Preparation and application of anti-novel influenza virus n protein human monoclonal antibody

Abstract

The application provides preparation and application of an anti-influenza N protein human monoclonal antibody, which comprises a heavy chain complementarity determining region of an amino acid sequence shown in SEQ ID NO. 1, 2 and 3 and a light chain complementarity determining region of an amino acid sequence shown in SEQ ID NO. 4, 5 and 6. The anti-influenza N protein human monoclonal antibody shows good sensitivity, anti-interference ability and specificity to influenza virus quality control products and clinical samples, and provides raw material guarantee for downstream development of high-quality diagnostic reagents.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to the preparation and application of a human monoclonal antibody against influenza A N protein. Background Technology

[0002] Influenza A virus (IAV) is a highly contagious pathogen that can cause seasonal epidemics and seriously threaten public health. Its nucleoprotein (N protein) is an essential structural protein for viral replication, highly conserved, and a core target for early diagnosis. Immunoassay methods based on the N protein (such as colloidal gold test strips, ELISA, and chemiluminescence) are widely used in clinical and field screening due to their speed and low cost. However, the sensitivity and specificity of existing detection technologies still face challenges, especially as they are susceptible to interference from endogenous antibodies in the sample, leading to false positive / false negative results.

[0003] Developing monoclonal antibodies, especially human monoclonal antibodies, that can effectively reduce endogenous interference is a feasible way to improve detection accuracy. Summary of the Invention

[0004] This application provides a method for preparing and applying a human monoclonal antibody against influenza A N protein to address the problems existing in related technologies. The technical solution is as follows:

[0005] In a first aspect, embodiments of this application provide a human monoclonal antibody against influenza A N protein, comprising a light chain variable region and a heavy chain variable region;

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

[0007] The amino acid sequence of VH-CDR1 is SEQ ID NO.1: GFTFSFYW; the encoding nucleotide sequence is SEQ ID NO.13: GGATTCACTTTTAGTTTCTATTGG.

[0008] The amino acid sequence of VH-CDR2 is SEQ ID NO.2: IKRDGSEK; the encoding nucleotide sequence is SEQ ID NO.14: ATAAAGCGAGATGGAAGTGAGAAA.

[0009] The amino acid sequence of VH-CDR3 is SEQ ID NO.3: ARDNDGDFQPHYDV; the encoding nucleotide sequence is SEQ ID NO.15: GCGAGAGACAATGACGGTGACTTCCAACCTCACTATGACGTC.

[0010] The amino acid sequence of Vλ-CDR1 is SEQ ID NO.4: NIGDKR; the encoding nucleotide sequence is SEQ ID NO.16: AACATTGGAGATAAACGT.

[0011] The amino acid sequence of Vλ-CDR2 is SEQ ID NO.5: FDQ; the encoding nucleotide sequence is SEQ ID NO.17: TTTGATCAG.

[0012] The amino acid sequence of Vλ-CDR3 is SEQ ID NO.6: QVWDDNSGHRM; the encoding nucleotide sequence is SEQ ID NO.18: CAGGTCTGGGATGATAATAGTGGTCATCGGATG.

[0013] In one embodiment, the heavy chain variable region amino acid sequence of the anti-influenza N protein human monoclonal antibody is shown in SEQ ID NO.7.

[0014] The encoding nucleotide sequence is shown in SEQ ID NO.11.

[0015] The amino acid sequence of the light chain variable region of the anti-influenza N protein human monoclonal antibody is shown in SEQ ID NO.8.

[0016] The encoding nucleotide sequence is shown in SEQ ID NO.12.

[0017] In one embodiment, the sequence of the anti-influenza N protein human monoclonal antibody is derived from a human sequence.

[0018] In one embodiment, the heavy chain amino acid sequence of the anti-influenza N protein human monoclonal antibody is shown in SEQ ID NO. 9.

[0019] The encoding nucleotide sequence is shown in SEQ ID NO.19.

[0020] The light chain amino acid sequence of the anti-influenza N protein human monoclonal antibody is shown in SEQ ID NO.10.

[0021] The encoding nucleotide sequence is shown in SEQ ID NO.20.

[0022] Secondly, embodiments of this application provide a nucleic acid that encodes any of the aforementioned anti-influenza N protein human monoclonal antibodies.

[0023] In one embodiment, the nucleotide sequence for encoding the heavy chain variable region is shown in SEQ ID NO.11; and the nucleotide sequence for encoding the light chain variable region is shown in SEQ ID NO.12.

[0024] In one embodiment, the nucleotide sequence for encoding the heavy chain is shown in SEQ ID NO.19; and the nucleotide sequence for encoding the light chain is shown in SEQ ID NO.20.

[0025] Thirdly, embodiments of this application provide a biological material comprising the nucleic acid described above; the biological material is an expression cassette, a vector, or a transgenic cell.

[0026] Fourthly, this application provides a method for preparing the above-mentioned anti-influenza N protein human monoclonal antibody, which involves gene expression of the above-mentioned nucleic acid; or culturing the above-mentioned transgenic cells.

[0027] Fifthly, the reagents or kits in the embodiments of this application include any of the above-described anti-influenza N protein human monoclonal antibodies, or the above-described nucleic acids, or the above-described biological materials.

[0028] In one embodiment, the reagent or kit is used to detect influenza A N protein.

[0029] The advantages or beneficial effects of the above technical solutions include at least the following:

[0030] The human monoclonal antibody against influenza A N protein in this application is derived from B cells in the blood of recovered influenza A patients. It is a fully human monoclonal antibody; its detection of influenza A N protein in the human body is free from species differences, thus avoiding functional problems caused by species specificity and solving issues such as rejection reactions and weak affinity associated with heterologous expression. Furthermore, it possesses both light and heavy chain variable regions, making it a human monoclonal antibody with strong anti-interference ability, high specificity, and high sensitivity in clinical influenza A detection.

[0031] The above overview 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 this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0032] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0033] Figure 1 SDS-PAGE image of influenza A N protein;

[0034] Figure 2 Electrophoresis diagram of gene amplification in the variable region of a single B cell;

[0035] Figure 3 Reference chart for immunochromatographic detection of paired antibody pairs against different concentrations of positive H1N1 virus control samples. Detailed Implementation

[0036] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0037] Example

[0038] 1. Prokaryotic expression of influenza A N protein

[0039] The N protein gene sequence of the H1N1 subtype of influenza A was obtained from the Uniport database (UniPort ID: P18070). The full-length N protein gene sequence (encoding protein sequence 1-498 aa) was selected as the expression gene. Codon optimization of the expression sequence in the *E. coli* expression system was performed using Beijing Qingke Biotechnology Co., Ltd., and the gene was synthesized. The synthesized N protein gene fragment was cloned into the pET28a+ prokaryotic expression vector via two restriction enzyme sites, Nco I and Xho I. The target protein has a 6*HIS tag at its C-terminus. The specific amino acid sequence and gene sequence are shown in SEQ ID NO.21 and SEQ ID NO.22.

[0040] The N protein expression plasmid was transformed into BL21 competent cells, and expression was induced at 30℃ for 5 h using 0.2 mM IPTG. The cells were collected by centrifugation in 300 ml of bacterial culture, resuspended in 20 ml of PBS, and then subjected to 1 mM PMSF. The cells were then disrupted using an ultrasonic homogenizer (output power 60%, working for 4 seconds, stopping for 7 seconds as one cycle, total time 15 min). The cells were then collected by centrifugation at 12000 g for 30 min. The supernatant was passed through Ni-NTA affinity resin (Invitrogen). ·Affinity chromatography purification was performed using (R90115). First, 1 ml of Ni-NTA was equilibrated with 10 ml Native Binding Buffer (10 mM imidazole). Then, the bacterial supernatant was slowly loaded through Ni-NTA. Impurities bound to Ni-NTA were washed with 10 ml Native Wash Buffer (20 mM imidazole). Finally, the target protein was eluted with 10 ml Native Elution Buffer (250 mM imidazole). The eluted target protein was concentrated by ultrafiltration to replace the PBS buffer, and then identified by SDS-PAGE. The electrophoresis image is shown below. Figure 1 The N protein has a molecular weight of approximately 56 kDa and a purity of >90%.

[0041] 2. N protein-coupled fluorescein

[0042] NHS-Biotin (BBI, C608212) provides a simple and effective method for biotinylation of antibodies, proteins, and other primary amine-containing macromolecules in solution. First, biotin is conjugated to influenza A N protein, and then SA-PE and SA-APC are used to bind the biotin. A 10 mM biotin reagent solution is prepared using dimethyl sulfoxide (DMSO) as the organic solvent. Then, 100 μL of 2 mg / mL N protein is labeled with a 20-fold molar excess of biotin reagent and incubated on ice for 2 h. The labeled protein is purified using a desalting column to remove unreacted biotin. Following a 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 30 min before proceeding directly to the next step of cell labeling.

[0043] 3. Flow cytometry sorting of influenza A N protein-specific memory B cells

[0044] Peripheral blood was collected from several recovered patients with H1N1 influenza at the Seventh Affiliated Hospital of Southern Medical University. Human lymphocyte separation medium (Solarbio, P8610) was used to separate PBMCs, and the PBMCs were obtained at a concentration of 5*10-1. 6 Cells / tubes were cryopreserved in liquid nitrogen for later use. After cryopreservation, PBMCs were rapidly thawed in a 37°C water bath. The thawed PBMCs were resuspended in FACS buffer (10mM PBS, 1mM EDTA, 2% FBS). 100µl of the resuspended cells (1*102) was then used. 6Fluorescent markers were added sequentially: DAPI, IgM-BV241, CD19-PE / cy7, CD27-APC / cy7, IgG-FITC, N-PE, and N-APC, and incubated on ice for 30 min. PBMCs were sorted using a BDFACSAria III flow cytometer, and a population of cells double-positive for DAPI- / IgM- / CD19+ / CD27+ / IgG+ and influenza A N protein was selected, yielding 88 single memory B cells specifically targeting influenza A N protein.

[0045] 4. Amplify the variable region genes of single B cells.

[0046] The amplification primers and amplification conditions were referenced 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). First, RT-PCR was performed using a mixture of heavy chain, Kappa chain, and Lambda chain primers, as shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] The aim is to cover all possible variable region (V) gene families. Nested PCR enriches variable region genes, amplifying DNA to a level sufficient to obtain heavy and light chain V gene sequences. Cloning PCR uses highly specific primers for each V gene family for amplification, designed to integrate cloning restriction sites and frame the VDJ heavy chain or VJ light chain genes with their respective signal peptide sequences and constant region genes within their cloning vectors. Gene amplification results are shown below. Figure 2 As shown, Heavy Chain 75 / 88 (89%), Kappa Chain (40 / 88, 45%), Lambda Chain (48 / 88, 54%).

[0051] 5. Construction of antibody expression plasmids

[0052] First, the gamma, kappa, or lambda cloning vectors were double-digested using restriction endonucleases Age I & Sal I, Age I & BsiWI, and Age I & Xho I, respectively. The cloning vector sequences are referenced 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). Digestion was performed at 37°C for 2 hours. The variable region gene products of the gamma, kappa, or lambda strands amplified in step 4 were purified and recovered using a standard DNA purification kit (TIANGEN, DP204-02). The double-digested cloning vector samples were subjected to 1% agarose gel electrophoresis; the vector length was approximately 5700 bp. Homologous recombination of the variable region gene fragment and the vector fragment was performed using the ClonExpress II One Step Cloning Kit (Vazyme, C112-01) at 37°C for 30 min. The recombination product was transformed into DH5α competent cells and evenly plated on plates containing Amp resistance, and incubated overnight at 37°C. The next day, three single clones from each plate were picked with a toothpick and placed in 500 μL of LB medium containing Amp resistance, and incubated at 37°C for 4–6 h. Colony PCR was performed using 2×Taq Master Mix (Vazyme, P112-01) to confirm successful insertion of the variable region into the vector. Positive clones were sequenced to confirm the accuracy of the variable region sequence.

[0053] 6. Small-batch expression of antibodies

[0054] The recombinant antibody plasmid was transfected into 293T cells for low-level expression. 24 hours before transfection, 293T cells were seeded into 96-well plates to maintain 90% confluence. The complete culture medium (DMEM + 10% FBS) in the 96-well plates was gently aspirated and replaced with serum-free medium (DMEM). A clean 96-well plate was prepared, and 30 ml of DMEM medium was mixed with 500 μL of PEI (1 mg / ml). This mixture was added to each well at a rate of 300 μL. Then, 10 ng each of light and heavy chains were added to the 96-well plates. The plates were incubated at room temperature for 15 min, and then added to the cells accordingly. After culturing at 37°C and 5% CO2 for 3 days, the cell supernatant was collected.

[0055] 7. Antibody ELISA specific screening

[0056] Dilute the N protein antigen to a final concentration of 1 μg / ml with carbonate coating buffer. Add 200 μl to each well and incubate overnight at 4°C. Wash the plate three times with 300 μl PBST for 3 min each time. Add 280 μl of 5% skim milk to each well and block at 37°C for 1 h. Wash the plate three times with 300 μl PBST for 3 min each time. Add 100 μl of cell supernatant to each well and incubate at 37°C for 40 min. Wash the plate three times with 300 μl PBST for 3 min each time. Add 100 μl of secondary antibody (5% skim milk diluted 1:10000) to each well and block at 37°C for 40 min. Wash the plate three times with 300 μl PBST for 3 min each time. Then, add 100 μl of TMB substrate to each well in the dark and incubate at room temperature for 10 min. Add 50 μl of 2M sulfuric acid to each well to stop the colorimetric reaction. Then, place the plate on a microplate reader and measure the OD450 value. The results are shown in Table 1. A total of 25 positive antibodies with OD>2 were screened from 88 samples. The results are shown in Table 2.

[0057] Table 2 Results of ELISA specificity screening for influenza A N protein

[0058]

[0059]

[0060] 8. Specific antibody expression

[0061] The selected specific antibodies were expressed on a small scale using 293F cells. The 293F cells were passaged one day before transfection, and on the day of transfection, cell viability was required to be >90%, and the cell density to be 3–4 × 10⁻⁴ cells / year. 6 cells / mL. Adjust the cell density to 1.0*10⁻⁶ cells / mL during transfection. 6 Cells / mL were transfected with PEI (1 mg / mL) and 80 μg plasmid DNA (H:L = 1:1) at room temperature for 10 min to form a cationic polymer (PEI:DNA = 3:1, mass ratio), which was then slowly added to 293F cells. Each antibody transfected 80 mL of cells, and the cell supernatant was collected for purification after 6 days. The cell supernatant after expression was purified using AT Protein ADiamond (Bestchrom, AA0272). The supernatant was washed with 10 mL of equilibration buffer, followed by antibody elution 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. The dialysate was changed every 2 h, and dialyzed 3 times. The purity and concentration of the antibody were identified by SDS-PAGE and UV absorption.

[0062] 9. Antibody pairing screening

[0063] Take 50 μg of each purified antibody and add 10 times the volume of starter solution and 50 μg of HRP sequentially. Incubate at 37°C for 2 hours. Add 1 / 10 volume of stop solution and incubate at room temperature for 1 hour. Dilute the 25 purified antibodies to a final concentration of 1 μg / ml, and add 100 μl to each well for coating. Incubate overnight at 4°C or 2 hours at 37°C. 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. Then dilute the N protein to a final concentration of 1 μg / ml and add 100 μl of diluted N protein to each well. 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 HRP-labeled antibody (diluted 1:10000 with 5% skim milk) to each well and block at 37°C for 40 minutes. Wash the plate three times with 300 μL PBST for 3 min each time. Then, add 100 μL of TMB substrate to each well in the dark and incubate at room temperature for 10 min. Add 50 μL of 2M sulfuric acid to each well and then place the plate on a microplate reader to measure the OD value at 450 nm. The best-paired antibodies were found to be 1B10 and 1E5, where 1B10 is the coating antibody and 1E5 is the labeling antibody.

[0064] The monoclonal antibody 1B10 was sequenced, and 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.19; 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.20.

[0065] This includes the variable region of the heavy chain and the variable region of the light chain;

[0066] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7.

[0067] SEQ ID NO.7:

[0068] AVQLLESGGGLVHPGGSLRLSCAASGFTFSFYWMSWVRQAPGKGLEW VASIKRDGSEKHYVDSVKGRFTISRDNGKTSVFLEMTSLRGEDTAVYYCARD NDGDFQPHYDVWGQGILVTVSS.

[0069] The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.11;

[0070] SEQ ID NO.11:

[0071] GCGGTGCAGTTGCTGGAGTCTGGGGGAGGCTTGGTCCACCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTTAGTTTCTATTGGATGTCGTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAGCATAAAGCGAGATGGAAGTGAGAAACACTATGT GGACTCTGTGAAGGGCCGATTCACCATTTCCAGGGACAACGGCAAGACTTCAGTCTTTCTGGAGATGACCAGTCTAAGAGGCGAGGACACGGCTGTTTATTACTGTGCGAGAGACAATGACGGTGACTTCCAACCTCACTATGACGTCTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAG.

[0072] Heavy chain variable region complementarity-determining region;

[0073] CDR1: Amino acid sequence GFTFSFYW (SEQ ID NO.1);

[0074] Nucleotide sequence GGATTCACTTTTAGTTTCTATTGG (SEQ ID NO.13);

[0075] CDR2: Amino acid sequence IKRDGSEK (SEQ ID NO.2);

[0076] Nucleotide sequence: ATAAAGCGAGATGGAAGTGAGAAA (SEQ ID NO.14); CDR3: amino acid sequence: ARDNDGDFQPHYDV (SEQ ID NO.3);

[0077] Nucleotide sequence: (SEQ ID NO.15)GCGAGAGACAATGACGGTGACTTCCAACCTCACTATGACGTC;

[0078] The amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8;

[0079] SEQ ID NO.8:

[0080] SYVLTQSPSVSVAPKTARITCGGNNIGDKRVHWYQKRSGQAPVMVMY FDQDRPSGIPERFSGSNSGNTATLTISRVEAVDEADYYCQVWDDNSGRMFG GGTKLTVL.

[0081] The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.12;

[0082] SEQ ID NO.12:

[0083] TCCTATGTGTTGACTCAGTCACCCTCAGTGTCAGTGGCCCCTGGAAAGACGGCCAGGATTACCTGTGGGGGAAACAACATTGGAGATAAACGTGTTCATTGGTACCAGAAGAGGTCAGGCCAGGCCCCTGTGATGGTCATGTATTTTGATCAGGACCGGCCCT CCGGCATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGTGGATGAGGCCGACTATTACTGTCAGGTCTGGGATGATAATAGTGGTCATCGGATGTTCGGCGGAGGGACCAAGCTGACCGTCCTAG.

[0084] Complementary determinant region of light chain variable region;

[0085] CDR1: Amino acid sequence NIGDKR (SEQ ID NO.4);

[0086] Nucleotide sequence AACATTGGAGATAAACGT (SEQ ID NO.16);

[0087] CDR2: Amino acid sequence FDQ (SEQ ID NO.5);

[0088] Nucleotide sequence TTTGATCAG (SEQ ID NO.17);

[0089] CDR3: Amino acid sequence QVWDDNSGHRM (SEQ ID NO.6);

[0090] Nucleotide sequence: CAGGTCTGGGATGATAATAGTGGTCATCGGATG (SEQ ID NO.18).

[0091] The monoclonal antibody 1E5 was sequenced, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.23; the encoding nucleotide sequence is shown in SEQ ID NO.39; the amino acid sequence of the light chain is shown in SEQ ID NO.24; and the encoding nucleotide sequence is shown in SEQ ID NO.40.

[0092] This includes the heavy chain variable region and the light chain variable region;

[0093] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.25;

[0094] SEQ ID NO.25:

[0095] QVQLVESGGGVVQPGRSLRLSCEASGLHFSTYAMHWVRQAPGKGLQW VAVISSDGTNKFYADSVTGRFTVSRDNSKNMLFLQADRLTTDDTAVYYCARD RFGHGERYHYFENWGQGTLVTVSS.

[0096] The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.41;

[0097] SEQ ID NO.41:

[0098] CAGGTGCAACTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGAAGCCTCTGGACTCCACTTCAGTACATACGCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGCAGTGGGTGGCAGTGATATCATCTGATGGAACTAATAAATTCTACGCAGA TTCCGTGACGGGCCGATTCACTGTCTCCAGAGACAATTCCAAGAATATGTTATTTCTACAAGCGGACAGACTGACAACTGACGACACGGCTGTATATTACTGTGCGAGAGATCGATTCGGGCACGGTGAGAGATATCACTACTTTGAAAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG.

[0099] Heavy chain variable region complementarity-determining region;

[0100] CDR1: Amino acid sequence GLHFSTYA (SEQ ID NO.27);

[0101] Nucleotide sequence: GGACTCCACTTCAGTACATACGCC (SEQ ID NO.33);

[0102] CDR2: Amino acid sequence ISSDGTNK (SEQ ID NO.1 = 28);

[0103] Nucleotide sequence: ATATCATCTGATGGAACTAATAAA (SEQ ID NO.34);

[0104] CDR3: Amino acid sequence ARDRFGHGERYHYFEN (SEQ ID NO.29);

[0105] Nucleotide sequence (SEQ ID NO.35)

[0106] GCGAGAGATCGATTCGGGCACGGTGAGAGATATCACTACTTTGAAAC.

[0107] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.26;

[0108] SEQ ID NO.26:

[0109] EVVLTQSPGTLSLSPGERATLSCRASQSIGSYLAWYQQKPGQAPRLLIYG ASNRATGIADRFTGSVSGTDFTLIISRMEPADFAVYYCHQYGNSRGTFGQGTK VEIK.

[0110] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.42;

[0111] SEQ ID NO.42:

[0112] GAAGTTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTGGCAGCTACTTAGCCTGGTACCAGCAGAAGCCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAATAG GGCCACTGGCATCGCAGACAGGTTCACTGGCAGTGTATCAGGGACAGACTTCACTCTCATCATCAGCAGAATGGAGCCTGCAGACTTTGCAGTGTATTACTGTCACCAATATGGAAACTCACGGGGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC.

[0113] Complementary determinant region of light chain variable region;

[0114] CDR1: Amino acid sequence QSIGSY (SEQ ID NO.30);

[0115] Nucleotide sequence CAGAGTATTGGCAGCTAC (SEQ ID NO.36);

[0116] CDR2: Amino acid sequence GAS (SEQ ID NO.31);

[0117] Nucleotide sequence GGTGCATCC (SEQ ID NO.37);

[0118] CDR3: Amino acid sequence HQYGNSRGT (SEQ ID NO.32);

[0119] Nucleotide sequence: CACCAATATGGAAACTCACGGGGGACG (SEQ ID NO.38).

[0120] 10. Performance testing of paired antibodies

[0121] Using the paired antibodies 1B10 and 1E5 obtained in the previous screening step, the coated antibody 1B10 was coupled to red polystyrene microspheres (Nanomicro, LDRNC-030). The specific coupling protocol was referenced in the microsphere instructions. The microspheres were then evenly added to the conjugation pad. The NC membrane was cut into strips, and the C-line and T-line were marked. 1E5 antibody was immobilized on the T-line of the NC membrane, and mouse anti-human specific IgG antibody was immobilized on the C-line. Immunochromatographic detection was performed on the positive control sample (ZeptoMetrix, NATFLUAH1N1-6MC) for influenza A (2009H1N1 pdm). The colorimetric card is shown below. Figure 3 As shown in Table 3, the results are as follows.

[0122] Table 3. Results of positive quality control samples

[0123]

[0124]

[0125] From Table 3 and Figure 3 It can be seen that the positive control sample, diluted 20,000 times, can still effectively detect the virus, indicating that the antibody has high sensitivity.

[0126] Immunochromatographic plates prepared using paired antibodies 1B10 and 1E5 were compared with plates made using a commercially available mouse antibody against the N protein of influenza A to detect clinical samples. A total of 43 positive samples and 25 negative samples were detected. The results of parallel comparisons are shown in Table 4.

[0127] Table 4. Results of clinical sample testing for paired antibodies against influenza A.

[0128]

[0129] As shown in Table 4, the overall concordance rate of the paired antibodies 1B10 and 1E5 for positive samples was 97.7%, and the overall concordance rate for negative samples was 100%. The overall concordance rate of the control antibody pair for positive samples was 93.0%, and the overall concordance rate for negative samples was 92%. This indicates that the 1B10 and 1E5 antibody pairs are significantly superior to the control mouse antibody pair in terms of detection specificity and anti-interference ability.

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0131] 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 indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0132] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A human monoclonal antibody against influenza A N protein, characterized in that, It includes variable regions for light chains and variable regions for heavy chains; The variable region of the heavy chain includes complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3; the variable region of the light chain includes complementarity-determining regions Vλ-CDR1, Vλ-CDR2, and Vλ-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 Vλ-CDR1 is shown in SEQ ID NO.4, the amino acid sequence of Vλ-CDR2 is FDQ, and the amino acid sequence of Vλ-CDR3 is shown in SEQ ID NO.

6.

2. The human monoclonal antibody against influenza A N protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the anti-influenza N protein human monoclonal antibody 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 human monoclonal antibody against influenza A N protein according to claim 1, characterized in that, The sequence of the anti-influenza N protein human monoclonal antibody is derived from a human sequence.

4. A nucleic acid, characterized in that, The nucleic acid encodes the human monoclonal antibody against influenza A N protein as described in any one of claims 1-3.

5. The nucleic acid according to claim 4, characterized in that, The nucleotide sequence used to encode the variable region of the heavy chain is shown in SEQ ID NO.11; the nucleotide sequence used to encode the variable region of the light chain is shown in SEQ ID NO.

12.

6. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid as described in claim 4 or 5; the biomaterial is an expression cassette, vector, or transgenic cell.

7. The method for preparing the human monoclonal antibody against influenza A N protein according to any one of claims 1-3, characterized in that, Gene expression is performed using the nucleic acid described in claim 4 or 5; or the transgenic cells described in claim 6 are cultured.

8. A reagent, characterized in that, The reagent is used to detect influenza A N protein, and the reagent includes the anti-influenza A N protein human monoclonal antibody according to any one of claims 1-3, or the nucleic acid according to claim 4 or 5, or the biological material according to claim 6.

9. A reagent kit, characterized in that, The kit comprises the human monoclonal antibody against influenza A N protein as described in any one of claims 1-3, or the nucleic acid as described in claim 4 or 5, or the biological material as described in claim 6, or the reagent as described in claim 8; the kit is used to detect influenza A N protein.

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