CN112111007A - Preparation method of novel coronavirus nucleocapsid protein monoclonal antibody - Google Patents
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Abstract
The invention discloses a preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody, which comprises the following steps: synthesizing S1 polypeptide, preparing S2 immunogen, preparing and screening antigen by S3, immunizing S4 animals, fusing S5 cells, screening and detecting S6, establishing S7 stable cell strain, producing S8 monoclonal antibody, coupling S9 antibody and screening S10 pairing antibody; the polypeptide is respectively synthesized by different antigen epitopes at two ends of the N protein to be used as the antigen, the prepared monoclonal antibody can realize direct pairing, in the process of polypeptide synthesis, a connecting bridge is added to reduce steric hindrance, so that the antigen epitopes are fully exposed on the surface of the carrier protein, the antigen can be quickly identified, the recombinant protein is selected as the detection antigen, the situation that the polypeptide antigen is only identified by the antibody obtained by screening, the risk of the N protein is not identified is avoided, and whether the novel coronavirus is contained in a sample can be directly detected by detecting the content of the N protein.
Description
Technical Field
The invention belongs to the technical field of monoclonal antibody preparation, and particularly relates to a preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody.
Background
Since 12 months in 2019, a plurality of cases of pneumonia with unknown reasons are discovered in succession, and the pneumonia is proved to be an acute respiratory infectious disease caused by novel coronavirus infection. Based on current epidemiological investigations, the latency period is generally 3-7 days, and the maximum period is not more than 14 days. The clinical manifestations of this epidemic disease mainly include fever, hypodynamia and dry cough. A few patients have nasal obstruction, watery nasal discharge, diarrhea, etc. Some patients only show low fever, slight hypodynamia and the like, have no appearance of the pneumonia, and recover after 1 week mostly, the world health organization names a pathogen causing the viral pneumonia case to be a 2019 novel coronavirus, namely SARS-CoV-2 virus, and nucleocapsid protein (N protein) is the most abundant protein in the coronavirus.
The detection antibody used in the existing novel coronavirus detection kit can not realize direct pairing, and in the preparation process of the detection antibody, because the antigen epitope is not fully exposed, the speed of antigen recognition is reduced, and simultaneously, when the detection antibody is prepared, coronavirus is used as the antigen, and the coronavirus is easily combined with N protein to form the spiral nucleocapsid protein, so that the accuracy of the detection result is reduced.
Disclosure of Invention
The invention aims to provide a preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody.
The technical problems to be solved by the invention are as follows:
the detection antibody used in the existing novel coronavirus detection kit can not realize direct pairing, and in the preparation process of the detection antibody, because the antigen epitope is not fully exposed, the speed of antigen recognition is reduced, and simultaneously, when the detection antibody is prepared, coronavirus is used as the antigen, and the coronavirus is easily combined with N protein to form the spiral nucleocapsid protein, so that the accuracy of the detection result is reduced.
The purpose of the invention can be realized by the following technical scheme:
a preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody specifically comprises the following steps:
1 polypeptide synthesis: synthesizing a polypeptide with 15 amino acid sequences at the N end of the N protein, adding a connecting bridge and a carrier protein binding site to prepare an NP-N polypeptide, synthesizing a polypeptide with an aa383-aa394 sequence at the C end of the N protein, and adding a connecting bridge and a carrier protein binding site to prepare an NP-C polypeptide;
2, immunogen preparation: after activating keyhole limpet hemocyanin by maleimide, respectively reacting NP-N polypeptide and NP-C polypeptide with the activated keyhole limpet hemocyanin for 2h at room temperature to prepare KLH-NP-N immunogen and KLH-NP-C immunogen;
3 preparation of screening antigen: constructing an N protein prokaryotic expression vector, adding 6-His tag to the C end of the expression vector, and expressing and purifying to prepare NP-His screening antigen;
4, animal immunization: respectively immunizing Balb/C mice with KLH-NP-N immunogen and KLH-NP-C immunogen, wherein each Balb/C mouse is immunized with 100ug each time for 3 times, and the interval time is two weeks each time;
5, cell fusion: 1 week after the third immunization, respectively taking spleens of mice in groups of KLH-NP-N and KLH-NP-C, separating splenocytes into single cell suspensions, mixing the single cell suspensions with mouse myeloma cells SP2/0 in logarithmic phase according to the ratio of 1:1, performing cell fusion by using a cell fusion instrument, plating fused cells in groups of KLH-NP-N and KLH-NP-C into a 96-hole cell culture plate, adding HAT screening reagent, and performing primary complete liquid exchange after culturing for 5 days;
6, screening and detecting: coating NP-His screening antigen into an enzyme label plate, respectively carrying out indirect ELISA screening on cell supernatant after KLH-NP-N, KLH-NP-C group fusion by using the NP-His screening antigen, and reserving positive clones;
7 establishment of stable cell lines: carrying out subcloning on the positive clone until the positive detection rate is 100%, carrying out expanded culture and freezing and preserving seeds to obtain a hybridoma stable cell strain;
8 monoclonal antibody production: culturing the hybridoma stable cell strain by serum-free fermentation, collecting cell culture supernatant, and performing affinity purification on the monoclonal antibody by Prontein A to obtain a primary N protein monoclonal antibody;
9 antibody coupling: coupling the monoclonal antibody with horseradish peroxidase by using a simple sodium periodate method to obtain a labeled antibody;
10 paired antibody screening: and (3) coating the unmarked antibody into an ELISA plate as a capture antibody, adding the diluted NP-His screening antigen into the ELISA plate, culturing, adding the labeled antibodies in pairs for pairing detection, and screening positive holes to obtain the nucleocapsid protein paired monoclonal antibody pair.
Further, the NP-N polypeptide sequence is as follows: MSDNGPQNQRNAPRIGGGGSC, NP-C polypeptide is listed as: PQRQKKQQTVTLGGGGSC, NP-N polypeptide and activated keyhole limpet hemocyanin at a ratio of 1:1, and NP-C polypeptide and activated keyhole limpet hemocyanin at a ratio of 1: 1.
Further, the specific steps of animal immunization are as follows: mixing KLH-NP-N immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare first mixed solution, mixing KLH-NP-C immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare second mixed solution, performing subcutaneous injection on 6-week-old Balb/C mice respectively by using the first mixed solution and the second mixed solution, wherein the number of injection sites is 6, and the injection amount is 100 mu g per time to obtain primary-immunized mice, mixing KLH-NP-N immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare third mixed solution, mixing KLH-NP-C immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare fourth mixed solution, repeating immunization on the mice which are immunized for two weeks for 2 times respectively by using the third mixed solution and the fourth mixed solution, two weeks apart.
Further, the specific steps of cell fusion are as follows: respectively taking spleens of mice in groups of KLH-NP-N and KLH-NP-C1 week after three times of immunization, putting the spleens of the mice into a culture dish containing 5ml of MEM liquid, crushing the spleens, filtering the spleen by using a nylon net to obtain single cell suspension, mixing the single cell suspension and cell liquid of mouse myeloma cells SP2/0 in a logarithmic phase according to the volume ratio of 1:1, performing cell fusion by using a cell fusion instrument, paving fused cells into a 96-hole cell culture plate, adding HAT screening reagent, and performing total liquid change after culturing for 5 days.
Further, the detection and screening comprises the following specific steps: the method comprises the steps of coating NP-His screening antigen into an enzyme label plate, carrying out overnight culture at the temperature of 4 ℃, washing with PBST (basic-bound protein) with the pH value of 7.4 for three times, adding a sealing solution at the temperature of 37 ℃ for sealing for 2 hours, then removing the sealing solution and drying at the temperature of 37 ℃, respectively adding 100 mu L of supernatant of KLH-NP-N and KLH-NP-C group fusion cells into the enzyme label plate per hole, carrying out incubation at the temperature of 37 ℃ for 60 minutes, then adding 100 mu L of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 thousandth into the enzyme label plate per hole, carrying out incubation at the temperature of 37 ℃ for 60 minutes, adding 100 mu L of TMB single-component developing solution per hole into the enzyme label plate, carrying out room-temperature action for 20 minutes, then adding 50 mu L of stop solution per hole, carrying out indirect ELISA screening, and retaining positive clones.
Further, the specific steps for establishing the stable cell strain are as follows: and (3) respectively carrying out cloning culture on the hybridoma cells with positive detection by using KLH-NP-N and KLH-NP-C group positive clones by using a limiting dilution method, and screening positive subclones until the positive detection rate is 100% to obtain a hybridoma stable cell strain.
Further, the specific steps of monoclonal antibody production are as follows: respectively culturing hybridoma stable cell strains of KLH-NP-N and KLH-NP-C groups by adopting serum-free fermentation, collecting cell culture supernatant, mixing the supernatant, ProntenA and a phosphate buffer solution with the pH of 7.4, adding the mixture into a chromatographic column, eluting an antibody by using a phosphate buffer solution with the pH of 7.4 as a balance solution and using a citric acid solution with the pH of 4.0, adjusting the pH value to 7.0 by using a Tris-HCl buffer solution with the pH of 9.0, and collecting the antibody to obtain the primary N protein monoclonal antibody.
Further, the specific steps of antibody coupling are as follows:
step A1: dissolving 5mg of horseradish peroxidase in 1mL of deionized beams to prepare a horseradish peroxidase solution, dissolving 241mg of sodium periodate in 10mL of deionized water to prepare a sodium periodate solution, adding the horseradish peroxidase solution into the sodium periodate solution, stirring for 20min at the rotation speed of 200r/min and at the lightproof room temperature to obtain a mixed solution a, adding the mixed solution a into a dialysis bag, and dialyzing a 1mM sodium acetate buffer solution with the pH value of 4.4 overnight at the temperature of 4 ℃ to obtain a mixed solution b;
step A2: adding 20 mu L of 0.2M carbonate buffer solution with the pH value of 9.5 into the mixed solution b prepared in the step A1 to enable the pH value of the hydroformylation HRP to be 9.5, respectively adding 1mL of 6 monoclonal antibodies dissolved in the 0.01M carbonate buffer solution, and stirring for 2h at the room temperature with the rotation speed of 60r/min and in a dark place to prepare 6 groups of mixed solution c 1-mixed solution c 6;
step A3: dissolving 4mg of sodium borohydride in 1mL of deionized water to prepare a sodium borohydride solution, adding the sodium borohydride solution into the mixed solution c 1-mixed solution c6, stirring uniformly, keeping the temperature for 2 hours at 4 ℃, putting the mixture into a dialysis bag, and dialyzing overnight in 0.15M PBS buffer solution with the pH value of 7.4 at 4 ℃ to obtain mixed solution d 1-mixed solution d 6;
step A4: adding an isosomal saturated ammonium sulfate aqueous solution into the mixed solution d 1-mixed solution d6, standing for 1h at the temperature of 4 ℃, centrifuging at the rotation speed of 3000r/min, removing supernatant, washing a substrate twice with a half-saturated ammonium sulfate aqueous solution, dissolving the substrate in PBS with the pH value of 7.4 at 0.15M to prepare a mixed solution e 1-mixed solution e6, respectively filling the mixed solution e 1-mixed solution e6 into dialysis bags, dialyzing with PB buffer saline with the pH value of 7.4 at 0.15M, removing ammonium ions, centrifuging at the rotation speed of 10000r/min for 30min, removing precipitates, and freeze-drying and storing the supernatant.
Further, the specific steps of paired antibody screening are as follows: coating an unlabeled antibody into an ELISA plate as a capture antibody, adding a diluted NP-His screening antigen with the concentration of 10ng/ml into the ELISA plate, incubating for 30min at the temperature of 37 ℃, washing away the unbound NP-His screening antigen by using PBST washing buffer solution, adding an HRP-labeled antibody into the ELISA plate as a detection antibody, incubating for 30min at the temperature of 37 ℃, washing away the unbound labeled antibody by using the PBST washing buffer solution, adding TMB developing solution into the ELISA plate, incubating for 5min at the temperature of 37 ℃, adding 2M sulfuric acid termination solution into the ELISA plate, measuring OD450 reading by using an ELISA reader, and selecting a positive hole to obtain the nucleocapsid protein monoclonal antibody capable of forming sandwich pairing.
The invention has the beneficial effects that: synthesizing polypeptide of 15 amino acid sequences at the N end of the N protein and polypeptide of aa383-aa394 sequences at the C end of the N protein, respectively adding a connecting bridge and a carrier protein combination site to obtain NP-N polypeptide and NP-C polypeptide, respectively reacting the NP-N polypeptide and NP-C polypeptide with activated keyhole limpet hemocyanin to obtain KLH-NP-N immunogen and KLH-NP-C immunogen, simultaneously constructing an N protein prokaryotic expression vector, adding 6 His tag at the C end of the expression vector, expressing and purifying to obtain NP-His screening antigen, respectively immunizing Balb/C mice aged for 6 weeks by using the KLH-NP-N immunogen and the KLH-NP-C immunogen as antigens, after 3 times of immunization, picking the spleen of the mice after one week of impact immunization, preparing spleen single cell suspension, carrying out cell fusion with mouse myeloma cells SP2/0, further culturing the fused cells, carrying out indirect ELISA screening on fused cell supernatant, keeping positive clones, carrying out subcloning on the positive clones to ensure that the positive detection rate is 100% to obtain stable hybridoma cell strains, culturing the stable hybridoma cell strains by serum-free fermentation, collecting cell culture supernatant, carrying out Prontein A affinity purification to obtain primary N protein monoclonal antibodies, coupling the primary N protein monoclonal antibodies with horseradish peroxidase to obtain labeled antibodies, and further carrying out pairing screening on the antibodies to obtain nucleocapsid protein monoclonal antibody pairs. The polypeptide is respectively synthesized by different antigen epitopes at two ends of the N protein to be used as the antigen, the prepared monoclonal antibody can realize direct pairing, in the process of polypeptide synthesis, a connecting bridge is added to reduce steric hindrance, so that the antigen epitopes are fully exposed on the surface of the carrier protein, the antigen can be quickly identified, the recombinant protein is selected as the detection antigen, the situation that the polypeptide antigen is only identified by the antibody obtained by screening, the risk of the N protein is not identified is avoided, and whether the novel coronavirus is contained in a sample can be directly detected by detecting the content of the N protein.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
A preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody specifically comprises the following steps:
1 polypeptide synthesis: synthesizing a polypeptide with 15 amino acid sequences at the N end of the N protein, adding a connecting bridge and a carrier protein binding site to prepare an NP-N polypeptide, synthesizing a polypeptide with an aa383-aa394 sequence at the C end of the N protein, and adding a connecting bridge and a carrier protein binding site to prepare an NP-C polypeptide;
2, immunogen preparation: after activating keyhole limpet hemocyanin by maleimide, respectively reacting NP-N polypeptide and NP-C polypeptide with the activated keyhole limpet hemocyanin for 2h at room temperature to prepare KLH-NP-N immunogen and KLH-NP-C immunogen;
3 preparation of screening antigen: constructing an N protein prokaryotic expression vector, adding 6-His tag to the C end of the expression vector, and expressing and purifying to prepare NP-His screening antigen;
4, animal immunization: mixing KLH-NP-N immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare first mixed solution, mixing KLH-NP-C immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare second mixed solution, performing subcutaneous injection on 6-week-old Balb/C mice respectively by using the first mixed solution and the second mixed solution, wherein the number of injection sites is 6, and the injection amount is 100 mu g per time to obtain primary-immunized mice, mixing KLH-NP-N immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare third mixed solution, mixing KLH-NP-C immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare fourth mixed solution, repeating immunization on the mice which are immunized for two weeks for 2 times respectively by using the third mixed solution and the fourth mixed solution, every two weeks;
5, cell fusion: respectively taking spleens of mice in groups of KLH-NP-N and KLH-NP-C1 week after three times of immunization, putting the spleens of the mice into a culture dish containing 5ml of MEM liquid, crushing the spleens, filtering the spleen by using a nylon net to obtain single cell suspension, mixing the single cell suspension and cell liquid of mouse myeloma cells SP2/0 in a logarithmic phase according to the volume ratio of 1:1, performing cell fusion by using a cell fusion instrument, paving fused cells into a 96-hole cell culture plate, adding an HAT screening reagent, and performing primary liquid change after culturing for 5 days;
6, screening and detecting: coating NP-His screening antigen into an enzyme label plate, performing overnight culture at the temperature of 4 ℃, washing with PBST (basic-bound protein) with the pH value of 7.4 for three times, adding a sealing solution at the temperature of 37 ℃ for sealing for 2 hours, then removing the sealing solution, drying at the temperature of 37 ℃, respectively adding 100 mu L of supernatant of KLH-NP-N and KLH-NP-C group fusion cells into the enzyme label plate per hole, incubating at the temperature of 37 ℃ for 60 minutes, then adding 100 mu L of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 per thousand into the enzyme label plate per hole, incubating at the temperature of 37 ℃ for 60 minutes, then adding 100 mu L of TMB single-component developing solution per hole into the enzyme label plate, acting at the room temperature for 20 minutes, adding 50 mu L of stop solution per hole, performing indirect ELISA screening, and keeping positive clones;
7 establishment of stable cell lines: respectively carrying out cloning culture on hybrid tumor cells with positive detection by using KLH-NP-N and KLH-NP-C group positive clones by using a limiting dilution method, and screening positive subclones until the positive detection rate is 100% to obtain a hybrid tumor stable cell strain;
8 monoclonal antibody production: respectively culturing hybridoma stable cell strains of KLH-NP-N and KLH-NP-C groups by adopting serum-free fermentation, collecting cell culture supernatant, mixing the supernatant, Prontein A and a phosphate buffer solution with the pH of 7.4, adding the mixture into a chromatographic column, eluting an antibody by using a phosphate buffer solution with the pH of 7.4 as a balance solution and using a citric acid solution with the pH of 4.0, adjusting the pH value to 7.0 by using a Tris-HCl buffer solution with the pH of 9.0, and collecting the antibody to obtain a primary N protein monoclonal antibody;
9 antibody coupling: step A1: dissolving 5mg of horseradish peroxidase in 1mL of deionized beams to prepare a horseradish peroxidase solution, dissolving 241mg of sodium periodate in 10mL of deionized water to prepare a sodium periodate solution, adding the horseradish peroxidase solution into the sodium periodate solution, stirring for 20min at the rotation speed of 200r/min and at the lightproof room temperature to obtain a mixed solution a, adding the mixed solution a into a dialysis bag, and dialyzing a 1mM sodium acetate buffer solution with the pH value of 4.4 overnight at the temperature of 4 ℃ to obtain a mixed solution b;
step A2: adding 20 mu L of 0.2M carbonate buffer solution with the pH value of 9.5 into the mixed solution b prepared in the step A1 to enable the pH value of the hydroformylation HRP to be 9.5, respectively adding 1mL of 6 monoclonal antibodies dissolved in the 0.01M carbonate buffer solution, and stirring for 2h at the room temperature with the rotation speed of 60r/min and in a dark place to prepare 6 groups of mixed solution c 1-mixed solution c 6;
step A3: dissolving 4mg of sodium borohydride in 1mL of deionized water to prepare a sodium borohydride solution, adding the sodium borohydride solution into the mixed solution c 1-mixed solution c6, stirring uniformly, keeping the temperature for 2 hours at 4 ℃, putting the mixture into a dialysis bag, and dialyzing overnight in 0.15M PBS buffer solution with the pH value of 7.4 at 4 ℃ to obtain mixed solution d 1-mixed solution d 6;
step A4: adding an isosomal saturated ammonium sulfate aqueous solution into the mixed solution d 1-mixed solution d6, standing for 1h at the temperature of 4 ℃, centrifuging at the rotation speed of 3000r/min, removing supernatant, washing a substrate twice by using a half-saturated ammonium sulfate aqueous solution, dissolving the substrate in PBS (0.15M and pH value of 7.4) to prepare a mixed solution e 1-mixed solution e6, respectively filling the mixed solution e 1-mixed solution e6 into dialysis bags, dialyzing at 0.15M and pH value of 7.4 by using PB buffer saline, removing ammonium ions, centrifuging at the rotation speed of 10000r/min for 30min, removing precipitates, and freeze-drying and storing the supernatant;
10 paired antibody screening: the method comprises the steps of coating an unlabeled antibody into an ELISA plate as a capture antibody, adding a diluted NP-His screening antigen with the concentration of 10ng/ml into the ELISA plate, incubating for 30min at the temperature of 37 ℃, washing away the unbound NP-His screening antigen by using PBST washing buffer, adding an HRP-labeled antibody into the ELISA plate as a detection antibody, incubating for 30min at the temperature of 37 ℃, washing away the unbound labeled antibody by using the PBST washing buffer, adding TMB developing solution into the ELISA plate, incubating for 5min at the temperature of 37 ℃, adding 2M sulfuric acid termination solution into the ELISA plate, measuring OD450 readings by using an ELISA reader, selecting positive holes, and recording corresponding antibody pairs, wherein the results are shown in the following table 1.
TABLE 1
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.
Claims (9)
1. A preparation method of a novel coronavirus nucleocapsid protein monoclonal antibody is characterized by comprising the following steps: the method specifically comprises the following steps:
1 polypeptide synthesis: synthesizing a polypeptide with 15 amino acid sequences at the N end of the N protein, adding a connecting bridge and a carrier protein binding site to prepare an NP-N polypeptide, synthesizing a polypeptide with an aa383-aa394 sequence at the C end of the N protein, and adding a connecting bridge and a carrier protein binding site to prepare an NP-C polypeptide;
2, immunogen preparation: after activating keyhole limpet hemocyanin by maleimide, respectively reacting NP-N polypeptide and NP-C polypeptide with the activated keyhole limpet hemocyanin for 2h at room temperature to prepare KLH-NP-N immunogen and KLH-NP-C immunogen;
3 preparation of screening antigen: constructing an N protein prokaryotic expression vector, adding 6-His tag to the C end of the expression vector, and expressing and purifying to prepare NP-His screening antigen;
4, animal immunization: respectively immunizing Balb/C mice with KLH-NP-N immunogen and KLH-NP-C immunogen, wherein each Balb/C mouse is immunized with 100ug each time for 3 times, and the interval time is two weeks each time;
5, cell fusion: 1 week after the third immunization, respectively taking spleens of mice in groups of KLH-NP-N and KLH-NP-C, separating splenocytes into single cell suspensions, mixing the single cell suspensions with mouse myeloma cells SP2/0 in logarithmic phase according to the ratio of 1:1, performing cell fusion by using a cell fusion instrument, plating fused cells in groups of KLH-NP-N and KLH-NP-C into a 96-hole cell culture plate, adding HAT screening reagent, and performing primary complete liquid exchange after culturing for 5 days;
6, screening and detecting: coating NP-His screening antigen into an enzyme label plate, respectively carrying out indirect ELISA screening on cell supernatant after KLH-NP-N, KLH-NP-C group fusion by using the NP-His screening antigen, and reserving positive clones;
7 establishment of stable cell lines: carrying out subcloning on the positive clone until the positive detection rate is 100%, carrying out expanded culture and freezing and preserving seeds to obtain a hybridoma stable cell strain;
8 monoclonal antibody production: culturing the hybridoma stable cell strain by serum-free fermentation, collecting cell culture supernatant, and performing affinity purification on the monoclonal antibody by Prontein A to obtain a primary N protein monoclonal antibody;
9 antibody coupling: coupling the monoclonal antibody with horseradish peroxidase by using a simple sodium periodate method to obtain a labeled antibody;
10 paired antibody screening: and (3) coating the unmarked antibody into an ELISA plate as a capture antibody, adding the diluted NP-His screening antigen into the ELISA plate, culturing, adding the labeled antibodies in pairs for pairing detection, and screening positive holes to obtain the nucleocapsid protein paired monoclonal antibody pair.
2. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the NP-N polypeptide sequence is as follows: MSDNGPQNQRNAPRIGGGGSC, NP-C polypeptide is listed as: PQRQKKQQTVTLGGGGSC, NP-N polypeptide and activated keyhole limpet hemocyanin at a ratio of 1:1, and NP-C polypeptide and activated keyhole limpet hemocyanin at a ratio of 1: 1.
3. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the specific steps of animal immunization are as follows: mixing KLH-NP-N immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare first mixed solution, mixing KLH-NP-C immunogen and Freund's complete adjuvant according to the mass ratio of 1:1 to prepare second mixed solution, performing subcutaneous injection on 6-week-old Balb/C mice respectively by using the first mixed solution and the second mixed solution, wherein the number of injection sites is 6, and the injection amount is 100 mu g per time to obtain primary-immunized mice, mixing KLH-NP-N immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare third mixed solution, mixing KLH-NP-C immunogen and Freund's incomplete adjuvant according to the mass ratio of 1:1 to prepare fourth mixed solution, repeating immunization on the mice which are immunized for two weeks for 2 times respectively by using the third mixed solution and the fourth mixed solution, two weeks apart.
4. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the specific steps of cell fusion are as follows: respectively taking spleens of mice in groups of KLH-NP-N and KLH-NP-C1 week after three times of immunization, putting the spleens of the mice into a culture dish containing 5ml of MEM liquid, crushing the spleens, filtering the spleen by using a nylon net to obtain single cell suspension, mixing the single cell suspension and cell liquid of mouse myeloma cells SP2/0 in a logarithmic phase according to the volume ratio of 1:1, performing cell fusion by using a cell fusion instrument, paving fused cells into a 96-hole cell culture plate, adding HAT screening reagent, and performing total liquid change after culturing for 5 days.
5. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the detection and screening method comprises the following specific steps: the method comprises the steps of coating NP-His screening antigen into an enzyme label plate, carrying out overnight culture at the temperature of 4 ℃, washing with PBST (basic-bound protein) with the pH value of 7.4 for three times, adding a sealing solution at the temperature of 37 ℃ for sealing for 2 hours, then removing the sealing solution and drying at the temperature of 37 ℃, respectively adding 100 mu L of supernatant of KLH-NP-N and KLH-NP-C group fusion cells into the enzyme label plate per hole, carrying out incubation at the temperature of 37 ℃ for 60 minutes, then adding 100 mu L of goat anti-mouse IgG enzyme-labeled secondary antibody with the mass fraction of 0.5 thousandth into the enzyme label plate per hole, carrying out incubation at the temperature of 37 ℃ for 60 minutes, adding 100 mu L of TMB single-component developing solution per hole into the enzyme label plate, carrying out room-temperature action for 20 minutes, then adding 50 mu L of stop solution per hole, carrying out indirect ELISA screening, and retaining positive clones.
6. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the specific steps for establishing the stable cell strain are as follows: and (3) respectively carrying out cloning culture on the hybridoma cells with positive detection by using KLH-NP-N and KLH-NP-C group positive clones by using a limiting dilution method, and screening positive subclones until the positive detection rate is 100% to obtain a hybridoma stable cell strain.
7. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the monoclonal antibody production method specifically comprises the following steps: respectively culturing hybridoma stable cell strains of KLH-NP-N and KLH-NP-C groups by adopting serum-free fermentation, collecting cell culture supernatant, mixing the supernatant, Prontein A and a phosphate buffer solution with the pH of 7.4, adding the mixture into a chromatographic column, eluting an antibody by using a phosphate buffer solution with the pH of 7.4 as a balance solution and using a citric acid solution with the pH of 4.0, adjusting the pH value to 7.0 by using a Tris-HCl buffer solution with the pH of 9.0, and collecting the antibody to obtain the primary N protein monoclonal antibody.
8. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the specific steps of antibody coupling are as follows:
step A1: dissolving 5mg of horseradish peroxidase in 1mL of deionized beams to prepare a horseradish peroxidase solution, dissolving 241mg of sodium periodate in 10mL of deionized water to prepare a sodium periodate solution, adding the horseradish peroxidase solution into the sodium periodate solution, stirring for 20min at the rotation speed of 200r/min and at the lightproof room temperature to obtain a mixed solution a, adding the mixed solution a into a dialysis bag, and dialyzing a 1mM sodium acetate buffer solution with the pH value of 4.4 overnight at the temperature of 4 ℃ to obtain a mixed solution b;
step A2: adding 20 mu L of 0.2M carbonate buffer solution with the pH value of 9.5 into the mixed solution b prepared in the step A1 to enable the pH value of the hydroformylation HRP to be 9.5, respectively adding 1mL of 6 monoclonal antibodies dissolved in the 0.01M carbonate buffer solution, and stirring for 2h at the room temperature with the rotation speed of 60r/min and in a dark place to prepare 6 groups of mixed solution c 1-mixed solution c 6;
step A3: dissolving 4mg of sodium borohydride in 1mL of deionized water to prepare a sodium borohydride solution, adding the sodium borohydride solution into the mixed solution c 1-mixed solution c6, stirring uniformly, keeping the temperature for 2 hours at 4 ℃, putting the mixture into a dialysis bag, and dialyzing overnight in 0.15M PBS buffer solution with the pH value of 7.4 at 4 ℃ to obtain mixed solution d 1-mixed solution d 6;
step A4: adding an isosomal saturated ammonium sulfate aqueous solution into the mixed solution d 1-mixed solution d6, standing for 1h at the temperature of 4 ℃, centrifuging at the rotation speed of 3000r/min, removing supernatant, washing a substrate twice with a half-saturated ammonium sulfate aqueous solution, dissolving the substrate in PBS with the pH value of 7.4 at 0.15M to prepare a mixed solution e 1-mixed solution e6, respectively filling the mixed solution e 1-mixed solution e6 into dialysis bags, dialyzing with PB buffer saline with the pH value of 7.4 at 0.15M, removing ammonium ions, centrifuging at the rotation speed of 10000r/min for 30min, removing precipitates, and freeze-drying and storing the supernatant.
9. The method for preparing the novel monoclonal antibody against coronavirus nucleocapsid protein according to claim 1, wherein: the specific steps of paired antibody screening are as follows: coating an unlabeled antibody into an ELISA plate as a capture antibody, adding a diluted NP-His screening antigen with the concentration of 10ng/ml into the ELISA plate, incubating for 30min at the temperature of 37 ℃, washing away the unbound NP-His screening antigen by using PBST washing buffer solution, adding an HRP-labeled antibody into the ELISA plate as a detection antibody, incubating for 30min at the temperature of 37 ℃, washing away the unbound labeled antibody by using the PBST washing buffer solution, adding TMB developing solution into the ELISA plate, incubating for 5min at the temperature of 37 ℃, adding 2M sulfuric acid termination solution into the ELISA plate, measuring OD450 reading by using an ELISA reader, and selecting a positive hole to obtain the nucleocapsid protein monoclonal antibody capable of forming sandwich pairing.
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