Anti-neocrown N protein human monoclonal antibody, enzyme-linked immunosorbent assay kit and application of enzyme-linked immunosorbent assay kit

Abstract

The invention provides an anti-neocrown N protein human monoclonal antibody, an enzyme-linked immunosorbent assay kit and application of the anti-neocrown N protein human monoclonal antibody and the enzyme-linked immunosorbent assay kit. The anti-neocoronal N protein human monoclonal antibody is obtained from N protein of human immune neocoronal virus, a heavy chain and a light chain of the anti-neocoronal N protein human monoclonal antibody have variable regions and complementary determining regions, and the anti-neocoronal N protein human monoclonal antibody is specifically combined with neocoronal N protein; and through paired use of the antibodies, the kit has strong anti-interference capability, good specificity and high sensitivity on clinical new crown detection.

Description

Technical Field

[0001] The present application relates to the field of immunoassay technology, and in particular to a human monoclonal antibody against the novel coronavirus N protein, an enzyme-linked immunosorbent assay kit, and its application. Background Art

[0002] The nucleoprotein of the new coronavirus (SARS-CoV-2) is a key structural protein for viral replication and assembly. It is highly conserved and is an important supplementary target for nucleic acid detection. At present, the monoclonal antibodies used in the new crown N protein detection reagents are mostly derived from heterologous animals such as mice or rabbits. Although these antibodies have high affinity, there are two major problems in their clinical application: first, endogenous antibody interference. Heterophilic antibodies (such as HAMA) in human samples can non-specifically bind to animal-derived antibodies and interfere with the detection signal. Secondly, it is easy to produce cross-reactions. Animal-derived antibodies may cross-react with non-target proteins (such as other coronavirus N proteins or host proteins), reducing the specificity of detection.

[0003] Human antibodies are homologous to endogenous immunoglobulins in the human body. Although studies have explored the application of human antibodies in the treatment of COVID-19 (such as monoclonal antibodies targeting the spike protein), their potential in the development of diagnostic reagents for the N protein has not been fully explored. Antibodies derived from human sources are more likely to recognize conserved epitopes naturally targeted by the human immune system, improving their specificity for the COVID-19 N protein. Therefore, it is imperative to develop human monoclonal antibodies that can effectively reduce endogenous interference. Summary of the Invention

[0004] The present application provides an anti-COVID-19 N protein human monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA) kit, and its application to solve the problems existing in the related technologies. The technical solutions are as follows:

[0005] In the first aspect, the embodiments of the present application provide a human monoclonal antibody against the novel coronavirus N protein, named 1B9, wherein 1B9 comprises a light chain variable region and a heavy chain variable region;

[0006] The heavy chain variable region includes complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3;

[0007] The amino acid sequence of VH-CDR1 is SEQ ID NO.1: GGSFSSYA; the encoding nucleotide sequence is SEQ ID NO.21: GGAGGCAGTTTCAGCAGTTATGCT.

[0008] The amino acid sequence of VH-CDR2 is SEQ ID NO. 2: IIPLLGKI; the encoding nucleotide sequence is SEQ ID NO. 22: ATCATCCCTCTTCTTGGCAAAATA.

[0009] The amino acid sequence of VH-CDR3 is SEQ ID NO. 3: ARGGWSTEVAGSAMDV; the encoding nucleotide sequence is SEQ ID NO. 23: GCGAGAGGTGGCTGGTCCACAGAAGTGGCTGGCTCAGCTATGGACGTC.

[0010] The light chain variable region includes complementarity determining regions VK-CDR1, VK-CDR2 and VK-CDR3;

[0011] The amino acid sequence of VK-CDR1 is SEQ ID NO.4: ENLLHTNGKNY; the encoding nucleotide sequence is SEQ ID NO.24: GAGAACCTCCTGCATACTAATGGAAAAAATTAT.

[0012] The amino acid sequence of VK-CDR2 is SEQ ID NO.5: VGS; the encoding nucleotide sequence is SEQ ID NO.25: GTGGGTTCT.

[0013] The amino acid sequence of VK-CDR3 is SEQ ID NO.6: MQALQMPYT; the encoding nucleotide sequence is SEQ ID NO.26: ATGCAGGCTCTACAAATGCCGTACACT.

[0014] In one embodiment, the amino acid sequence of the heavy chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1B9 is as shown in SEQ ID NO.7;

[0015] SEQ ID NO.7:

[0016] QVQLVQSGSEVKKPGSSVKVSCQASGGSFSSYAVNWVRQAPGQGLEW MGRIIPLLGKINYGQNFQGRVTITADESSGTAYMEMTRLRPADTAVYFCARGGWSTEVAGSAMDVWGPGTTITVS.

[0017] The encoding nucleotide sequence is shown in SEQ ID NO.27;

[0018] SEQ ID NO.27:

[0019] CAGGTGCAGCTGGTGCAGTCTGGGAGTGAGGTGAAAAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCCAGGCCTCGGGAGGCAGTTTCAGCAGTTATGCTGTCAACTGGGTGCGACAGGCTCCTGGACAAGGGCTTGAGTGGATGGGACGCATCATCCCTCTTCTTGGCAAAATAAACTACGGACA AAACTTCCAGGGCAGAGTCACCATCACCGCGGACGAATCCTCGGGCACAGCCTACATGGAGATGACTAGACTGAGACCTGCGGACACGGCCGTGTATTTTTGTGCGAGAGGTGGCTGGTCCACAGAAGTGGCTGGCTCAGCTATGGACGTCTGGGGCCCAGGGACCACGATCACTGTCTCCCCAG.

[0020] The amino acid sequence of the light chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1B9 is shown in SEQ ID NO.8;

[0021] SEQ ID NO.8:

[0022] DIVVTQSPLSLSVTPGEPAAISCRSSENLLHTNGKNYLDWYLQKPGQSP QLLIYVGSTRASGVSGRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQMP YTFGQGTKLEIK.

[0023] The encoding nucleotide sequence is shown in SEQ ID NO.28;

[0024] SEQ ID NO.28:

[0025] GATATTGTGGTGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCCGGCCGCCATCTCCTGCAGGTCTAGTGAGAACCTCCTGCATACTAATGGAAAAAAATTATTTGGATTGGTACTTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATGTGGGTT CTACTCGGGCCTCCGGGGTCTCTGGCAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTACTGCATGCAGGCTCTACAAATGCCGTACACTTTTGGCCAGGGGACCAAACTGGAGATCAAAC.

[0026] In one embodiment, the sequence of the anti-COVID-19 N protein human monoclonal antibody 1B9 is derived from a human sequence.

[0027] In one embodiment, the heavy chain amino acid sequence of the anti-COVID-19 N protein human monoclonal antibody 1B9 is as shown in SEQ ID NO.9.

[0028] The encoding nucleotide sequence is shown in SEQ ID NO.29.

[0029] The light chain amino acid sequence of the anti-COVID-19 N protein human monoclonal antibody 1B9 is shown in SEQ ID NO.10.

[0030] The encoding nucleotide sequence is shown in SEQ ID NO.30.

[0031] In the second aspect, the embodiments of the present application provide the use of the anti-COVID-19 N protein human monoclonal antibody in the preparation of a COVID-19 N protein detection reagent or kit.

[0032] In a third aspect, an embodiment of the present application provides a novel coronavirus N protein enzyme-linked immunosorbent assay kit, comprising two anti-novel coronavirus N protein human monoclonal antibodies, 1B9 and 1H5, wherein the monoclonal antibody 1B9 is used as a coating antibody and the monoclonal antibody 1H5 is used as a detection antibody;

[0033] The anti-COVID-19 N protein human monoclonal antibody 1H5 comprises a light chain variable region and a heavy chain variable region;

[0034] The heavy chain variable region includes complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3;

[0035] The amino acid sequence of VH-CDR1 is SEQ ID NO.11: NDSVNSYF; the encoding nucleotide sequence is SEQ ID NO.31: AATGACTCCGTCAATTCTTACTTC.

[0036] The amino acid sequence of VH-CDR2 is SEQ ID NO.12: IYSSGTT; the encoding nucleotide sequence is SEQ ID NO.32: ATCTACTCTAGTGGGACCACC.

[0037] The amino acid sequence of VH-CDR3 is SEQ ID NO.13: ARGLDRSKSANWFDP; the encoding nucleotide sequence is SEQ ID NO.33: GCGAGAGGACTCGATAGGTCTAAGAGCGCCAACTGGTTCGACCCC.

[0038] The light chain variable region includes complementarity determining regions VK-CDR1, VK-CDR2 and VK-CDR3;

[0039] The amino acid sequence of VK-CDR1 is SEQ ID NO.14: QSLLFSSNSKNY; the encoding nucleotide sequence is SEQ ID NO.34: CAGAGTCTTTTATTCAGCTCCAACAGTAAGAACTAC.

[0040] The amino acid sequence of VK-CDR2 is SEQ ID NO.15: WAS; the encoding nucleotide sequence is SEQ ID NO.35: TGGGCATCT.

[0041] The amino acid sequence of VK-CDR3 is SEQ ID NO.16: QQYYNTPFT; the encoding nucleotide sequence is SEQ ID NO.36: CAGCAATATTATAATACTCCATTCACT.

[0042] In one embodiment, the amino acid sequence of the heavy chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1H5 is shown in SEQ ID NO.17;

[0043] SEQ ID NO.17:

[0044] QVQLQESGPGLVKPSETLSLTCTVSNDSVNSYFWSWIRQPPGKGLEWIG YIYSSGTTNYNPSLKSRVTISIDASRNQFSLKLNSVTAADTAMYYCARGLDRS KSANWFDPWGQGILVTVSS.

[0045] The encoding nucleotide sequence is shown in SEQ ID NO.37;

[0046] SEQ ID NO.37:

[0047] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTAATGACTCCGTCAATTCTTACTTCTGGAGCTGGATCCGGCAGCCCCAGGGAAGGGACTGGAGTGGATTGGCTATATCTACTCTAGTGGGACCACCAATTATAACCC CTCCCTTAAGAGTCGAGTTACCATATCCATAGACGCGTCCAGGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCAGCGGACACGGCCATGTATTACTGTGCGAGAGGACTCGATAGGTCTAAGAGCGCCAACTGGTTCGACCCCTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG.

[0048] The amino acid sequence of the light chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1H5 is shown in SEQ ID NO.18.

[0049] SEQ ID NO.18:

[0050] DIVMTQSPDSLAVSLGGRATINCRSSQSLLFSSNKNYLAWYQQKPGQP PKLLMYWASTRESGVPDRFSGSGSGTDFTLTVTSLQAEDVAVYYCQQYYNTPFTFGPGTKVDIK.

[0051] The encoding nucleotide sequence is shown in SEQ ID NO.38;

[0052] SEQ ID NO.38:

[0053] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGGGAGGGCCACCATCAACTGCAGGTCCAGCCAGAGTCTTTTATTCAGCTCCAACAGTAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTGCTCATGTACTGGGC ATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCGTCACCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAATACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC.

[0054] In one embodiment, the sequence of the anti-COVID-19 N protein human monoclonal antibody 1H5 is derived from a human sequence.

[0055] In one embodiment, the heavy chain amino acid sequence of the anti-COVID-19 N protein human monoclonal antibody 1H5 is as shown in SEQ ID NO.19.

[0056] The encoding nucleotide sequence is shown in SEQ ID NO.39.

[0057] The amino acid sequence of the light chain of the anti-COVID-19 N protein human monoclonal antibody 1H5 is shown in SEQ ID NO.20.

[0058] The encoding nucleotide sequence is shown in SEQ ID NO.40.

[0059] In one embodiment, the anti-COVID-19 N protein human monoclonal antibody 1B9 and the anti-COVID-19 N protein human monoclonal antibody 1H5 are obtained by human immunization with the N protein of the new coronavirus, and the amino acid sequence of the N protein of the new coronavirus is shown in SEQ ID NO.41; the encoding nucleotide sequence is shown in SEQ ID NO.42.

[0060] The advantages or beneficial effects of the above technical solution include at least:

[0061] The anti-COVID-19 N protein human monoclonal antibody of the present application is a human antibody that can specifically bind to the COVID-19 N protein with high specificity. Furthermore, by pairing the antibodies, a COVID-19 N protein enzyme-linked immunosorbent assay (ELISA) kit is constructed, which has strong anti-interference ability, good specificity, and high sensitivity for clinical COVID-19 detection.

[0062] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0064] Figure 1 This is the SDS-PAGE image of the new coronavirus N protein;

[0065] Figure 2 This is the electrophoresis diagram of variable region gene amplification of B cells;

[0066] Figure 3 This is a reference chart for the colorimetric card for immunochromatographic detection of different concentrations of novel coronavirus positive quality control products using paired antibodies. DETAILED DESCRIPTION

[0067] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0068] Example

[0069] 1. Prokaryotic expression of the novel coronavirus N protein

[0070] The amino acid sequence of the new crown N protein is derived from the Uniprot database (UniPort ID: P0DTC9). The full-length gene sequence of the N protein was selected as the expression gene (protein sequence encoding 1-419aa). The expression sequence was codon-optimized for the Escherichia coli expression system by Beijing Qingke Biotechnology Co., Ltd. and gene synthesis was performed. The synthesized N protein gene fragment was cloned into the pET28a+ prokaryotic expression vector through the two restriction sites of Nco I and Xho I. The C-terminus of the target protein was carrying a 6*HIS tag. The specific amino acid sequence and gene sequence are shown in SEQ ID NO.41 and SEQ ID NO.42.

[0071] The N protein expression plasmid was transformed into BL21 competent cells and induced with 0.2 mM IPTG at 30°C for 5 h. 300 ml of bacterial suspension was centrifuged and harvested, resuspended in 20 ml of PBS, and 1 mM PMSF was added. The cells were disrupted using an ultrasonic disruptor (output power 60%, 4 s on, 7 s off as one cycle, total time 15 min). The cells were harvested by centrifugation at 12000 g for 30 min, and the supernatant was passed through Ni-NTA affinity resin (Invitrogen TM , R90115) for affinity chromatography purification. First, 10ml NativeBinding Buffer (10mM imidazole) was used to balance 1ml Ni-NTA, then the bacterial supernatant was slowly loaded and flowed through Ni-NTA, 10ml NativeWash Buffer (20mM imidazole) was used to wash the impurities bound to Ni-NTA, and finally 10ml NativeElution Buffer (250mM imidazole) was used to elute the target protein. The eluted target protein was concentrated by ultrafiltration tube to replace PBS buffer, and then SDS-PAGE was performed for identification. The electrophoresis pattern is shown as follows: Figure 1 The molecular weight of N protein is about 57 kDa and the purity is >90%.

[0072] 2. Novel coronavirus N protein coupled to fluorescein

[0073] NHS-Biotin (BBI, C608212) can simply and effectively biotinylate antibodies, proteins and other macromolecules containing primary amines in solution. First, biotin is coupled to the new crown N protein, and then SA-PE and SA-APC are used to connect to biotin. Use dimethyl sulfoxide (DMSO) organic solvent to prepare a 10mM biotin reagent solution. Then add a 20-fold molar excess of biotin reagent to label 100ul 2mg / mL N protein and incubate on ice for 2h. Use a desalting column to purify the labeled protein and remove unreacted biotin. According to the binding molar ratio of SA:biotin=1:4, the biotinylated N protein is mixed with SA-PE and SA-APC respectively, and incubated on ice for 30min before proceeding directly to the next step of cell labeling.

[0074] 3. Flow cytometry sorting of single memory B cells

[0075] Peripheral blood was collected from several patients with COVID-19 infection who recovered from the Seventh Affiliated Hospital of Southern Medical University. PBMCs were isolated using human lymphocyte separation medium (Solarbio, P8610) and cultured at a concentration of 5 × 10 6The frozen PBMCs were placed in a 37°C water bath for rapid thawing and resuscitation. The resuscitated PBMCs were resuspended in FACS buffer (10mM PBS, 1mM EDTA, 2% FBS). 100ul of the resuspended cells (1*10 6 ) Fluorescent markers were added in sequence: DAPI, IgM-BV241, CD19-PE / cy7, CD27-APC / cy7, IgG-FITC, N-PE, and N-APC, and incubated on ice for 30 minutes. PBMCs were sorted using a BD FACSAria III flow cytometer, and cell populations that were DAPI- / IgM- / CD19+ / CD27+ / IgG+ and double-positive for the novel coronavirus N protein were selected. 88 single memory B cells specific for the novel coronavirus N protein were sorted.

[0076] 4. Amplify variable region genes of single B cells

[0077] Amplification primers and conditions were as described in the literature (Smith, K., Garman, L., Wrammert, J. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4, 372-384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3). RT-PCR was first performed using a mixture of heavy chain, kappa chain, and lambda chain primers, as shown in Table 1:

[0078] Table 1

[0079]

[0080]

[0081] The goal is to cover all possible variable region (V) gene families. Nested PCR enriches for variable region genes and amplifies DNA to a sufficient level to obtain heavy and light chain V gene sequences. Cloning PCR uses highly specific primers for each V gene family to amplify, aiming to incorporate cloning restriction sites and place the VDJ heavy chain or VJ light chain gene in frame with the signal peptide sequence and constant region gene within the respective cloning vector. Gene amplification results are shown in Figure 2. Figure 2 As shown, Heavy chain 81 / 88 (92%), Kappa chain (59 / 88, 67%), Lambda chain (27 / 88, 31%)

[0082] 5. Antibody expression plasmid construction

[0083] First, double-digest the gamma, kappa, or lambda cloning vector with restriction endonucleases Age I & Sal I, Age I & BsiW I, or Age I & Xho I, respectively. Refer to the reference for the cloning vector sequence (Smith, K., Garman, L., Wrammert, J. et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 4, 372-384 (2009). https: / / doi.org / 10.1038 / nprot.2009.3) at 37°C for 2 hours. Purify and recover the gamma, kappa, or lambda chain variable region gene product amplified in step 4 using a standard DNA purification kit (TIANGEN, DP204-02). Submit the double-digested cloning vector sample for electrophoresis on a 1% agarose gel. The vector is approximately 5700 bp in length. The variable region gene fragment and the vector fragment were homologously recombined using the ClonExpress II One Step Cloning Kit (Vazyme, C112-01) and reacted at 37°C for 30 minutes. The recombinant product was transformed into DH5a competent cells, evenly spread on a plate containing Amp resistance, and cultured at 37°C overnight. The next day, single clones on 3 plates were picked with a toothpick and placed in 500ul of LB medium containing Amp resistance and cultured at 37°C for 4-6 hours. 2×Taq Master Mix (Vazyme, P112-01) was used for bacterial liquid PCR verification to confirm whether the variable region was successfully inserted into the vector. The positive clones were sequenced to confirm the accuracy of the variable region sequence.

[0084] 6. Antibody small batch expression

[0085] Use 293T cells to transfect the recombinant antibody plasmid for small-scale expression. 24h before transfection, inoculate 293T cells into 96-well plates so that the confluence of the cells in the 96-well plates is maintained at 90% before transfection. Gently aspirate the complete culture medium (DMEM + 10% FBS) in the 96-well plate cells and replace it with serum-free culture medium (DMEM). Prepare another clean 96-well plate, take 30ml of DMEM culture medium + 500ul of PEI (1mg / ml) and mix thoroughly, add 300ul / well to the 96-well plate, then add 10ng of light chain and heavy chain to the 96-well plate, incubate at room temperature for 15min, and then add them to the cells accordingly. After culturing for 3 days at 37°C and 5% CO2, recover the cell supernatant.

[0086] 7. Antibody ELISA specificity screening

[0087] Dilute N protein antigen with carbonate coating buffer to a final concentration of 1 μg / ml. 200 μl of the solution was added to each ELISA plate and incubated at 4°C overnight. Wash the plate three times with 300 μl of PBST for 3 minutes each time. Add 280 μl of 5% skim milk to each well and block at 37°C for 1 hour. Wash the plate three times with 300 μl of PBST for 3 minutes each time. Add 100 μl of cell supernatant to each well and incubate at 37°C for 40 minutes. Wash the plate three times with 300 μl of PBST for 3 minutes each time. Add 100 μl of secondary antibody (1:10,000 dilution in 5% skim milk) to each well and block at 37°C for 40 minutes. Wash the plate three times with 300 μl of PBST for 3 minutes each time. Then, add 100 μl of TMB substrate to each well and incubate at room temperature for 10 minutes in the dark. Stop the color reaction by adding 50 μl of 2M sulfuric acid to each well. The plate was then placed on a microplate reader and the OD450 value was measured. The results showed that 41 positive antibodies with OD>2 were screened out of 88 samples; as shown in Table 2.

[0088] Table 2 Specificity screening results of SARS-CoV-2 N protein ELISA

[0089]

[0090]

[0091] 8. Specific Antibody Expression

[0092] 293F cells were used to express the specific antibodies screened in a small scale. The day before transfection, 293F cells were subcultured. On the day of transfection, the cell activity was required to be >90% and the cell density was 3-4*10 6 cells / mL. During transfection, the cell density was adjusted to 1.0*10 6 cells / mL, PEI (1 mg / mL) and 80 μg of plasmid DNA (H:L = 1:1) were mixed and incubated at room temperature for 10 minutes to form a cationic polymer (PEI:DNA = 3:1, mass ratio). This polymer was then slowly added to 293F cells, with 80 ml of cells transfected with each antibody. Six days later, the cell supernatant was collected for purification. Antibodies were purified using AT Protein ADiamond (Bestchrom, AA0272) after expression. The supernatant was washed with 10 ml of equilibration buffer and then eluted with 5 ml of 0.1 M glycine. The eluted antibody solution was immediately neutralized with 500 μl of 1 M Tris-HCl and dialyzed against PBS at 4°C, with the dialysate replaced every 2 hours for three cycles. Antibody purity and concentration were assessed by SDS-PAGE and UV absorption.

[0093] 9. Antibody pairing screening

[0094] 50 μg of each purified antibody was added to 10 volumes of starter buffer and 50 μg of HRP, followed by reaction at 37°C for 2 hours. Stop buffer (1 / 10 volume of the reaction mixture) was added and allowed to react at room temperature for 1 hour. 41 purified antibodies were diluted to a final concentration of 1 μg / ml, and 100 μl of each was added to the ELISA plate for coating. The plate was incubated overnight at 4°C or for 2 hours at 37°C. The plate was washed three times with 300 μl of PBST for 3 minutes each time. 280 μl of 5% skim milk was added to each well and blocked at 37°C for 1 hour. The plate was washed three times with 300 μl of PBST for 3 minutes each time. Then, N protein was diluted to a final concentration of 1 μg / ml. 100 μl of the diluted N protein was added to each well and incubated at 37°C for 40 minutes. The plate was washed three times with 300 μl of PBST for 3 minutes each time. 100 μl of HRP-conjugated antibody (1:10,000 dilution in 5% skim milk) was added to each well and blocked at 37°C for 40 minutes. Wash the plate three times with 300 μl of PBST for 3 minutes each wash. Then, add 100 μl of TMB substrate to each well in the dark and incubate at room temperature for 10 minutes. Add 50 μl of 2M sulfuric acid to each well and read the plate on a microplate reader at 450 nm. The results showed that 1B9 and 1H5 were one of the best antibody pairs, with 1B9 acting as the coating antibody and 1H5 as the labeling antibody.

[0095] Monoclonal antibody 1B9 was sequenced, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.9; the encoding nucleotide sequence is shown in SEQ ID NO.29; the amino acid sequence of the light chain is shown in SEQ ID NO.10; and the encoding nucleotide sequence is shown in SEQ ID NO.30.

[0096] Among them, including heavy chain variable region and light chain variable region;

[0097] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7,

[0098] SEQ ID NO.7:

[0099] QVQLVQSGSEVKKPGSSVKVSCQASGGSFSSYAVNWVRQAPGQGLEW MGRIIPLLGKINYGQNFQGRVTITADESSGTAYMEMTRLRPADTAVYFCARGGWSTEVAGSAMDVWGPGTTITVS.

[0100] The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 27;

[0101] SEQ ID NO.27:

[0102] CAGGTGCAGCTGGTGCAGTCTGGGAGTGAGGTGAAAAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCCAGGCCTCGGGAGGCAGTTTCAGCAGTTATGCTGTCAACTGGGTGCGACAGGCTCCTGGACAAGGGCTTGAGTGGATGGGACGCATCATCCCTCTTCTTGGCAAAATAAACTACGGACA AAACTTCCAGGGCAGAGTCACCATCACCGCGGACGAATCCTCGGGCACAGCCTACATGGAGATGACTAGACTGAGACCTGCGGACACGGCCGTGTATTTTTGTGCGAGAGGTGGCTGGTCCACAGAAGTGGCTGGCTCAGCTATGGACGTCTGGGGCCCAGGGACCACGATCACTGTCTCCCCAG.

[0103] heavy chain variable region complementarity determining region;

[0104] CDR1: amino acid sequence GGSFSSYA (SEQ ID NO. 1);

[0105] Nucleotide sequence GGAGGCAGTTTCAGCAGTTATGCT (SEQ ID NO. 21); CDR2: amino acid sequence IIPLLGKI (SEQ ID NO. 2);

[0106] Nucleotide sequence ATCATCCCTCTTCTTGGCAAAATA (SEQ ID NO. 22); CDR3: amino acid sequence ARGGWSTEVAGSAMDV (SEQ ID NO. 3);

[0107] Nucleotide sequence: (SEQ ID NO. 23) GCGAGAGGTGGCTGGTCCACAGAAGTGGCTGGCTCAGCTATGGACGTC;

[0108] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.8;

[0109] SEQ ID NO.8:

[0110] DIVVTQSPLSLSVTPGEPAAISCRSSENLLHTNGKNYLDWYLQKPGQSP QLLIYVGSTRASGVSGRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQMP YTFGQGTKLEIK.

[0111] The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 28;

[0112] SEQ ID NO.28:

[0113] GATATTGTGGTGACTCAGTCTCCACTCTCCCTGTCCGTCACCCCTGGAGAGCCGGCCGCCATCTCCTGCAGGTCTAGTGAGAACCTCCTGCATACTAATGGAAAAAAATTATTTGGATTGGTACTTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATGTGGGTT CTACTCGGGCCTCCGGGGTCTCTGGCAGGTTCAGTGGCAGTGGATCAGGTACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTACTGCATGCAGGCTCTACAAATGCCGTACACTTTTGGCCAGGGGACCAAACTGGAGATCAAAC.

[0114] light chain variable region complementarity-determining region;

[0115] CDR1: amino acid sequence ENLLHTNGKNY (SEQ ID NO. 4);

[0116] Nucleotide sequence GAGAACCTCCTGCATACTAATGGAAAAAATTAT (SEQ ID NO. 14);

[0117] CDR2: amino acid sequence VGS (SEQ ID NO. 5);

[0118] Nucleotide sequence GTGGGTTCT (SEQ ID NO. 15);

[0119] CDR3: amino acid sequence MQALQMPYT (SEQ ID NO. 6);

[0120] Nucleotide sequence ATGCAGGCTCTACAAATGCCGTACACT (SEQ ID NO. 16).

[0121] Monoclonal antibody 1H5 was sequenced, wherein the amino acid sequence of the heavy chain is shown in SEQ ID NO.19; the encoding nucleotide sequence is shown in SEQ ID NO.31B99; the amino acid sequence of the light chain is shown in SEQ ID NO.20; the encoding nucleotide sequence is shown in SEQ ID NO.40.

[0122] Among them, among them, include heavy chain variable region and light chain variable region;

[0123] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 17;

[0124] SEQ ID NO.17:

[0125] QVQLQESGPGLVKPSETLSLTCTVSNDSVNSYFWSWIRQPPGKGLEWIG YIYSSGTTNYNPSLKSRVTISIDASRNQFSLKLNSVTAADTAMYYCARGLDRS KSANWFDPWGQGILVTVSS.

[0126] The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 37;

[0127] SEQ ID NO.37:

[0128] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTAATGACTCCGTCAATTCTTACTTCTGGAGCTGGATCCGGCAGCCCCAGGGAAGGGACTGGAGTGGATTGGCTATATCTACTCTAGTGGGACCACCAATTATAACCC CTCCCTTAAGAGTCGAGTTACCATATCCATAGACGCGTCCAGGAACCAGTTCTCCCTGAAGCTGAACTCTGTGACCGCAGCGGACACGGCCATGTATTACTGTGCGAGAGGACTCGATAGGTCTAAGAGCGCCAACTGGTTCGACCCCTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG.

[0129] heavy chain variable region complementarity determining region;

[0130] CDR1: amino acid sequence NDSVNSYF (SEQ ID NO. 11);

[0131] Nucleotide sequence: AATGACTCCGTCAATTCTTACTTC (SEQ ID NO. 31); CDR2: amino acid sequence: IYSSGTT (SEQ ID NO. 1=12);

[0132] Nucleotide sequence ATCTACTCTAGTGGGACCACC (SEQ ID NO. 32);

[0133] CDR3: amino acid sequence ARGLDRSKSANWFDP (SEQ ID NO. 13);

[0134] Nucleotide sequence (SEQ ID NO. 33): GCGAGAGGACTCGATAGGTCTAAGAGCGCCAACTGGTTCGACCCC.

[0135] The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 18;

[0136] SEQ ID NO.18:

[0137] DIVMTQSPDSLAVSLGGRATINCRSSQSLLFSSNKNYLAWYQQKPGQP PKLLMYWASTRESGVPDRFSGSGSGTDFTLTVTSLQAEDVAVYYCQQYYNTPFTFGPGTKVDIK.

[0138] The nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.38;

[0139] SEQ ID NO.38:

[0140] GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGGGAGGGCCACCATCAACTGCAGGTCCAGCCAGAGTCTTTTATTCAGCTCCAACAGTAAGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAACTGCTCATGTACTGGGC ATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCGTCACCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAATATTATAATACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC.

[0141] light chain variable region complementarity-determining region;

[0142] CDR1: amino acid sequence QSLLFSSNSKNY (SEQ ID NO. 14);

[0143] Nucleotide sequence CAGAGTCTTTTATTCAGCTCCAACAGTAAGAACTAC (SEQ ID NO. 34);

[0144] CDR2: amino acid sequence WAS (SEQ ID NO. 15);

[0145] Nucleotide sequence TGGGCATCT (SEQ ID NO. 35);

[0146] CDR3: amino acid sequence QQYYNTPFT (SEQ ID NO. 16);

[0147] Nucleotide sequence CAGCAATATTATAATACTCCATTCACT (SEQ ID NO. 36).

[0148] 10. Paired Antibody Performance Testing

[0149] Using the paired antibodies 1B9 and 1H5 obtained in the previous step, the coated antibody 1B9 was coupled to red polystyrene microspheres (Nanomicro, LDRNC-030). The specific coupling scheme was referred to the microsphere instructions, and then the microspheres were evenly added to the conjugate pad. The NC membrane was cut into strips, and the C and T line positions were marked. The 1H5 antibody was fixed on the T line of the NC membrane, and the mouse anti-human specific IgG antibody was fixed on the C line. The positive quality control product of the new coronavirus (SARS-CoV-2) (ZeptoMetrix, NATSARS (COV2) -ERC-IVD) was tested by immunochromatography. The colorimetric card was referenced as follows: Figure 3 The results are shown in Table 3.

[0150] Table 3 Test results of positive quality control products

[0151]

[0152] From Table 3 and Figure 3 It can be seen that the positive control product can still effectively detect the virus after being diluted 20,000 times, indicating that the antibody has a high sensitivity.

[0153] Clinical sample testing was conducted using immunochromatographic panels prepared using paired antibodies 1B9 and 1H5, compared to panels prepared using a mainstream SARS-CoV-2 mouse antibody pair against the N protein. A total of 40 positive samples and 22 negative samples were detected. The results of this parallel comparison are shown in Table 3.

[0154] Table 4 Detection results of clinical samples of SARS-CoV-2 paired antibodies

[0155]

[0156] As shown in Table 4, the paired antibodies 1B9 and 1H5 showed a total coincidence rate of 95% for positive samples and 100% for negative samples. The control antibody pair showed a total coincidence rate of 95% for positive samples and 95.4% for negative samples. This indicates that the 1B9 and 1H5 antibody pair significantly outperformed the control mouse antibody pair in terms of detection specificity and anti-interference ability.

[0157] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials, or characteristics described may be combined in any appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0158] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0159] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A human monoclonal antibody against the novel coronavirus N protein, characterized in that: Designated as 1B9, the 1B9 comprises a light chain variable region and a heavy chain variable region; The heavy chain variable region includes complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region includes complementarity determining regions VK-CDR1, VK-CDR2 and VK-CDR3; The amino acid sequence of VH-CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO. 3; The amino acid sequence of VK-CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VK-CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of VK-CDR3 is shown in SEQ ID NO.

6.

2. The anti-COVID-19 N protein human monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1B9 is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

8.

3. The anti-COVID-19 N protein human monoclonal antibody according to claim 1, characterized in that The sequence of the anti-COVID-19 N protein human monoclonal antibody 1B9 is derived from a human sequence.

4. The anti-COVID-19 N protein human monoclonal antibody according to claim 1, characterized in that The heavy chain amino acid sequence of the anti-COVID-19 N protein human monoclonal antibody 1B9 is shown in SEQ ID NO.9; the light chain amino acid sequence is shown in SEQ ID NO.

10.

5. Use of the anti-COVID-19 N protein human monoclonal antibody according to any one of claims 1 to 4 in the preparation of a COVID-19 N protein detection reagent or kit.

6. A novel coronavirus N enzyme-linked immunosorbent assay kit, characterized in that: The kit includes two anti-COVID-19 N protein human monoclonal antibodies, 1B9 and 1H5, wherein the monoclonal antibody 1B9 is used as a coating antibody and the monoclonal antibody 1H5 is used as a detection antibody; The anti-COVID-19 N protein human monoclonal antibody 1H5 comprises a light chain variable region and a heavy chain variable region; The heavy chain variable region includes complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3; the light chain variable region includes complementarity determining regions VK-CDR1, VK-CDR2 and VK-CDR3; The amino acid sequence of VH-CDR1 is shown in SEQ ID NO.11, the amino acid sequence of VH-CDR2 is shown in SEQ ID NO.12, and the amino acid sequence of VH-CDR3 is shown in SEQ ID NO.13; The amino acid sequence of VK-CDR1 is shown in SEQ ID NO.14, the amino acid sequence of VK-CDR2 is shown in SEQ ID NO.15, and the amino acid sequence of VK-CDR3 is shown in SEQ ID NO.

16.

7. The PCR-ELISA kit for COVID-19 according to claim 6, wherein: The amino acid sequence of the heavy chain variable region of the anti-COVID-19 N protein human monoclonal antibody 1H5 is shown in SEQ ID NO.17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

18.

8. The PCR-ELISA kit for COVID-19 according to claim 6, wherein: The sequence of the anti-COVID-19 N protein human monoclonal antibody 1H5 is derived from a human sequence.

9. The PCR-ELISA kit for COVID-19 according to claim 6, wherein: The heavy chain amino acid sequence of the anti-COVID-19 N protein human monoclonal antibody 1H5 is shown in SEQ ID NO.19; the light chain amino acid sequence is shown in SEQ ID NO.

20.

10. The PCR-ELISA kit for COVID-19 according to claim 6, wherein: The anti-COVID-19 N protein human monoclonal antibody 1B9 and the anti-COVID-19 N protein human monoclonal antibody 1H5 are obtained by human immunization with the N protein of the new coronavirus, and the amino acid sequence of the N protein of the new coronavirus is shown in SEQ ID NO.41.

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