CN111474345A

CN111474345A - SARS-CoV-2 antibody detection method

Info

Publication number: CN111474345A
Application number: CN202010218584.5A
Authority: CN
Inventors: 赵风强; 吴妤; 邬晓乐
Original assignee: Beijing Biosynthesis Biotechnology Co ltd
Current assignee: Beijing Biosynthesis Biotechnology Co ltd
Priority date: 2020-03-25
Filing date: 2020-03-25
Publication date: 2020-07-31

Abstract

A process for detecting the antibody of SARS-CoV-2 includes such steps as labelling recombinant SARS-CoV-2 nucleomapped protein with horse radish peroxidase, coating enzyme linked plate with Anti-SARS-CoV-2 antibody or dominant antibody, closing, adding the sample to be detected and enzyme-labeled antigen to the test hole of enzyme linked plate, incubating at room temp, washing, adding TMB substrate, coloration, stopping reaction, measuring the light absorption at 450nm, and calculating the antibody inhibiting rate.

Description

SARS-CoV-2 antibody detection method

Technical Field

The invention relates to a SARS-CoV-2 antibody detection method, belonging to the antibody detection technical field.

Background

SARS-CoV-2 belongs to β genus of coronavirus family, scientific research team finds that SARS-CoV-2 complete genome has about 76% sequence homology with SARS-CoV (severe acute respiratory syndrome), and has up to 96% sequence homology with Bat coronavirus BatCoVRaTG13 in Hepialus Jujuba, and the team confirms that SARS-CoV-2 and SARS-CoV enter into cell receptor as ACE2 by comparing 7 conserved non-structural proteins.

The structure of coronaviruses consists of a double-layered lipid Envelope containing Spike protein (S), Envelope protein (E), Membrane protein (M), and Nucleocapsid protein (N). Among them, the S protein and the N protein are the most important target proteins. The S protein is located on the outermost layer of the virus and is associated with the infectious capacity of the virus. The S protein has the functions of binding virus and host cell membrane receptor and fusing membrane, and is the key target of host neutralizing antibody and vaccine design. The N protein is the most abundant protein in the coronavirus and has high conservation. The N protein is a highly immunogenic phosphoprotein that binds to viral RNA during virion assembly to form a helical nucleocapsid; and is related to virus genome replication and regulation of cell signal pathways, so that the N protein is often used as a coronavirus diagnosis and detection tool and is a core raw material of an immunological rapid diagnosis reagent.

Clinically, patients with Novel Coronavirus Pneumonia (NCP) have a latency period of 1 to 14 days, mostly 3 to 7 days, and are mainly manifested by fever, fatigue and dry cough, and a few patients have symptoms such as nasal obstruction, watery nasal discharge, pharyngalgia and diarrhea. Severe patients often have dyspnea and/or hypoxemia after 7 days of morbidity, severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis and hemorrhagic and blood coagulation dysfunction which are difficult to correct, and the like, and it is worth noting that severe and critical patients can show moderate-low fever in the course of disease, even have no obvious fever.

The current detection technologies for the novel coronavirus mainly comprise: nucleic acid detection, immunological detection (antigen or antibody detection), virus isolation and identification, and the like. The detection tools are comprehensively used, the rapid and efficient detection is realized, and the diagnosis period of the novel coronavirus pneumonia patient can be effectively shortened by combining the clinical imaging examination result. In a plurality of detection technologies, the detection of the specific antibody can be carried out by detecting the contents of IgM and IgG of SARS-CoV-2N protein antibodies in samples such as blood, serum and the like, and the method has the advantages of relatively convenient sampling, short detection time, low requirement on detection environment and the like. However, colloidal gold chromatography is mostly adopted for detecting IgM and IgG of anti-SARS-CoV-2N protein antibody at present, but SARS-CoV-2 novel coronavirus has high homology with congeneric coronavirus, and cross and non-specific reaction easily exists. Therefore, the research and development and optimization of the novel coronavirus antibody detection method have important application prospects and scientific research values.

Disclosure of Invention

Aiming at the prior art, the invention provides a SARS-CoV-2 antibody detection method.

The invention is realized by the following technical scheme:

a method for detecting SARS-CoV-2 antibody, which is a non-diagnosis or treatment purpose detection method, comprises the following steps:

the recombinant SARS-CoV-2Nucleocapsid protein (antigen) is labeled by Horse Radish Peroxidase (HRP), Anti-SARS-CoV-2 antibody or dominant antibody is selected to coat the enzyme linked plate and seal, according to the competitive E L ISA principle, the sample to be tested and enzyme labeled antigen are added into the test hole of the enzyme linked plate, incubated at room temperature, after the plate is thoroughly washed, substrate TMB is added, color development is carried out in dark, reaction is finally stopped and the light absorption value at 450nm is measured, and the titer of the antibody in the sample is evaluated by calculating the antibody inhibition rate.

The amino acid sequence of the recombinant SARS-CoV-2Nucleocapsid protein is shown in SEQ ID NO. 1.

The recombinant SARS-CoV-2Nucleocapsid protein is prepared by the following method: human SARS-CoV-2Nucleocapsid gene (nucleotide sequence is shown in SEQ ID NO.2) is inserted into expression vector pET28a, pET28a-SARS-CoV-2-N fusion expression plasmid is constructed, colibacillus is transformed, then IPTG is used for induction, purification and renaturation, and the recombinant SARS-CoV-2Nucleocapsid protein is obtained. The preparation method is also described in another patent application of the applicant, the application date of which is 26.02/2020, and the application number is 2020101203494.

Further, horseradish peroxidase (HRP) labeling was performed using sodium periodate oxidation.

Furthermore, the Anti-SARS-CoV-2 antibody is prepared by immunizing a BA L B/c mouse with a recombinant SARS-CoV-2Nucleocapsid protein as an immunogen, then carrying out fusion and subcloning of mouse spleen cells and mouse myeloma cells, carrying out specific screening and domestication of SARS-CoV-2Nucleocapsid and MERS Nucleocapsid to finally obtain a monoclonal cell strain, and then carrying out ascites preparation and purification to obtain the Anti-SARS-CoV-2 antibody.

Furthermore, the Anti-SARS-CoV-2 dominant antibody and enzyme-labeled antigen can be subjected to high-throughput screening by an E L ISA chessboard competition method, so that the dominant antibody clone and the working concentration of related raw materials are obtained.

Further, the competitive antibody titer was positively correlated with the inhibition rate according to the principle of competitive E L ISA, and the antibody inhibition rate was calculated by the formula of (%) inhibition rate (%) (negative control OD value-sample OD value) × 100%/negative control OD value.

Further, the test sample, selected from serum or cell culture supernatant, can be evaluated for matrix interference effects prior to testing.

The invention relates to a SARS-CoV-2 antibody detection method, which is a detection method for non-diagnosis or treatment, such as laboratory research, research on protective effect evaluation of new coronavirus vaccine, epidemiological investigation of new coronavirus, etc. compared with the prior art, the invention has the advantages that the invention realizes the detection of SARS-CoV-2 specific antibody in serum or cell supernatant samples in a shorter time by carrying out horseradish peroxidase (HRP) marking on recombinant SARS-CoV-2 nucleomapped protein (antigen), and simultaneously adopts Anti-SARS-CoV-2N protein dominant antibody to coat an enzyme linked plate, and evaluates the titer of the antibody in the samples by calculating inhibition rate according to the competitive E L ISA principle and steps, thereby avoiding cross reaction with the sample matrix to a great extent, compared with the traditional antibody detection method, realizing faster, more convenient, more sensitive completion, more conservative N protein and specific antibody in the SARS-CoV-2 as raw materials, and having important specificity, precision and sensitivity, and specificity and application prospect of SARS-CoV-2.

The invention carries out Horse Radish Peroxidase (HRP) marking on recombinant SARS-CoV-2Nucleocapsid protein (antigen), simultaneously adopts specific Anti-SARS-CoV-2Nucleocapsid protein monoclonal antibody coating enzyme linked plate combined with antigen dominant epitope, realizes the detection of SARS-CoV-2 specific antibody in serum, blood and other samples in a short time according to the competitive E L ISA principle and steps, and evaluates the titer of antibody in the samples by calculating inhibition rate.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.

The amino acid sequence of SARS-CoV-2Nucleocapsid recombinant protein (SEQ ID NO. 1):

MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINT NSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAA IVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSG KGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFG MSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLP AADLDDFSKQLQQSMSSADSTQA。

nucleotide sequence of human SARS-CoV-2Nucleocapsid gene (SEQ ID NO. 2):

atgtctgataatggaccccaaaatcagcgaaatgcaccccgcattacgtttggtggaccctcagattcaactggcagt aaccagaatggagaacgcagtggggcgcgatcaaaacaacgtcggccccaaggtttacccaataatactgcgtcttggtt caccgctctcactcaacatggcaaggaagaccttaaattccctcgaggacaaggcgttccaattaacaccaatagcagtc cagatgaccaaattggctactaccgaagagctaccagacgaattcgtggtggtgacggtaaaatgaaagatctcagtcca agatggtatttctactacctaggaactgggccagaagctggacttccctatggtgctaacaaagacggcatcatatgggt tgcaactgagggagccttgaatacaccaaaagatcacattggcacccgcaatcctgctaacaatgctgcaatcgtgctac aacttcctcaaggaacaacattgccaaaaggcttctacgcagaagggagcagaggcggcagtcaagcctcttctcgttcc tcatcacgtagtcgcaacagttcaagaaattcaactccaggcagcagtaggggaacttctcctgctagaatggctggcaa tggcggtgatgctgctcttgctttgctgctgcttgacagattgaaccagcttgagagcaaaatgtctggtaaaggccaac aacaacaaggccaaactgtcactaagaaatctgctgctgaggcttctaagaagcctcggcaaaaacgtactgccactaaa gcatacaatgtaacacaagctttcggcagacgtggtccagaacaaacccaaggaaattttggggaccaggaactaatcag acaaggaactgattacaaacattggccgcaaattgcacaatttgcccccagcgcttcagcgttcttcggaatgtcgcgca ttggcatggaagtcacaccttcgggaacgtggttgacctacacaggtgccatcaaattggatgacaaagatccaaatttc aaagatcaagtcattttgctgaataagcatattgacgcatacaaaacattcccaccaacagagcctaaaaaggacaaaaagaagaaggctgatgaaactcaagccttaccgcagagacagaagaaacagcaaactgtgactcttcttcctgctgcagatt tggatgatttctccaaacaattgcaacaatccatgagcagtgctgactcaactcaggcctaa。

drawings

FIG. 1: and (4) a potency identification result is shown schematically.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1: antigen Horse Radish Peroxidase (HRP) labeling

Antigen horseradish peroxidase (HRP) labeling:

(1) 6.4mg of HRP dry powder is taken, 0.6ml of double distilled water is added, 60 mu l of sodium periodate (0.1M) is slowly and dropwise added into the HRP, the mixture is incubated for 20min at room temperature, and a sodium acetate solution (1mM, pH4.4) is dialyzed overnight;

(2) adding 1.6mg of antigen protein into the dialyzed HRP solution, and incubating for 2h at room temperature;

the amino acid sequence of the antigen protein is shown as SEQ ID NO.1, and the antigen protein is prepared by the following method: inserting human SARS-CoV nucleoapsid gene (nucleotide sequence is shown in SEQ ID NO.2) into expression vector pET28a, constructing pET28a-SARS-CoV-N fusion expression plasmid, transforming colibacillus, then inducing with IPTG, purifying and renaturing to obtain recombinant SARS-CoV-2 nucleoapsid protein;

(3) adding 60 mu l of sodium borohydride (4mg/ml) into the HRP and antigen complex, and incubating for 2h at room temperature;

(4) centrifuging, precipitating with saturated ammonium sulfate solution for 2h, centrifuging again, removing supernatant, and dissolving precipitate again;

(5) and (3) detecting the molar ratio of the fluorescent protein by using an enzyme-labeling instrument, and referring to the detection result of the titer of the enzyme-labeled antigen by the E L ISA method.

(II) E L ISA method enzyme-labeled antigen titer detection:

(1) coating: all Anti-SARS-CoV-2 specific antibodies were prepared at 2ug/ml in carbonate buffer (50mM pH9.6), 100. mu.l/well, coated on an ELISA plate, and left overnight at 4 ℃;

the Anti-SARS-CoV-2 specific antibody is prepared by taking recombinant SARS-CoV-2Nucleocapsid protein as immunogen, immunizing a BA L B/c mouse, then carrying out fusion and subcloning of mouse spleen cells and mouse myeloma cells, and carrying out specific screening and domestication of SARS-CoV-2Nucleocapsid and MERS Nucleocapsid to finally obtain a monoclonal cell strain, and then carrying out ascites preparation and purification to obtain the Anti-SARS-CoV-2 antibody;

(2) and (3) sealing: preparing phosphate buffer solution (10mM pH7.4) containing 5% bovine serum albumin, sealing at 300 μ l/well, and incubating at 37 deg.C for 120 min;

(3) and (3) detection: diluting enzyme-labeled antigen in proportion, wherein the diluent is phosphate buffer (10mM pH7.4) of 1% bovine serum albumin, adding sample at 100 μ l/hole, and incubating at 37 deg.C for 60 min;

(4) color development: after the plate is thoroughly washed, adding a substrate TMB, and developing for 5-10 min at 37 ℃ in a dark place;

(5) and (4) terminating: the absorbance at 450nM was measured by a microplate reader using 50. mu.l of stop solution (2M sulfuric acid solution) and the results are shown in Table 1.

TABLE 1 enzyme-labeled antigen titer E L ISA detection result

Example 2: screening for Competition experiments

Competitive assay screening was performed by checkerboard titration, first, all Anti-SARS-CoV-2 nucleomapped protein specific antibodies were prepared to concentrations of 2. mu.g/ml and 1. mu.g/ml and prepared into coated plates, which were then coated and sealed in the process described in example 1. An experiment is designed according to a chessboard titration method, Anti-SARS-CoV-2 nucleocapsitdprotein antibody is prepared into 20ug/ml and 0ug/ml solution to be used as competitive antibodies, enzyme-labeled antigen is prepared into 2ug/ml and 1ug/ml concentration, 50ul of each of the two is used for competitive detection, the incubation is carried out for 15min at room temperature, and the rest detection, color development and termination processes are shown in reference example 1. According to the experimental result, selecting the antibody clone with the maximum inhibition rate and the working concentration of the corresponding raw material, wherein the antibody clone is 1C7, the working concentration is 2ug/ml, and the working concentration of the enzyme-labeled antigen is 1 ug/ml. Some of the best results are shown in table 2.

Note that the inhibition ratio is expressed as the inhibition ratio (%) - (blank OD value-sample OD value) × 100%/blank OD value, wherein the sample OD value is the OD value of 20ug/ml of the competitive antibody and the enzyme-labeled antigen in this case, and the negative control OD value is the OD value of 0ug/ml of the competitive antibody and the enzyme-labeled antigen in the experiment well.

TABLE 2 chessboard titration method for screening competitive antibody test results

Example 3: matrix effect detection of samples

According to the experimental result obtained in example 2, dominant antibody clones and the working concentrations of the relevant raw materials are selected to further detect the matrix effect of the sample, and the specific steps are as follows:

(1) serum matrix effect assay

a. Sample source: the rabbit is immunized by adopting the recombinant SARS-CoV-2Nucleocapsid protein, and serum after immunization is taken as an experimental positive sample.

b. Coating: the 1C7 antibody was coated at a concentration of 2ug/ml, and the rest of the procedure was the same as in example 1.

And (3) sealing: the method was the same as in embodiment 1.

Sample dilution: normal human serum was diluted with 1% bovine serum albumin in phosphate buffer, and the dilution gradient was as shown in table 3, while 1% bovine serum albumin in phosphate buffer was used as the negative control. Subsequently, 87.5. mu.l of each of the serum dilution and the negative control was taken, and 12.5. mu.l of the positive sample was added thereto to prepare a corresponding positive control.

And (3) detection: the diluted serum, negative control and corresponding positive control were added to the experimental wells in an amount of 50. mu.l each, and 50ul enzyme-labeled antigen (working concentration 1ug/ml) was added to each well, and the rest of the procedure was the same as in example 1.

The coloring and terminating methods were the same as in example 1.

And (4) statistics of experimental results: according to the change of the experimental OD value and the inhibition rates corresponding to different serum dilutions, the serum is diluted by at least 1:10, so that the requirement of avoiding the serum matrix effect can be met.

TABLE 3 serum matrix Effect assay

(2) Cell supernatant matrix effect assay

The sample source, coating, sealing, detection, visualization and termination methods are the same as described above.

Sample dilution: the cell culture supernatant was diluted with 1% bovine serum albumin phosphate buffer, and the dilution gradient was as shown in table 4, while 1% bovine serum albumin phosphate buffer was used as the negative control. Subsequently, 87.5. mu.l of each of the supernatant of each dilution and the negative control was taken, and 12.5. mu.l of the positive sample was added thereto to prepare a corresponding positive control.

And (4) statistics of experimental results: according to the change of the experimental OD value and the inhibition rate corresponding to different dilutions of the supernatant, the cell supernatant stock solution (undiluted) can meet the requirement of avoiding the matrix effect of the cell supernatant.

TABLE 4 supernatant substrate Effect assay

Example 4: detection of anti-SARS-CoV-2Nucleocapsid protein antibody in serum sample

According to the experimental results obtained in the embodiment 2 and the embodiment 3, the dominant antibody clone and the related raw material working concentration are selected, an Anti-SARS-CoV-2Nucleocapsid protein antibody detection method is established, and the Anti-SARS-CoV-2Nucleocapsid protein antibody in serum is detected, the specific steps are as follows:

sample source: 10 rabbits were immunized with recombinant SARS-CoV-2Nucleocapsid protein, and the pre-and post-immune sera were taken as negative and positive sera.

The coating and sealing method was the same as in embodiment 3.

Sample dilution: positive and negative serum samples were taken and diluted 1:10 with 1% bovine serum albumin phosphate buffer (10mM pH7.4) and a blank 1% bovine serum albumin phosphate buffer was used as a negative control.

And (3) detection: the diluted positive and negative serum samples and the experimental negative control were added to the experimental wells at 50. mu.l each, and 50ul enzyme-labeled antigen (working concentration 1ug/ml) was added to each experimental well, and the rest of the procedure was the same as in example 3.

The coloring and terminating methods were the same as those in embodiment 3.

The results of the experiment were counted, and the inhibition ratio of the antibody in each serum sample was calculated as (inhibition ratio (%)) - (blank control OD value-sample OD value) × 100%/blank control OD value.

And (4) judging a result: by calculating the inhibition rate of the antibody in each serum sample in the laboratory, the positive and negative determination criteria (CUTOFF +2SD, NC is negative serum) of the antibody in the serum sample in the laboratory can be established, and in this example, the inhibition rate determination value of the antibody is: 26.39%, i.e., a sample having an inhibition rate above this value is defined as a positive serum sample, and below this value is defined as a negative serum sample. Meanwhile, the titer of Anti-SARS-CoV-2Nucleocapsid protein antibody in positive serum sample can be evaluated by comparing the serum antibody inhibition rate.

The specific results are shown in Table 5.

TABLE 5 serum samples anti-SARS-CoV-2Nucleocapsid protein antibody detection results

Example 5: optimization of antibody detection method against SARS-CoV-2Nucleocapsid protein in serum sample

The Anti-SARS-CoV-2 nucleomapped protein antibody detection method established in the embodiment 4, and the reaction time of the method is optimized, the specific steps are as follows:

sample source: the 10 rabbits were immunized with recombinant SARS-CoV-2Nucleocapsid protein, and the serum before and after immunization was taken as negative serum and positive serum.

The coating and sealing methods were the same as in example 4.

And (3) detection: the 8# positive and negative serum samples and blank controls of example 4 were selected and 50. mu.l each was added to the assay wells, 50ul of enzyme-labeled antigen (working concentration 1ug/ml) was added to each assay well, and incubation was carried out at 37 ℃ for 30min, 25min, 20min, 15min, and the rest of the procedure was the same as in example 4.

The coloring and terminating methods were the same as in example 4.

The results of the experiment were counted, and the inhibition ratio of the antibody in each serum sample was calculated as (inhibition ratio (%)) - (blank OD value-sample OD value) × 100%/blank OD value.

And (4) judging a result:

by adopting the anti-SARS-CoV-2Nucleocapsid protein antibody detection method established in example 4, the result difference between 30min and 15min of incubation at 37 ℃ is not obvious, but the time can be reduced by half, thereby meeting the requirement of rapid diagnosis and realizing high flux.

The specific results are shown in Table 6.

TABLE 6

Example 6: method for detecting antibody against SARS-CoV-2Nucleocapsid protein and its sensitivity analysis

Using the reaction time of 15min determined in example 5 and using the coated antibody 1C7 as a blocking standard, the same volume of enzyme-labeled antigen was added to a pre-prepared plate at the same time at concentrations of 10ug/ml, 2.5ug/ml, 0.625ug/ml, 0.156ug/ml, 0.039ug/ml, 0.010ug/ml and 0.002ug/ml, and incubated at 37 ℃ for 15min, in the same manner as in example 5.

The coloring and terminating methods were the same as in example 5.

And (4) judging a result: as shown in FIG. 1, the reference example shows that the inhibition ratio Cutoff is 26.39%, the sensitivity of the invention can reach 0.625ug/ml, and the antibody against SARS-CoV-2Nucleocapsid protein in serum sample can be quantitatively detected.

The Nucleocapsid protein is most abundant in the new type coronavirus SARS-CoV-2, and after human infection, it can quickly produce antibody against SARS-CoV-2Nucleocapsid protein. The invention selects specific anti-SARS-CoV-2Nucleocapsid protein dominant antibody (the example takes 1C7 antibody as an example for explanation, but not limited to the antibody, other antibodies obtained by the method of the invention can also implement the invention), through competitive inhibition method, anti-SARS-CoV-2Nucleocapsid protein dominant epitope antibody in serum can be detected, thereby judging whether to infect novel coronavirus SARS-CoV-2 and organism infection degree (according to the height of inhibition titer), and simultaneously can detect the anti-SARS-CoV-2Nucleocapsid protein dominant antibody level in serum sample in recovery period, which is helpful to extract antibody which can be used for treating novel coronavirus SARS-CoV-2 from serum in recovery period in large quantity. The invention is original and has important significance for the clinical detection and tracking of novel coronavirus SARS-CoV-2 antibody.

The above examples are provided to enable those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art will be within the scope of the appended claims.

Sequence listing

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atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60

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Claims

1. A SARS-CoV-2 antibody detecting method is characterized by that it makes horseradish peroxidase labeling on recombined SARS-CoV-2 nucleocapsitdproteins, at the same time selects Anti-SARS-CoV-2 antibody or dominant antibody to coat enzyme linked plate and seal, according to the competitive E L ISA principle, adds the sample to be detected and enzyme labeled antigen into the test hole of enzyme linked plate, incubate at room temp., after thoroughly washing plate, add substrate TMB, avoid light to develop color, finally terminate reaction and measure light absorption value at 450nm, and utilizes calculation of antibody inhibition rate to evaluate antibody titer in sample.

2. The method for detecting SARS-CoV-2 antibody according to claim 1, wherein: the amino acid sequence of the recombinant SARS-CoV-2Nucleocapsid protein is shown in SEQ ID NO. 1.

3. The method for detecting SARS-CoV-2 antibody according to claim 1 or 2, wherein: the recombinant SARS-CoV-2Nucleocapsid protein is prepared by the following method: human SARS-CoV nucleoapsid gene whose nucleotide sequence is shown in SEQ ID No.2 is inserted into expression vector pET28a to construct pET28a-SARS-CoV-N fusion expression plasmid, and then the colibacillus is transformed, and then the recombinant SARS-CoV-2 nucleoapsid protein is obtained by using IPTG induction, purification and renaturation.

4. The method for detecting SARS-CoV-2 antibody according to claim 1, wherein: and (3) adopting a sodium periodate oxidation method to carry out horseradish peroxidase labeling.

5. The SARS-CoV-2 antibody detection method as claimed in claim 1, 2 or 3, wherein the Anti-SARS-CoV-2 antibody is prepared by immunizing a BA L B/c mouse with a recombinant SARS-CoV-2 nucleoapsed protein as an immunogen, performing fusion and subcloning of mouse spleen cells and mouse myeloma cells, specifically screening and domesticating SARS-CoV-2 nucleoapsed and MERS nucleoapsed to obtain a monoclonal cell strain, and preparing and purifying ascites to obtain the Anti-SARS-CoV-2 antibody.

6. The method for detecting SARS-CoV-2 antibody as claimed in claim 1, wherein the Anti-SARS-CoV-2 dominant antibody and enzyme-labeled antigen are subjected to high throughput screening by E L ISA chessboard competition method to obtain the working concentration of dominant antibody clone and related raw materials.

7. A method for detecting SARS-CoV-2 antibody according to claim 1 or 6, wherein the antibody inhibition ratio is calculated as inhibition ratio (%) - (negative control OD value-sample OD value) × 100%/negative control OD value.

8. The method for detecting SARS-CoV-2 antibody according to claim 1, wherein: the sample to be detected is selected from serum or cell culture supernatant.