belonging to the genus Coronavirus of the family Coronaviridae of the order Nidovirales. The transmission route of the new coronavirus is mainly through respiratory droplets and close contact, and there is also the possibility of aerosol transmission in a relatively closed environment. The new coronavirus is highly contagious, with an incubation period of 1-14 days after infection. There are clinically asymptomatic infections, and there are currently no effective vaccines and therapeutic drugs. Therefore, it is extremely important to diagnose and treat the novel coronavirus early, cut off the source of transmission, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/569G01N33/577G01N33/558
CPCC07K16/10G01N33/56983G01N33/577G01N33/558C07K2317/35C07K2317/92C07K2317/33G01N2333/165
Inventor 马岚吴峰岑瑜
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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