CN113603769A - Hybridoma cell strain capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody and establishment method and application thereof - Google Patents
Hybridoma cell strain capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody and establishment method and application thereof Download PDFInfo
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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 monoclonal antibodies against novel coronavirus nucleocapsid proteins, and an establishment method and application thereof; the recombinant SARS-COV-2 virus N protein is expressed in colibacillus and purified, the purified recombinant nucleoprotein is used for animal immunization, so that hybridoma cell strain nCoVN3G6 capable of stably secreting monoclonal antibody resisting SARS-CoV-2 is obtained, and nCoVN3G6 monoclonal antibody is used for establishing new crown capture method IgM ELISA, double antibody sandwich method ELISA kit for detecting new crown virus antigen, colloidal gold kit for detecting new crown virus antigen and time-resolved fluorescence chromatography kit for detecting new crown virus antigen, so that the method lays a foundation for the diagnosis of SARS-COV-2 virus infection, the infection mechanism and the research of vaccine development.
Description
Technical Field
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 a novel coronavirus nucleocapsid protein, and an establishment method and application thereof.
Background
The novel coronavirus (SARS-CoV-2) is a new strain of coronavirus that has not been previously discovered in humans. SARS-CoV-2 belongs to the genus beta coronavirus in the family Coronaviridae, is a positive strand single stranded RNA virus with a diameter of about 80-120nm, and the viral genome contains 29844 bases, and the encoded virus structural proteins mainly include Spike (Spike, S) protein, membrane (M) protein and Nucleocapsid (N) protein. The disease caused by the new type of coronavirus infection is called 2019 coronavirus disease (COVID-19), the symptoms of the infection can range from mild or minimal respiratory symptoms to Acute Respiratory Distress Syndrome (ARDS) and even death, and no specific treatment method for the COVID-19 exists at present. The early rapid diagnosis is the key of prevention and treatment, and is vital to controlling nosocomial infection, cutting off disease transmission and the like.
At present, RT-PCR has become the gold standard for diagnosing COVID-19, but studies have shown that this method has high requirements on operators and laboratory conditions, and in addition, asymptomatic infectors are present, and the viral load is low in some patients after the onset of disease, so that an immunological detection method based on the antibody of the COVID-19 virus is urgently needed as a supplement.
The prior patent document with the application number of CN202010564744.1 discloses a hybridoma cell and a monoclonal antibody capable of secreting a novel coronavirus N protein monoclonal antibody, which is to dilute the N protein of SARS-CoV-2 by using normal saline, mix the diluted N protein with Quick Anti-body-Mouse 3W adjuvant, inject the mixture into an immune Balb/c Mouse, take serum to detect the serum titer, fuse spleen cells and myeloma cells, culture the fused cells, clone, freeze-store, purify, dialyze and screen to obtain a hybridoma cell strain.
The patent document with application number of CN202010986023.X discloses a monoclonal antibody for detecting SARS-CoV-2 virus N protein and its application, and it also adopts recombinant N protein to obtain hybridoma cell through animal immunization, cell fusion and hybridoma cell culture and screening, but the adopted recombinant N protein is a complete open reading frame region synthesized by nucleotide sequence of coding N protein in new coronavirus genome sequence, and clones it to plasmid vector pUC57, designs upstream primer and downstream primer, and introduces EcoRI/XhoI double enzyme cutting site, and after cell transfection and culture, the recombinant N protein with histidine label is obtained after purification.
Monoclonal antibodies are single antibodies against a single epitope produced by a single B lymphocyte, and are widely used because they are derived from a single B lymphocyte and have high specificity. Many of them are secreted and produced by hybridoma cells formed by fusing B lymphocytes having an antibody-producing function with myeloma cells having an immortal function. Therefore, different hybridoma cell strains have different B lymphocyte sources, and the secreted monoclonal antibodies have only single reactivity, namely, the method can be only used for one detection method, and the screening of the hybridoma cell strains secreting the monoclonal antibodies with multiple reactivities is a great challenge. Meanwhile, hybridoma cell strains often cause the reduction and even loss of the ability of secreting monoclonal antibodies in the process of passage and the process of cryopreservation and recovery, and obtaining cell strains stably secreting monoclonal antibodies is also a challenge.
Disclosure of Invention
The invention provides a hybridoma cell strain for stably secreting a monoclonal antibody against a novel coronavirus nucleocapsid protein to solve the problems, and an establishment method and application thereof.
The method is realized by the following technical scheme:
a hybridoma cell strain secreting monoclonal antibody against novel coronavirus SARS-CoV-2 nucleocapsid protein, named nCoVN3G6, is deposited in China center for type culture Collection CCTCC with addresses of: the preservation date of Wuhan university in Wuhan, China is 2020, 12 and 01 months, and the preservation number is CCTCC NO: C2020255.
Secondly, the method for establishing the hybridoma cell strain secreting the anti-novel coronavirus nucleocapsid protein monoclonal antibody comprises the following steps:
1. recombinant nucleoproteins
According to the published SARS-CoV-2 genome sequence, a gene sequence encoding SARS-CoV-2 nucleocapsid protein suitable for expression in E.coli was artificially synthesized and cloned into expression vector pET28 a. Transforming competent Escherichia coli BL-21 with the recombinant expression vector, coating LB kanamycin flat plate, standing and culturing overnight at 37 ℃, respectively picking single colony the next day, inoculating in 3ml LB culture solution, shaking and culturing at 37 ℃ for 16h, extracting plasmid, and performing gene sequencing. The BL-21 strain transformed by the recombinant expression plasmid with correct sequencing is inoculated into LB culture medium containing 50 mug/ml kanamycin, shaking culture is carried out overnight at 37 ℃, then the strain is respectively inoculated into 200ml LB culture medium triangular flasks containing 50 mug/ml kanamycin according to the inoculum size of 1 percent for expanding culture, shaking culture is carried out at 37 ℃ until the A600 value of the strain is about 0.6, IPTG with the final concentration of 0.5M is added, and shaking culture induction is carried out at 37 ℃ for 4 hours. Collecting the thalli, carrying out ultrasonication and centrifugation, taking supernatant, purifying by using a Ni2+ affinity chromatography column, and identifying and analyzing a purified expression product by 12% SDS-PAGE and Western blot.
2. Cell fusion
Immunizing BALB/C mice with purified recombinant SARS-COV-2 nucleoprotein, immunizing at a dose of 100 micrograms per mouse, emulsifying with Freund's complete adjuvant for the first immunization, emulsifying with incomplete adjuvant for the second and third immunizations, immunizing at an interval of two weeks, and collecting blood from the tail of the mouse after 7 days of the third immunizations to determine the serum antibody titer. 3d, strengthening immunity once before fusion, taking splenocytes of the immunized mice and SP2/0 to perform cell fusion according to a conventional method, and screening hybridoma cells through HAT selective culture.
3. Screening
Screening positive hybridoma cells by using an indirect ELISA method, and after 3 times of subcloning by using a limiting dilution method, obtaining 1 hybridoma cell strain which stably secretes the anti-SARS-COV-2-N protein monoclonal antibody and is named as nCoVN3G 6.
4. Identification of monoclonal antibody secreted by hybridoma cell strain
The monoclonal antibody secreted by the hybridoma cell strain nCoVN3G6 reacts with SARS-COV-2 infected cells, influenza virus infected cells, EV71 virus infected cells and Coxsacky virus infected cells respectively, and specificity identification is carried out by using an indirect immunofluorescence method.
Inoculating the hybridoma cell secreting monoclonal antibody of SARS-CoV-2 nucleocapsid protein to abdominal cavity of mouse to prepare ascites, and performing ELISA and immunofluorescence titer on ascites, specificity of monoclonal antibody and subclass identification on secreted monoclonal antibody.
5. Detection of stability of monoclonal antibody secreted by hybridoma cell strain
Culturing the hybridoma cell strain nCoVN3G6 in a 1640 culture medium containing 10% fetal calf serum, adding a fresh culture medium according to the growth condition of the cells at a ratio of 1:4 after 3-4 days, subculturing, continuously subculturing for 30 generations, taking the supernatant of the cultured cells, and detecting the secreted antibody by immunofluorescence, ELISA and Western-blot detection.
Meanwhile, the hybridoma cells for passage are taken, after the cells are collected by centrifugation, cell freezing medium is added, after the cells are frozen for 3 months, 6 months and 12 months in a refrigerator with the temperature of 80 ℃ below zero and a liquid nitrogen tank, the frozen cells are taken, after recovery, the culture medium is added for culture, and the stability of the secreted monoclonal antibody is detected.
Application method of monoclonal antibody secreted by hybridoma cell strain nCoVN3G6
1. New crown IgM antibody detection ELISA kit by using nCoVN3G6 monoclonal antibody capture method
Adding 100 μ L of goat anti-human IgM antibody diluted with 1:500 phosphate buffer solution (PBS pH7.2) to a 96-well ELISA reaction plate per well, coating overnight at 4 deg.C, adding 100 μ L of 3% BSA per well, blocking at room temperature for 1 hr, PBS-Tween20 washes the ELISA plate 3 times, adds 100. mu.L 1:100 diluted patient serum, reacts at 37 ℃ for 1 hour, PBS-Tween20 washes the ELISA plate 3 times, adds 100. mu.L (25ng) of recombinant SARS-CoV-2-N protein, reacts for 1 hour at 37 ℃, PBS-Tween20 washing ELISA plate 3 times, diluted horseradish peroxidase (HRP) labeled nCoVN3G6 monoclonal antibody, 37 degrees C reaction for 1 hours, washing the ELISA plate 3 times with PBS-Tween20, adding HRP substrate (ABTS or TMB or OPD), developing at 37 deg.C for 30 min, terminating the reaction, and determining the absorbance OD value of each sample well with microplate reader, wherein the OD value is more than 2 times higher than that of the negative control, and determining the sample is positive.
2. ELISA kit for detecting SARS-CoV-2 virus antigen by double-antibody sandwich method using nCoVN3G6 monoclonal antibody
Adding 100 mu L of nCoVN3G6 monoclonal antibody diluted by 1:10000 phosphate buffer solution (PBS pH7.2) into each well, adding 96-well ELISA reaction plates into each well, coating overnight at 4 ℃, adding 100 mu L of 3% BSA into each well, sealing for 1 hour at room temperature, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of patient nasopharyngeal swab sample preserving fluid, reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of diluted nCoVN3G6 monoclonal antibody labeled by Horse Radish Peroxidase (HRP), reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding HRP substrates (ABTS or TMB or OPD), performing chromogenic reaction at 37 ℃ for 30 minutes, stopping the reaction, measuring the absorbance OD value of each sample by a microplate reader, and judging that the OD value is more than 2 times higher than that of the negative control to be positive.
3. Kit for detecting new coronavirus antigen by using colloidal gold of nCoVN3G6 monoclonal antibody
The kit is an immune colloidal gold test strip, and the test strip sequentially comprises the following components in connection order: the sample pad, the colloidal gold pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate which is positioned below and used as an assembly platform.
The nitrocellulose membrane and the colloidal gold pad are in lap joint, the nitrocellulose membrane and the absorbent paper are in lap joint, the colloidal gold pad is composed of a glass cellulose membrane which adsorbs an nCoVN3G6 monoclonal antibody which is labeled by colloidal gold and is used for resisting new coronavirus, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-mouse IgG polyclonal antibody and a detection line coated by the nCoVN3G6 monoclonal antibody.
The quality control line was obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating the nCoVN3G6 monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
The detection method of the kit for SFTS virus detection comprises the following steps: dripping 200 mu L of a test sample into the sample dripping hole, reacting for 10-15min and observing the result; the quality control line and the detection line are positive if red strips appear; a red strip appears only on the quality control line, and the strip is negative; if no strip appears in the quality control line, the result is judged to be invalid no matter whether the detection line has a red strip or not.
4. Kit for detecting neocoronavirus antigen by using time-resolved fluorescence chromatography of nCoVN3G6 monoclonal antibody
The kit is a time-resolved fluorescence chromatography test strip, and the test strip sequentially comprises the following components in connection order: the sample pad, the fluorescence ball pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate which is arranged below and used as an assembly platform.
The nitrocellulose membrane is overlapped with the fluorescent ball pad, the nitrocellulose membrane is overlapped with the absorbent paper, the fluorescent ball pad is composed of a glass cellulose membrane which adsorbs a nCoVN3G6 monoclonal antibody which is labeled by a time-resolved fluorescent ball and is used for resisting the new coronavirus, and the nitrocellulose membrane is provided with a quality control line coated by a goat anti-mouse IgG polyclonal antibody and a detection line coated by the nCoVN3G6 monoclonal antibody.
The quality control line was obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating the nCoVN3G6 monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
The detection method of the kit for SFTS virus detection comprises the following steps: dripping 200 mu L of a test sample into the sample dripping hole, reacting for 10-15min and observing the result; the quality control line and the detection line are detected by a dry fluorescence immunoassay analyzer to generate fluorescence signals, and the fluorescence signals are positive; the fluorescence signal appears only on the quality control line, and the signal is negative; if the quality control line has no fluorescence signal, the result is judged to be invalid no matter whether the detection line has the fluorescence signal or not.
In conclusion, the beneficial effects of the invention are as follows: the invention expresses recombinant SARS-COV-2 virus N protein in colon bacillus and purifies, adopts purified recombinant nucleoprotein to carry out animal immunity, establishes hybridoma cell strain nCoVN3G6 which can stably secrete monoclonal antibody (MAb) for resisting SARS-CoV-2, and utilizes nCoVN3G6 monoclonal antibody to establish IgM of new crown capture method, ELISA of double antibody sandwich method for detecting new crown virus antigen, reagent kit for detecting new crown virus antigen by colloidal gold, reagent kit for detecting new crown virus antigen by time-resolved fluorescence chromatography, and lays a foundation for the diagnosis of SARS-COV-2 virus infection, infection mechanism and research of vaccine development in the future.
Since the N protein of SARS-CoV-2 is the most abundant protein in coronavirus, it is located in the interior of virus and has high conservation. The N protein is a highly immunogenic phosphorylated protein that binds to viral RNA during virion assembly to form a helical nucleocapsid and is involved in viral genome replication and regulation of cellular signaling pathways. Therefore, the monoclonal antibody of SARS-CoV-2 can be stably obtained by using the N protein of SARS-CoV-2 to establish hybridoma cell strain, and further, the monoclonal antibody of SARS-CoV-2 can be applied to a detection kit, and can also be used for timely diagnosing asymptomatic infectors, the currently adopted pharynx swab nucleic acid detection needs to detect a plurality of times for partial asymptomatic infectors to detect positive reaction, and the pharynx swab detection has a new corona nucleic acid positive rate of not 100%, if the patient has epidemiological history and has fever symptoms, the routine chest CT and blood examination is perfected to carry out comprehensive judgment. An immunological detection method of a virus antibody is used as a supplementary diagnosis method, and the virus antibody can be quickly and efficiently detected in the early latent period of the virus, so that the infection is effectively controlled, and the disease transmission is cut off.
Hybridoma cell strains obtained in different laboratories are cell strains prepared independently, and the characteristics, cell stability and application range of the secreted monoclonal antibody may differ. The hybridoma cell strain prepared by the method can stably secrete the monoclonal antibody of the anti-new coronavirus N protein through repeated freezing storage and recovery passage of liquid nitrogen, and the secreted monoclonal antibody can be used for detecting the new coronavirus antigen by multiple methods such as immunofluorescence, ELISA, Western-blot, colloidal gold test strips and the like, and has wide application.
Drawings
FIG. 1 shows the SDS-PAGE analysis of recombinant SARS-COV-2-N protein; wherein Lane M is a standard molecular weight protein, and Lane 1 is a supernatant obtained after ultrasonic disruption of Escherichia coli; lane 2 is bacterial sediment after the ultrasonic disruption of escherichia coli; lane 3 is a purified recombinant protein; lane4 is a purified recombinant protein.
FIG. 2 shows the Western-blot analysis result of purified recombinant SARS-COV-2-N protein; wherein Lane M is a standard molecular weight protein, and Lane 1 is a purified recombinant protein.
FIG. 3 shows the results of immunofluorescence analysis of nCoVN3G6 mab; wherein, A picture is the result of analyzing uninfected Vero cells; and B is the analysis result of the new coronavirus infected Vero cell.
FIG. 4 shows the Western-blot analysis result of the reaction of nCoVN3G6 monoclonal antibody and recombinant SARS-CoV-2-N protein.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
Establishing a hybridoma cell strain of the anti-neocoronavirus nucleocapsid protein monoclonal antibody:
1. recombinant nucleoproteins
According to the published SARS-CoV-2 genome sequence, a gene sequence encoding SARS-CoV-2 nucleocapsid protein suitable for expression in E.coli was artificially synthesized and cloned into expression vector pET28 a. Transforming competent Escherichia coli BL-21 with the recombinant expression vector, coating LB kanamycin flat plate, standing and culturing overnight at 37 ℃, respectively picking single colony the next day, inoculating in 3ml LB culture solution, shaking and culturing at 37 ℃ for 16h, extracting plasmid, and performing gene sequencing. The BL-21 strain transformed by the recombinant expression plasmid with correct sequencing is inoculated into LB culture medium containing 50 mug/ml kanamycin, shaking culture is carried out overnight at 37 ℃, then the strain is respectively inoculated into 200ml LB culture medium triangular flasks containing 50 mug/ml kanamycin according to the inoculum size of 1 percent for expanding culture, shaking culture is carried out at 37 ℃ until the A600 value of the strain is about 0.6, IPTG with the final concentration of 0.5M is added, and shaking culture induction is carried out at 37 ℃ for 4 hours. The cells were collected, sonicated, centrifuged, and the supernatant was purified by Ni2+ affinity column chromatography.
The purified expression product was analyzed by 12% SDS-PAGE, and a specific protein band having a relative molecular weight of about 50kD was observed, the size of which was consistent with the expected size, as shown in FIG. 1.
Western blot analysis shows that the purified recombinant protein is separated through SDS-PAGE electrophoresis, electrophoretically transferred to PADF fibrous membrane to react with serum of new coronary patient in recovery period, and the specific reaction between the purified recombinant protein and the serum of new coronary patient proves that the expressed protein is SARS-COV-2-N protein as shown in figure 2.
2. Cell fusion
Immunizing BALB/C mice with purified recombinant SARS-COV-2 nucleoprotein, immunizing at a dose of 100 micrograms per mouse, emulsifying with Freund's complete adjuvant for the first immunization, emulsifying with incomplete adjuvant for the second and third immunizations, immunizing at an interval of two weeks, and collecting blood from the tail of the mouse after 7 days of the third immunizations to determine the serum antibody titer. 3d, strengthening immunity once before fusion, taking splenocytes of the immunized mice and SP2/0 to perform cell fusion according to a conventional method, and screening hybridoma cells through HAT selective culture.
3. Screening
Screening positive hybridoma cells by using an indirect ELISA method, and after 3 times of subcloning by using a limiting dilution method, obtaining 1 hybridoma cell strain which stably secretes the anti-SARS-COV-2-N protein monoclonal antibody and is named as nCoVN3G 6.
4. Identification of monoclonal antibody secreted by hybridoma cell strain
(1) Detecting by adopting an indirect immunofluorescence method:
preparation and inactivation of green monkey kidney (Vero) cell sheet infected with novel coronavirus (SARS-CoV-2) were carried out in the Biosafety class 3 (P3) laboratory of the institute of tropical medicine, university of Nagasaki, Japan. The main operation is as follows:
a novel Japanese isolate of coronory TY-WK-521/20202(Genebank No.: LC522975) was inoculated with Vero cells and cultured at 37 ℃ for 4 days in an Eagle basal medium containing 2% fetal bovine serum. The virus-infected cells were digested with 0.25% trypsin, washed 2 times with phosphate buffered saline (PBS, pH7.2), centrifuged at 2000 rpm to collect the cells, and suspended in a small amount of PBS. Vero was not infected and treated similarly. Virus infected and uninfected Vero were dropped onto 8-well slides (MP Bio-chemicals, CA, USA), cell sheets were blown dry, uv-inactivated, pre-cooled acetone fixed at 4 ℃ and further inactivated. Blocking virus infected and uninfected Vero cell slides by 3% BSA at room temperature for 1 hour, adding nCoVN3G6 hybridoma cell culture supernatant or diluting hybridoma cell ascites, reacting at 37 ℃ for 1 hour, washing the slides with PBS buffer, drying the slides by blowing, adding 1:50 diluted FITC-labeled goat anti-mouse IgG antibody, reacting at 37 ℃ for 1 hour, washing the slides with PBS buffer, drying the slides by blowing, adding a fluorescent protective agent (vector laboratories, Inc.), placing the slides on a cover slip, mounting the slides, and observing and recording the result under a fluorescent microscope (Olympus, Japan) as shown in FIG. 3.
(2) Western-blot analysis of nCoVN3G6 mab:
the analysis result shows that the nCoVN3G6 monoclonal antibody can specifically recognize the recombinant SARS-CoV-2-N protein, as shown in FIG. 4. Suggesting that the nCoVN3G6 monoclonal antibody can be used for western-blot analysis of the new coronavirus.
(3) ELISA and immunofluorescence titer identification:
injecting hybridoma nCoVN3G6 into mouse abdominal cavity to prepare monoclonal antibody titer of ascites, and measuring by indirect ELISA method of coating recombinant nucleoprotein, wherein the titer is 1:100, 000; the ascites titer is 1:100000 determined by indirect immunofluorescence; are obviously higher than the titer of immune serum.
(4) And (3) monoclonal antibody subclass identification:
the monoclonal antibody of the strain is identified to have a heavy chain of IgG2a and a light chain of kappa chain.
5. Determination of stability of monoclonal antibody secreted by hybridoma cell strain
Culturing the hybridoma cell strain nCoVN3G6 in a 1640 culture medium containing 10% fetal calf serum, adding a fresh culture medium according to the growth condition of the cells at 1:4 after 4 days, subculturing, continuously subculturing for 30 generations, taking the supernatant of the cultured cells, and detecting the secreted antibody by immunofluorescence, ELISA and Western-blot.
Meanwhile, the hybridoma cells for passage are taken, after the cells are collected by centrifugation, cell freezing medium is added, after the cells are frozen for 3 months, 6 months and 12 months in a refrigerator with the temperature of 80 ℃ below zero and a liquid nitrogen tank, the frozen cells are taken, after recovery, the culture medium is added for culture, and the stability of the secreted monoclonal antibody is detected.
The result shows that the hybridoma cell strain nCoVN3G6 can stably secrete the monoclonal antibody against the new coronavirus N protein after continuous subculture for more than 30 generations, repeated cryopreservation and resuscitation at different time periods, and is a hybridoma cell strain capable of stably secreting the monoclonal antibody.
Example 2
New crown IgM antibody detection ELISA kit by using nCoVN3G6 monoclonal antibody capture method
Adding 100 μ L of goat anti-human IgM antibody diluted with 1:500 phosphate buffer solution (PBS pH7.2) to a 96-well ELISA reaction plate per well, coating overnight at 4 deg.C, adding 100 μ L of 3% BSA per well, blocking at room temperature for 1 hr, PBS-Tween20 washes the ELISA plate 3 times, adds 100. mu.L 1:100 diluted patient serum, reacts at 37 ℃ for 1 hour, PBS-Tween20 washes the ELISA plate 3 times, adds 100. mu.L (25ng) of recombinant SARS-CoV-2-N protein, reacts for 1 hour at 37 ℃, PBS-Tween20 washing ELISA plate 3 times, diluted horseradish peroxidase (HRP) labeled nCoVN3G6 monoclonal antibody, 37 degrees C reaction for 1 hours, washing the ELISA plate 3 times with PBS-Tween20, adding HRP substrate (ABTS or TMB or OPD), developing at 37 deg.C for 30 min, terminating the reaction, and determining the absorbance OD value of each sample well with microplate reader, wherein the OD value is more than 2 times higher than that of the negative control, and determining the sample is positive.
The positive SARS-CoV-2 virus specific IgM antibody can diagnose the recent SARS-CoV-2 virus infection, and is the main auxiliary diagnosis means of COVID-19.
Example 3
ELISA kit for detecting SARS-CoV-2 virus antigen by double-antibody sandwich method using nCoVN3G6 monoclonal antibody
Adding 100 mu L of nCoVN3G6 monoclonal antibody diluted by 1:10000 phosphate buffer solution (PBS pH7.2) into each well, adding 96-well ELISA reaction plates into each well, coating overnight at 4 ℃, adding 100 mu L of 3% BSA into each well, sealing for 1 hour at room temperature, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of patient nasopharyngeal swab sample preserving fluid, reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding 100 mu L of diluted nCoVN3G6 monoclonal antibody labeled by Horse Radish Peroxidase (HRP), reacting for 1 hour at 37 ℃, washing the ELISA plates by PBS-Tween20 for 3 times, adding HRP substrates (ABTS or TMB or OPD), performing chromogenic reaction at 37 ℃ for 30 minutes, stopping the reaction, measuring the absorbance OD value of each sample by a microplate reader, and judging that the OD value is more than 2 times higher than that of the negative control to be positive.
Example 4
Kit for detecting new coronavirus antigen by using colloidal gold of nCoVN3G6 monoclonal antibody
The kit is an immune colloidal gold test strip, and the test strip sequentially comprises the following components in connection order: the sample pad, the colloidal gold pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate which is positioned below and used as an assembly platform.
The nitrocellulose membrane and the colloidal gold pad are in lap joint, the nitrocellulose membrane and the absorbent paper are in lap joint, the colloidal gold pad is composed of a glass cellulose membrane which adsorbs an nCoVN3G6 monoclonal antibody which is labeled by colloidal gold and is used for resisting new coronavirus, and the nitrocellulose membrane is provided with a quality control line coated by goat anti-mouse IgG polyclonal antibody and a detection line coated by the nCoVN3G6 monoclonal antibody.
The quality control line was obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating the nCoVN3G6 monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
The detection method of the kit for SFTS virus detection comprises the following steps: dripping 200 mu L of a test sample into the sample dripping hole, reacting for 10-15min and observing the result; the quality control line and the detection line are positive if red strips appear; a red strip appears only on the quality control line, and the strip is negative; if no strip appears in the quality control line, the result is judged to be invalid no matter whether the detection line has a red strip or not.
The test paper strip is adopted to test new coronavirus, influenza virus H1N1, enterovirus EV71 and coxsackievirus CA16 respectively, and the result shows that the test paper strip specificity based on the nCoVN3G6 monoclonal antibody reacts with the new coronavirus, and has no cross reaction with the influenza virus H1N1, the enterovirus EV71 and the coxsackievirus CA 16.
Example 5
Kit for detecting neocoronavirus antigen by using time-resolved fluorescence chromatography of nCoVN3G6 monoclonal antibody
The kit is a time-resolved fluorescence chromatography test strip, and the test strip sequentially comprises the following components in connection order: the sample pad, the fluorescence ball pad, the nitrocellulose membrane, the absorbent paper and the PVC bottom plate which is arranged below and used as an assembly platform.
The nitrocellulose membrane is overlapped with the fluorescent ball pad, the nitrocellulose membrane is overlapped with the absorbent paper, the fluorescent ball pad is composed of a glass cellulose membrane which adsorbs a nCoVN3G6 monoclonal antibody which is labeled by a time-resolved fluorescent ball and is used for resisting the new coronavirus, and the nitrocellulose membrane is provided with a quality control line coated by a goat anti-mouse IgG polyclonal antibody and a detection line coated by the nCoVN3G6 monoclonal antibody.
The quality control line was obtained by coating goat anti-mouse IgG polyclonal antibody on nitrocellulose membrane at a concentration of 0.8 mg/ml; the test line was obtained by coating the nCoVN3G6 monoclonal antibody on a nitrocellulose membrane at a concentration of 0.5 mg/ml.
The detection method of the kit for SFTS virus detection comprises the following steps: dripping 200 mu L of a test sample into the sample dripping hole, reacting for 10-15min and observing the result; the quality control line and the detection line are detected by a dry fluorescence immunoassay analyzer to generate fluorescence signals, and the fluorescence signals are positive; the fluorescence signal appears only on the quality control line, and the signal is negative; if the quality control line has no fluorescence signal, the result is judged to be invalid no matter whether the detection line has the fluorescence signal or not.
The test paper strip is adopted to test new coronavirus, influenza virus H1N1, enterovirus EV71 and coxsackievirus CA16 respectively, and the results show that the test paper strip specificity based on the nCoVN3G6 monoclonal antibody reacts with the new coronavirus protein and has no cross reaction with the influenza virus H1N1, the enterovirus EV71 and the coxsackievirus CA 16.
Claims (10)
1. A hybridoma cell strain for stably secreting monoclonal antibodies against novel coronavirus SARS-CoV-2 nucleocapsid protein is characterized in that the hybridoma cell strain is named as nCoVN3G6 and is preserved in China center for type culture Collection CCTCC with the address as follows: the preservation date of Wuhan university in Wuhan, China is 2020, 12 and 01 months, and the preservation number is CCTCC NO: C2020255.
2. The hybridoma cell line ncouv 3G6 that stably secretes a monoclonal antibody against the novel coronavirus SARS-CoV-2 nucleocapsid protein of claim 1, wherein the heavy chain of the monoclonal antibody secreted by the hybridoma cell line is IgG2a and the light chain is kappa chain.
3. The hybridoma cell line ncouv 3G6 secreting monoclonal antibodies against the novel coronavirus SARS-CoV-2 nucleocapsid protein according to claim 1 or 2, wherein the hybridoma cell line is obtained by expressing the recombinant SARS-CoV-2 virus N protein in escherichia coli, purification, animal immunization, cell fusion, screening, identification process.
4. A method for establishing a hybridoma cell strain nCoVN3G6 capable of stably secreting anti-novel coronavirus nucleocapsid protein monoclonal antibody is characterized by comprising the following steps:
(1) recombinant nucleoprotein: expressing recombinant SARS-CoV-2 virus N protein in colibacillus, and purifying with Ni2+ affinity chromatographic column;
(2) cell fusion: immunizing a mouse by using the purified recombinant nucleoprotein, fusing immune mouse spleen lymphocytes and mouse myeloma SP2/0 cells, and culturing to obtain hybridoma cells;
(3) screening: detecting the supernatant of the hybridoma cell by adopting an indirect ELISA and an indirect immunofluorescence method, and screening out a hybridoma cell strain nCoVN3G6 secreting the monoclonal antibody against SARS-CoV-2 nucleocapsid protein;
(4) and (3) identification: the reaction between the monoclonal antibody secreted by the hybridoma cell strain nCoVN3G6 and SARS-COV-2 infected cells, influenza virus infected cells, EV71 virus infected cells and coxsackie virus infected cells is specifically identified by adopting an indirect immunofluorescence method.
5. The method for establishing a hybridoma cell line nCoVN3G6 according to claim 4, comprising the following steps:
(1) recombinant nucleoprotein: artificially synthesizing a gene sequence which codes SARS-CoV-2 nucleocapsid protein and is suitable for expression in escherichia coli according to a published SARS-CoV-2 genome sequence, cloning the gene sequence onto an expression vector pET28a, transforming competent escherichia coli BL-21 by a recombinant expression vector for culture, extracting plasmids for gene sequencing, transforming the recombinant expression plasmids with correct sequencing into the competent escherichia coli BL-21, culturing until the value of a bacterial liquid A600 is about 0.6, adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.5M, culturing and inducing for 4 hours, collecting thalli, performing ultrasonic disruption and centrifugation, taking supernatant, purifying by using a Ni2+ affinity chromatography column, and identifying and analyzing a purified expression product;
(2) cell fusion: immunizing a BALB/C mouse by using the purified recombinant nucleoprotein according to the dose of 100 micrograms per mouse, taking splenic lymphocytes of the immunized mouse and myeloma SP2/0 cells of the mouse after three times of immunization and one time of boosting immunization, and culturing to obtain hybridoma cells;
(3) screening: screening positive hybridoma cells by adopting an indirect ELISA and an indirect immunofluorescence method, and after 3 times of subcloning by a limiting dilution method, obtaining 1 hybridoma cell strain nCoVN3G6 which stably secretes an anti-SARS-CoV-2 nucleocapsid protein monoclonal antibody;
(4) and (3) identification: the reaction between the monoclonal antibody secreted by the hybridoma cell strain nCoVN3G6 and SARS-COV-2 infected cells, influenza virus infected cells, EV71 virus infected cells and coxsackie virus infected cells is specifically identified by adopting an indirect immunofluorescence method; and inoculating the hybridoma cells secreting monoclonal antibody of anti-SARS-CoV-2 nucleocapsid protein to abdominal cavity of mouse to prepare ascites, and identifying ELISA and immunofluorescence titer of ascites, specificity of monoclonal antibody and subclass of secreted monoclonal antibody.
6. Use of a hybridoma cell line nCoVN3G6 according to any one of claims 1 to 3 or of a hybridoma cell line nCoVN3G6 obtained by the method of construction according to any one of claims 4 to 6 in the detection of novel coronaviruses.
7. The application of claim 6, wherein the application is: the hybridoma cell strain nCoVN3G6 is applied to the acquisition method of nCoVN3G6 monoclonal antibody to obtain the application of the ELISA kit for detecting the novel coronavirus IgM antibody.
8. The application of claim 6, wherein the application is: the hybridoma cell strain nCoVN3G6 is applied to a double-antibody sandwich method using nCoVN3G6 monoclonal antibody to obtain an ELISA kit for detecting novel coronavirus SARS-CoV-2 antigen.
9. The application of claim 6, wherein the application is: the hybridoma cell strain nCoVN3G6 is applied to a kit for detecting the novel coronavirus antigen by using a colloidal gold method of nCoVN3G6 monoclonal antibody.
10. The application of claim 6, wherein the application is: the hybridoma cell strain nCoVN3G6 is applied to a time-resolved fluorescence chromatography method using nCoVN3G6 monoclonal antibody to obtain a kit for detecting novel coronavirus SARS-CoV-2 antigen.
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