Hybridoma cell strain capable of stably secreting monoclonal antibody against novel coronavirus nucleocapsid protein as well as establishment method and application of hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, applied in the field of hybridoma cell line and its establishment, can solve the problems of loss, decreased ability to secrete monoclonal antibody, monoclonal antibody single reactivity, etc.

Pending Publication Date: 2021-11-05
GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, due to the different sources of B lymphocytes of different hybridoma cell lines, most of the monoclonal antibodies secreted have only a single reactivity, that is, they can only be used for one detection method to screen out the hybrids that secrete polyreactive monoclonal antibodies. Tumor cell lines are a huge challenge
At the same time, hybridoma cell lines often decrease or even lose the ability to secrete monoclonal antibodies during the passage process, cryopreservation and recovery process, and it is also a challenge to obtain cell lines that stably secrete monoclonal antibodies

Method used

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  • Hybridoma cell strain capable of stably secreting monoclonal antibody against novel coronavirus nucleocapsid protein as well as establishment method and application of hybridoma cell strain
  • Hybridoma cell strain capable of stably secreting monoclonal antibody against novel coronavirus nucleocapsid protein as well as establishment method and application of hybridoma cell strain
  • Hybridoma cell strain capable of stably secreting monoclonal antibody against novel coronavirus nucleocapsid protein as well as establishment method and application of hybridoma cell strain

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Embodiment 1

[0047] Establishment of hybridoma cell lines for anti-new coronavirus nucleocapsid protein monoclonal antibody:

[0048] 1. Recombinant nucleoprotein

[0049] According to the published SARS-CoV-2 genome sequence, a gene sequence encoding the SARS-CoV-2 nucleocapsid protein suitable for expression in Escherichia coli was artificially synthesized and cloned into the expression vector pET28a. Transform competent Escherichia coli BL-21 with the recombinant expression vector, apply LB kanamycin plate, and culture overnight at 37°C, pick a single colony the next day, inoculate in 3ml LB culture medium, and culture at 37°C Shake culture for 16 hours, extract the plasmid, and carry out gene sequencing. Transform the recombinant expression plasmid with correct sequencing into BL-21 bacteria, inoculate it into LB medium tubes containing 50 μg / ml kanamycin, culture it with shaking at 37°C overnight, and then inoculate 1% of the inoculum into the tubes containing 50μg / ml Kanamycin 200m...

Embodiment 2

[0071] ELISA kit for detection of new crown IgM antibody using nCoVN3G6 monoclonal antibody

[0072] Dilute goat anti-human IgM antibody with 1:500 phosphate buffer solution (PBS pH7.2), add 100 μL per well to a 96-well ELISA reaction plate, coat at 4°C overnight, then add 100 μL 3% BSA to each well to block for 1 hour at room temperature , wash the ELISA plate 3 times with PBS-Tween20, add 100 μL of 1:100 diluted patient serum, react at 37°C for 1 hour, wash the ELISA plate 3 times with PBS-Tween20, add 100 μL (25ng) of recombinant SARS-CoV-2--N protein, 37 React for 1 hour at ℃, wash the ELISA plate 3 times with PBS-Tween20, dilute horseradish peroxidase (HRP)-labeled nCoVN3G6 monoclonal antibody, react for 1 hour at 37℃, wash the ELISA plate 3 times with PBS-Tween20, add HRP base (ABTS or TMB or OPD), stop the reaction after 30 minutes of chromogenic reaction at 37°C, measure the absorbance OD value of each sample well with a microplate reader, if the OD value is more than ...

Embodiment 3

[0075] ELISA kit for detection of SARS-CoV-2 virus antigen by double antibody sandwich method using nCoVN3G6 monoclonal antibody

[0076] Add nCoVN3G6 monoclonal antibody diluted with 1:10000 phosphate buffered saline (PBS pH7.2), add 100 μL per well to a 96-well ELISA reaction plate, coat at 4°C overnight, then add 100 μL 3% BSA to each well to block at room temperature for 1 hour, Wash the ELISA plate 3 times with PBS-Tween20, add 100 μL patient’s nasopharyngeal swab sample preservation solution, react at 37°C for 1 hour, wash the ELISA plate 3 times with PBS-Tween20, add 100 μL diluted horseradish peroxidase (HRP)-labeled nCoVN3G6 monoclonal antibody, react at 37°C for 1 hour, wash the ELISA plate 3 times with PBS-Tween20, add HRP substrate (ABTS or TMB or OPD), stop the reaction after 30 minutes of color reaction at 37°C, and measure each sample with a microplate reader Well absorbance OD value, OD value more than 2 times higher than the negative control was judged as posi...

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Abstract

The invention belongs to the technical field of biological immunity, and particularly relates to a hybridoma cell strain capable of stably secreting a monoclonal antibody against novel coronavirus nucleocapsid protein as well as an establishment method and application of the hybridoma cell strain. The hybridoma cell strain nCoVN3G6 capable of stably secreting the anti-SARS-CoV-2 monoclonal antibody is obtained by expressing and purifying a recombinant SARS-COV-2 virus N protein in escherichia coli and performing animal immunization by adopting the purified recombinant nucleoprotein. The nCoVN3G6 monoclonal antibody is utilized to establish a kit for detecting novel coronavirus antigens through a novel coronavirus capture method IgM ELISA, a kit for detecting the novel coronavirus antigens through a double-antibody sandwich method ELISA, a kit for detecting the novel coronavirus antigens through colloidal gold and a kit for detecting the novel coronavirus antigens through time resolution fluorescence chromatography, and a foundation is laid for diagnosis of SARS-COV-2 virus infection and research of infection mechanisms and vaccine development in the future.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and in particular relates to a hybridoma cell line stably secreting monoclonal antibody against nucleocapsid protein of novel coronavirus and its establishment method and application. Background technique [0002] The novel coronavirus (SARS-CoV-2) is a new strain of coronavirus that has never been found in humans before. SARS-CoV-2 belongs to the genus Betacoronavirus of the Coronaviridae family. It is a packaged positive-sense single-stranded RNA virus with a diameter of about 80-120nm. The viral genome contains 29844 bases, and the encoded viral structural protein mainly has spikes. (Spike, S) protein, membrane (membrane, M) protein and nucleocapsid (Nucleocapsid, N) protein. The disease caused by the new coronavirus infection is called coronavirus disease 2019 (COVID-19). Symptoms of infection can range from mild or minimal respiratory symptoms to acute respiratory distress syndr...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N5/20G01N33/569G01N33/577
CPCC07K16/10G01N33/56983G01N33/577G01N2333/165
Inventor 余福勋查艳詹琳刘琳杨斌杨丽
Owner GUIZHOU PROVINCIAL PEOPLES HOSPITAL
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