Anti-influenza virus high-sensitivity antibody, and preparation method and application thereof

Abstract

The application discloses an anti-influenza virus high-sensitivity antibody and a preparation method and application thereof. The application provides an antibody for binding to an influenza virus, named VHH108-Fc-Nluc antibody, which comprises three segments, namely, a VHH108 antibody, Fc and NLuc luciferase; the VHH108 antibody has a segment shown in positions 3-125 in SEQ ID NO:1. The application further provides a kit for detecting the influenza virus, which comprises the VHH108-Fc-Nluc antibody and a VHH57 antibody. The VHH57 antibody has a segment shown in positions 230-350 in SEQ ID NO:3. The application is suitable for detecting various subtypes of influenza A viruses, has the advantages of high sensitivity and strong specificity, and has market value and application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and relates to an anti-influenza virus high-sensitivity antibody and a preparation method and application thereof. BACKGROUND

[0002] Influenza virus is a single-stranded negative-sense RNA virus with an envelope belonging to the Orthomyxoviridae family; it is spherical in appearance, about 80-120 nm in diameter, and can be divided into three parts from the outside in structure: envelope, matrix protein (M protein), and core protein (NP protein); it is composed of 7-8 RNA gene fragments encoding different proteins in terms of genetic composition; and it can be divided into four virus types, including type A (also referred to as type A), type B (also referred to as type B), type C (also referred to as type C), and type D, according to the antigenic differences of the nucleoprotein and matrix protein.

[0003] Among the above influenza virus types, type A influenza virus is the most harmful to humans, poultry, and mammals, and is the most common seasonal influenza virus, which is easy to cause a pandemic; type A influenza virus can be divided into different subtypes according to the antigenic differences of hemagglutinin (HA) and neuraminidase (NA) embedded on the surface of the virus particle; as of now, type A influenza virus is divided into 18 HA subtypes and 11 NA subtypes; the most common type A influenza virus subtypes include H1N1, H3N2, H5N1, H7N9, and H9N2, etc.

[0004] Most type A influenza viruses are transmitted in birds, and some subtypes can be transmitted across species; currently, the seasonal type A influenza viruses transmitted in humans are mostly H1N1 and H3N2 subtypes, and high-pathogenicity avian influenza viruses such as H7N9 and H5N1 also cause fatal infections in humans. Therefore, it is of great significance to establish a detection technology that can detect multiple subtypes of type A influenza virus for the control of infection of the virus.

[0005] Antigen detection is an important technical means for identifying type A influenza virus. Specific detection methods include enzyme-linked immunosorbent assay, immunocolloidal gold technology, and immunofluorescence, etc. Regardless of which technical method, the preparation of sensitive and specific antibodies is a prerequisite for the development of detection technology.

[0006] The traditional antibody preparation method is mainly hybridoma antibody preparation technology, which uses specific antigen to immunize mice, stimulates B lymphocyte activation, proliferation and differentiation into sensitized lymphocytes, collects mouse spleen cells, and fuses and monoclonalizes myeloma cells to screen and isolate cell strains that can secrete specific monoclonal antibodies. However, during the subculture of the hybridoma cell strain, the antibody gene often has the risk of loss. Although the molecular biology technology can realize the gene expression and intracellular culture of the hybridoma antibody, it can overcome the problem of antibody gene loss, but the in vitro expression of the antibody gene needs to be carried out in eukaryotic cells, and the expression cost is high and the expression amount is low, so the antibody preparation technology needs to be improved.

[0007] Single-chain antibody (VHH antibody), also known as nanobody, is a new type of antibody found in alpaca. Although the nanobody is smaller than the traditional antibody IgG in volume, the complementarity determining regions CDR1 and CDR3 of the nanobody are longer than the CDR regions of the traditional antibody in structure, can penetrate into the interior of the antigen, and can be combined with the hidden target binding site that cannot be combined by the traditional antibody; and then the antigen binding affinity of the nanobody is increased. The nanobody has the advantages of small molecular weight, simple structure, no glycosylation and other modification sites, and can be expressed in a large amount in a prokaryotic system, and the preparation cost is low. This solves the problem of high preparation cost of the traditional antibody. In summary, compared with the traditional antibody, the nanobody has the advantages of small molecular weight (about 12-15 kDa), high chemical resistance, good stability, high affinity, easy expression and the like. Developing the nanobody as a detection antibody is expected to develop an antigen detection method with higher sensitivity. SUMMARY

[0008] The purpose of the present application is to provide an anti-influenza virus antibody with high sensitivity and a preparation method and application thereof.

[0009] The present application provides an antibody binding to influenza virus, named VHH108-Fc-Nluc antibody, comprising the following three segments: VHH108 antibody, Fc and NLuc luciferase; the VHH108 antibody has the segment shown in SEQ ID NO: 1 3-125.

[0010] Specifically, the VHH108-Fc-Nluc antibody comprises the following three segments in turn from N-terminal to C-terminal: VHH108 antibody, Fc and NLuc luciferase.

[0011] Specifically, the VHH108 antibody is shown in SEQ ID NO: 7 2-124.

[0012] Specifically, the Fc is shown in SEQ ID NO: 7 125-355.

[0013] Specifically, the NLuc luciferase is shown in SEQ ID NO: 7 at positions 371-540.

[0014] Specifically, the VHH108-Fc-Nluc antibody is shown in SEQ ID NO: 7.

[0015] The present application provides an antibody binding to influenza virus, named VHH108-Fc antibody, comprising two segments: VHH108 antibody and Fc; the VHH108 antibody has a segment shown in SEQ ID NO: 1 at positions 3-125.

[0016] Specifically, the VHH108-Fc antibody comprises two segments in order from N-terminus to C-terminus: VHH108 antibody and Fc.

[0017] Specifically, the VHH108 antibody is shown in SEQ ID NO: 5 at positions 2-124.

[0018] Specifically, the Fc is shown in SEQ ID NO: 5 at positions 125-355.

[0019] Specifically, the VHH108-Fc antibody is shown in SEQ ID NO: 5.

[0020] The present application provides an antibody binding to influenza virus, named VHH108 antibody, which has a segment shown in SEQ ID NO: 1 at positions 3-125.

[0021] Specifically, the VHH108 antibody is shown in SEQ ID NO: 1 at positions 3-125.

[0022] Specifically, the VHH108 antibody is shown in SEQ ID NO: 1 at positions 1-125.

[0023] Specifically, the VHH108 antibody further has a protein purification tag.

[0024] The protein purification tag is located at the N-terminus or C-terminus.

[0025] Illustratively, the protein purification tag is a flag tag.

[0026] Specifically, the VHH108 antibody is shown in SEQ ID NO: 1.

[0027] The present application provides an antibody binding to influenza virus, named VHH57 antibody, which has a segment shown in SEQ ID NO: 3 at positions 230-350.

[0028] Specifically, the VHH57 antibody is as shown in SEQ ID NO: 3 at positions 230-350.

[0029] Specifically, the VHH57 antibody further has a protein purification tag.

[0030] The protein purification tag is located at the N-terminus or C-terminus.

[0031] Illustratively, the protein purification tag is a GST tag.

[0032] Specifically, the VHH57 antibody is as shown in SEQ ID NO: 3.

[0033] The application also protects the use of any of the above-mentioned VHH108-Fc-Nluc antibodies in the preparation of a kit for detecting influenza virus.

[0034] The application also protects the use of any of the above-mentioned VHH108-Fc antibodies in the preparation of a kit for detecting influenza virus.

[0035] The application also protects the use of any of the above-mentioned VHH108 antibodies in the preparation of a kit for detecting influenza virus.

[0036] The application also protects the use of any of the above-mentioned VHH57 antibodies in the preparation of a kit for detecting influenza virus.

[0037] The application also protects the use of any of the above-mentioned VHH108-Fc-Nluc antibodies and any of the above-mentioned VHH57 antibodies in the preparation of a kit for detecting influenza virus.

[0038] The application also protects the use of any of the above-mentioned VHH108-Fc antibodies and any of the above-mentioned VHH57 antibodies in the preparation of a kit for detecting influenza virus.

[0039] The application also protects the use of any of the above-mentioned VHH108 antibodies and any of the above-mentioned VHH57 antibodies in the preparation of a kit for detecting influenza virus.

[0040] The application also provides a kit for detecting influenza virus, comprising a VHH108-Fc-Nluc antibody and / or a VHH108-Fc antibody and / or a VHH108 antibody and / or a VHH57 antibody.

[0041] Specifically, the kit comprises a VHH108-Fc-Nluc antibody and a VHH57 antibody.

[0042] Specifically, the kit comprises a VHH108-Fc antibody and a VHH57 antibody.

[0043] Specifically, the kit comprises the VHH108 antibody and the VHH57 antibody.

[0044] The present application also protects antibody-related biological materials.

[0045] The antibody-related biological materials are VHH108-Fc-Nluc antibody-related biological materials and / or VHH108-Fc antibody-related biological materials and / or VHH108 antibody-related biological materials and / or VHH57 antibody-related biological materials.

[0046] The present application also protects the use of antibody-related biological materials in the preparation of a kit for detecting influenza viruses.

[0047] The antibody-related biological materials are VHH108-Fc-Nluc antibody-related biological materials and / or VHH108-Fc antibody-related biological materials and / or VHH108 antibody-related biological materials and / or VHH57 antibody-related biological materials.

[0048] The present application also provides a kit for detecting influenza viruses, comprising VHH108-Fc-Nluc antibody-related biological materials and / or VHH108-Fc antibody-related biological materials and / or VHH108 antibody-related biological materials and / or VHH57 antibody-related biological materials.

[0049] Any of the above-mentioned related biological materials is a nucleic acid molecule expressing an antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule.

[0050] The VHH108-Fc-Nluc antibody-related biological materials are nucleic acid molecules expressing VHH108-Fc-Nluc antibodies, expression cassettes, recombinant vectors, recombinant microorganisms, or recombinant cells. Specifically, the nucleic acid molecule is shown in SEQ ID NO: 8, positions 7-1629. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between AflII and EcoRI enzyme recognition sites in the vector pcDNA3.1(+) with a double-stranded DNA molecule shown in SEQ ID NO: 8, positions 7-1629. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21(DE3).

[0051] The VHH108-Fc antibody-related biological material is a nucleic acid molecule expressing the VHH108-Fc antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule. Specifically, the nucleic acid molecule is as shown in SEQ ID NO: 6 from the 7th to the 1074th position. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between the AflII and EcoRI enzyme recognition sites in the vector pcDNA3.1(+) with a double-stranded DNA molecule as shown in SEQ ID NO: 6 from the 7th to the 1072nd position. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21(DE3).

[0052] The VHH108 antibody-related biological material is a nucleic acid molecule expressing the VHH108 antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule. Specifically, the nucleic acid molecule is as shown in SEQ ID NO: 4 from the 1st to the 1053rd position. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between the EcoRI and XhoI enzyme recognition sites in the vector pGEX-4T-1 with a double-stranded DNA molecule as shown in SEQ ID NO: 4 from the 688th to the 1053rd position. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21(DE3).

[0053] The VHH57 antibody-related biological material is a nucleic acid molecule expressing the VHH57 antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule. Specifically, the nucleic acid molecule is as shown in SEQ ID NO: 2 from the 3rd to the 404th position. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between the NcoI and XhoI enzyme recognition sites in the vector pET-28a(+) with a double-stranded DNA molecule as shown in SEQ ID NO: 2 from the 7th to the 404th position. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21(DE3).

[0054] Fc, i.e., antibody FC fragment, also known as antibody constant region.

[0055] Any of the above-mentioned kits is an ELISA kit.

[0056] Any of the above-mentioned kits is an ELISA kit based on a double-antibody sandwich method for detecting influenza virus.

[0057] In the kit, the VHH108-Fc-Nluc antibody serves as a detection antibody.

[0058] The VHH108-Fc antibody is a biotin-labeled VHH108-Fc antibody.

[0059] In the kit, the VHH108-Fc antibody serves as a detection antibody.

[0060] The VHH108 antibody is a biotin-labeled VHH108 antibody.

[0061] In the kit, the VHH108 antibody serves as a detection antibody.

[0062] The VHH57 antibody is a biotin-labeled VHH57 antibody.

[0063] In the kit, the VHH57 antibody serves as a detection antibody.

[0064] In the kit, the VHH57 antibody serves as a capture antibody.

[0065] Any of the above influenza viruses can be an influenza A virus (type A influenza virus).

[0066] Any of the above influenza viruses can be an N1 subtype influenza virus, an N2 subtype influenza virus, or an N9 subtype influenza virus.

[0067] Any of the above influenza viruses can be an H1 subtype influenza virus, an H3 subtype influenza virus, an H5 subtype influenza virus, or an H7 subtype influenza virus.

[0068] Any of the above influenza viruses can be an H1N1 subtype influenza virus, an H3N2 subtype influenza virus, an H5N1 subtype influenza virus, or an H7N9 subtype influenza virus.

[0069] In response to the current situation of insufficient detection reagents for influenza A antigens that can detect multiple subtypes, the present invention has developed and screened nanoantibodies VHH108 and VHH57 that can bind to multiple subtypes of influenza viruses. Furthermore, in the development of a double-antibody sandwich ELISA test, it was found that when VHH nanoantibodies were used as capture antibodies, they showed a more sensitive detection effect. However, the sensitivity of nanoantibodies as detection antibodies is not high, significantly lower than that of traditional IgG antibodies. The main reason is that small-molecule nanoantibodies are not as structurally stable as long-fragment nanoantibodies. Furthermore, the present invention optimized the structure of the VHH108 nanoantibody. By recombinantly expressing the Fc fragment at the end of the antibody, the two VHH antibodies spontaneously formed a disulfide bond through the cysteine ​​residues on the Fc after expression, thereby forming a bivalent VHH-FC antibody. The results of affinity analysis showed that the modified VHH108-FC antibody had a 10-fold increase in affinity compared to the original VHH108 nanoantibody; in the double-antibody sandwich ELISA experiment, the detection sensitivity was also significantly improved. Furthermore, the present invention recombined the sensitive detection marker Nluc at the C-terminus of the VHH-FC antibody to establish a highly sensitive Nluc nanobody ELISA, which has a detection sensitivity 100 times higher than that of traditional ELISA.

[0070] The present invention is applicable to the detection of multiple subtypes of influenza A viruses, has the advantages of high sensitivity and strong specificity, and has market value and promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is the result diagram of electrophoresis detection in step 6 of Example 1.

[0072] Figure 2 Detect OD for step 1 of Example 2 450 Result graph of the values.

[0073] Figure 3 Detect OD for step 2 of Example 2 450 Result graph of the value (NP antigen of H1 subtype influenza virus).

[0074] Figure 4 Detect OD for step 2 of Example 2 450 Result graph of the values ​​(NP antigen of H3 subtype influenza virus).

[0075] Figure 5 Detect OD for step 2 of Example 2 450 Result graph of the value (NP antigen of H5 subtype influenza virus).

[0076] Figure 6 Detect OD for step 2 of Example 2 450 Result graph of the value (NP antigen of H7 subtype influenza virus).

[0077] Figure 7 Figure of results for affinity detection for step three of Example 2 (VHH108 antibody).

[0078] Figure 8 Figure of results for affinity detection for step three of Example 2 (VHH108-Fc antibody).

[0079] Figure 9 Figure of results for OD detection for Example 3. 450

[0080] Figure 10 Figure of results for OD detection for step one of Example 4. 450

[0081] Figure 11 Figure of results for OD detection for step two of Example 4. 450

[0082] Figure 12 Figure of results for affinity detection for step three of Example 4.

[0083] Figure 13 Figure of results for OD detection for Example 5. 450

[0084] Figure 14 Figure of results for fluorescence value detection for Example 6. DETAILED DESCRIPTION

[0085] The present application will be further described in conjunction with the specific embodiments, which are presented only for the purpose of illustrating the present application and not for the purpose of limiting the same. The following examples are provided as a guide to further improve the present application for those skilled in the art, and do not in any way constitute a limitation on the present application.

[0086] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified. The quantitative tests in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified. Fc: antibody constant region.

[0087] COS7 cells, also known as COS-7 cells, are commercially available cell lines derived from African green monkey kidney fibroblasts and transformed by SV40 virus genes. EZ-Link TM ​​​​Sulfo-NHS Biotinylation Kit: Thermo Scientific, Cat# 21425. Coating solution: Beijing Solabio Technology Co., Ltd., Cat# C1055. HRP-labeled anti-Flag antibody: Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 100233-MM01-H. HRP-labeled anti-His antibody: Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 105327-MM02T-H. MDCK cells (dog kidney cells): ATCC, Cat# CCL-34. TMB substrate solution: Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat# PA107-01. Horseradish peroxidase-labeled streptavidin (product specification: liquid, 1 mg / ml): Beijing Boao Sun Biotechnology Co., Ltd., Cat# bs-0437p-HRP.

[0088] H1N1-NP protein, full name: Influenza A H1N1 (A / Guangdong-Maonan / SWL1536 / 2019) Nucleoprotein / NP Protein (His Tag): Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 40723-V08B. H3N2-NP protein, full name: Influenza A H3N2 (A / Hong Kong / 2671 / 2019) Nucleoprotein / NP Protein (His Tag): Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 40753-V08B. H5N1-NP protein, full name: Influenza A H5N1 (A / Cambodia / NPH230032 / 2023) Nucleoprotein / NP Protein (His Tag): Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 40947-V08B. H7N9-NP protein, full name: Influenza A H7N9 (A / Anhui / 1-BALF_RG6 / 2013) Nucleoprotein / NP Protein (His Tag): Beijing Yiqioshenzhou Technology Co., Ltd., Cat# 40110-V08B.

[0089] Example 1, Preparation of Antibodies

[0090] I. Construction of Recombinant Plasmid

[0091] The small fragment between Ncol and Xhol enzyme recognition sites in the vector pET-28a(+) was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 2, to obtain the recombinant plasmid pET28a-VHH108. The recombinant plasmid pET28a-VHH108 has the DNA molecule shown in SEQ ID NO: 2. The nucleotides 3-404 in SEQ ID NO: 2 constitute an open reading frame, which encodes the protein shown in SEQ ID NO: 1 (named VHH108-Flag fusion protein, i.e. VHH108 antibody with flag tag at C-terminus, referred to as VHH108 antibody). In SEQ ID NO: 1, the amino acid residues 3-125 constitute the VHH108 antibody, and the amino acid residues 126-133 constitute the flag tag.

[0092] The small fragment between EcoRI and Xhol enzyme recognition sites in the vector pGEX-4T-1 was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 4, to obtain the recombinant plasmid pGEX-4T-1-VHH57. The recombinant plasmid pGEX-4T-1-VHH57 has the DNA molecule shown in SEQ ID NO: 4. The nucleotides 1-1053 in SEQ ID NO: 4 constitute an open reading frame, which encodes the protein shown in SEQ ID NO: 3 (named GST-VHH57 fusion protein, i.e. VHH57 antibody with GST tag at N-terminus, referred to as VHH57 antibody). In SEQ ID NO: 3, the amino acid residues 1-229 constitute the GST tag, and the amino acid residues 230-350 constitute the VHH57 antibody.

[0093] The small fragment between AflII and EcoRI enzyme recognition sites in the vector pcDNA3.1(+) was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 6, to obtain the recombinant plasmid pcDNA3.1-VHH108-Fc. The recombinant plasmid pcDNA3.1-VHH108-Fc has the DNA molecule shown in SEQ ID NO: 6. In SEQ ID NO: 6, the nucleotides 7-1074 constitute an open reading frame, which encodes the protein shown in SEQ ID NO: 5 (named VHH108-Fc fusion protein, also referred to as VHH108-Fc antibody). In SEQ ID NO: 5, the amino acid residues 2-124 constitute the VHH108 antibody, and the amino acid residues 125-355 constitute the Fc.

[0094] The small fragment between the AflII and EcoRI enzyme recognition sites in the vector pcDNA3.1(+) was replaced with the double-stranded DNA molecule shown in SEQ ID NO: 8 at positions 7-1629 to obtain the recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc. The recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc has the DNA molecule shown in SEQ ID NO: 8. In SEQ ID NO: 8, nucleotides at positions 7-1629 constitute an open reading frame, which encodes the protein shown in SEQ ID NO: 7 (named VHH108-Fc-Nluc fusion protein, also referred to as VHH108-Fc-Nluc antibody). In SEQ ID NO: 7, amino acid residues at positions 2-124 constitute the VHH108 antibody, amino acid residues at positions 125-355 constitute the Fc, and amino acid residues at positions 371-540 constitute the NLuc luciferase.

[0095] II. Expression and purification of VHH108 antibody

[0096] 1. The recombinant plasmid pET28a-VHH108 was introduced into E. coli BL21(DE3) competent cells to obtain a recombinant bacterium.

[0097] 2. The recombinant bacterium obtained in step 1 was inoculated into liquid LB medium and cultured at 37°C with shaking at 200 rpm until the system OD 600nm value was 0.6, then IPTG was added to a concentration of 1 mM, and then the culture was continued at 37°C with shaking at 200 rpm for 4-5 hours.

[0098] 3. After step 2 was completed, the bacterial cells were collected, the cells were broken, the supernatant was collected, and protein purification was performed using Anti-Flag affinity purification gel (Yixing Biotech (Shanghai) Co., Ltd., product catalog number 20584ES08, according to the instructions). Then, the ultrafiltration tube was used for concentration and buffer exchange (so that the buffer system was PBS buffer), to obtain an antibody solution, which was named VHH108 antibody solution.

[0099] III. Expression and purification of VHH57 antibody

[0100] 1. The recombinant plasmid pGEX-4T-1-VHH57 was introduced into E. coli BL21(DE3) competent cells to obtain a recombinant bacterium.

[0101] 2. The recombinant bacterium obtained in step 1 was inoculated into liquid LB medium and cultured at 37°C with shaking at 200 rpm until the system OD 600nm value was 0.6, then IPTG was added to a concentration of 1 mM, and then the culture was continued at 37°C with shaking at 200 rpm for 4-5 hours.

[0102] 3、After step 2, collect the bacteria, and then collect the supernatant after the bacteria are broken. Then, use Anti-GST affinity purification gel (Yixing Biotech (Shanghai) Co., Ltd., product catalog number 20507ES10, and follow the instructions) to purify the protein. Then, use ultrafiltration tube to concentrate and change the buffer (so that the buffer system is PBS buffer), to obtain the antibody solution, which is named VHH57 antibody solution.

[0103] Four, expression and purification of VHH108-Fc antibody

[0104] 1、COS7 cells are inoculated into a 24-well plate and cultured in DMEM medium containing 10% FBS to a confluence of 80%-90%. Then, the recombinant plasmid pcDNA3.1-VHH108-Fc is transfected (with the help of Lipofectamine 3000 transfection reagent and following the instructions) and cultured for 3-4 days. Then, the supernatant is collected. TM

[0105] 2、Take 20 mL of the supernatant obtained in step 1, dilute it to 200 mL with PBS buffer, and then add 20 mg of PureProteome Protein A / G Mix Magnetic Beads (Millipore, product catalog number LSKMAGAG10). Incubate at room temperature for 60 min in a rotary shaker at 80 rpm.

[0106] 3、After step 2, collect the magnetic beads, wash them with PBST solution, and then add 2 mL of pH 3.0 citrate buffer to the magnetic beads. Mix well by vortexing for 5 min, then discard the magnetic beads and collect the supernatant.

[0107] 4、Take the supernatant obtained in step 3, add 1M Tris buffer to adjust the pH to 7.0, and then obtain the antibody solution, which is named VHH108-Fc antibody solution.

[0108] When the recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc is used in the above steps, the obtained antibody solution is named VHH108-Fc-Nluc antibody solution.

[0109] Five, expression and purification of VHH108-Fc-Nluc antibody

[0110] Replace the recombinant plasmid pcDNA3.1-VHH108-Fc with the recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc, and follow the same steps as step four. Obtain the antibody solution, which is named VHH108-Fc-Nluc antibody solution.

[0111] Six, electrophoresis detection​

[0112] The test samples were: VHH108 antibody solution prepared in Step Two, VHH57 antibody solution prepared in Step Three, VHH108-Fc antibody solution prepared in Step Four, or VHH108-Fc-Nluc antibody solution prepared in Step Five.

[0113] 1. Reducing SDS-PAGE

[0114] 20 μL of sample was taken, 5 μL of 5x protein loading buffer (Beijing Jin Pulei Biological Technology Co., Ltd., product catalog number P06M18) was added, and boiled for 10 min, and then loaded after cooling. The sample and standard protein Marker (molecular weight 10-170 kDa; Thermo Scientific, product catalog number 26616) were both loaded at 10 μL. A 15% separation gel (Shanghai Shenguo Biological Engineering Co., Ltd., product catalog number C651103-0001) was used. During electrophoresis, the concentrated gel condition was set at 80 V for 30 min, and the separation gel condition was set at 120 V for 40 min. After electrophoresis, Protein Show-G250 protein rapid staining reagent (Kangwei Reagent, product catalog number CW0023S, according to the instructions) was used for operation, and then a gel imaging instrument was used to collect information.

[0115] The reducing SDS-PAGE result can destroy the disulfide bond between the peptide segments, so as to accurately display the protein molecular weight. The reducing SDS-PAGE electropherogram is shown in Figure 1 A of FIG. 1. Figure 1 A of FIG. 1: Lane M: Maker; Lane 1: VHH108-Fc (44 kDa); Lane 2: VHH108-Fc-Nluc (65 kDa); Lane 3: VHH57 (39 kDa); Lane 4: VHH108 (15 kDa). The results show that the band size is consistent with the expected molecular weight of the antibody.

[0116] 2. Non-reducing SDS-PAGE

[0117] Take 10 μL, add 10 μL 2x Native protein loading buffer (Shanghai Genechem Co., Ltd., product catalog number C506025-0003). The sample loading amount is 20 μL, and the standard protein Marker (molecular weight 10-170 kDa; Thermo Scientific, product catalog number 26616) loading amount is 10 μL. 12% separation gel (Shanghai Genechem Co., Ltd., product catalog number C651102-0001) is used. During electrophoresis, the concentrated gel condition is set to 80V, 30min, and the separation gel condition is set to 120V, 40min. After electrophoresis, Protein Show-G250 protein rapid staining reagent (Kangwei reagent product, product catalog number CW0023S, according to the instruction) is used for operation, and then a gel imaging instrument is used to collect information.

[0118] Non-reducing SDS-PAGE gel electrophoresis maintains the natural structure of proteins, does not destroy the disulfide bond structure between peptide segments, and can reflect the size of multimeric proteins in solution. The non-reducing SDS-PAGE electropherogram is shown in Figure 1 B. Figure 1 In B of the above table: Lane M: Maker; Lane 1: VHH108-Fc (88kDa); Lane 2: VHH108-Fc-Nluc (130kDa); Lane 3: VHH57 (39kDa); Lane 4: VHH108 (15kDa). The protein band size of VHH108 is consistent with the band size in reducing SDS-PAGE, indicating that the naturally occurring form of VHH108 is a monomer. The protein band size of VHH57 is consistent with the band size in reducing SDS-PAGE, indicating that the naturally occurring form of VHH57 is a monomer. The molecular weight of VHH108-Fc and VHH108-Fc-Nluc in non-reducing SDS-PAGE is twice that in reducing SDS-PAGE, indicating that the naturally occurring form of VHH108-Fc and VHH108-Fc-Nluc is a dimer. The results show that the antibody in the form of a dimer is prepared by connecting Fc to the C-terminal of VHH108 antibody.

[0119] Seven, biotin labeling of VHH108 antibody and VHH108-Fc antibody

[0120] Take the VHH108 antibody solution prepared in step two, and use the EZ-Link TM Sulfo-NHS Biotinylation Kit according to the instruction to obtain biotin-labeled VHH108 antibody.

[0121] Take the VHH57 antibody solution prepared in step three, and use the EZ-Link TMThe Sulfo-NHS Biotinylation Kit was operated according to the instructions to obtain the biotin-labeled VHH57 antibody.

[0122] The VHH108-Fc antibody solution prepared in Step Four was used to prepare the biotin-labeled VHH108-Fc antibody according to the EZ-Link Sulfo-NHS Biotinylation Kit. TM The Sulfo-NHS Biotinylation Kit was operated according to the instructions to obtain the biotin-labeled VHH108-Fc antibody.

[0123] Example 2, Comparison of the binding activity of VHH108 antibody and VHH108-Fc antibody

[0124] I. Comparison of the capture effect of the capture antibody

[0125] The capture antibody was respectively VHH108 antibody (provided by the VHH108 antibody solution prepared in Example 1) or VHH108-Fc antibody (provided by the VHH108-Fc antibody solution prepared in Example 1).

[0126] The antigen was respectively H1N1-NP protein or H3N2-NP protein.

[0127] The 96-well ELISA microplate was taken, 100 μL of coating solution containing 2 μg / mL of capture antibody was added to each well, and coated overnight at 4°C. Then, blocking was performed. Then, 100 μL of antigen diluent (the concentration of antigen in the antigen diluent was 8 μg / mL-0.000512 μg / mL, 5-fold gradient) was added to each well, incubated at 37°C for 1.5 h, and washed. Then, 100 μL of HRP-labeled anti-His antibody was added to each well, incubated at 37°C for 1.5 h, and washed. Then, TMB substrate solution was added for color development, and the OD 450 value was determined.

[0128] The OD 450 >0.2 was used as the cut off value for judging as positive, and the lowest concentration of multi-subtype influenza virus NP antigen that could be detected by the antibody was compared. The lower the detectable antigen concentration, the higher the sensitivity of the antibody.

[0129] The results are shown in Figure 2 . The two forms of antibodies were respectively used as capture antibodies to detect NP antigen, and the results were similar. The lowest detection limit for H1N1-NP antigen was 0.32 μg / mL, and the lowest detection limit for H3N2-NP antigen was 0.0128 μg / mL.

[0130] II. Comparison of the detection effect of the detection antibody

[0131] The detection antibody was respectively biotin-labeled VHH108 antibody prepared in Example 1 or biotin-labeled VHH108-Fc antibody prepared in Example 1.

[0132] Take 96-well ELISA microplates, add 100 μL of coating solution containing 1 μg / mL of antigen (antigens are H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein, respectively) to each well, and coat at 4°C overnight. Then, blocking is performed. Then, add 100 μL of antibody diluent to each well (the concentration of the detection antibody in the antibody diluent is 8 μg / mL-0.000512 μg / mL, with a 5-fold gradient), incubate at 37°C for 1.5 h, and wash. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 1 h, and wash. Then, add TMB substrate solution for color development, and measure OD 450 values.

[0133] Take 96-well ELISA microplates, add 100 μL of coating solution containing 1 μg / mL of antigen (antigens are H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein, respectively) to each well, and coat at 4°C overnight. Then, blocking is performed. Then, add 100 μL of antibody diluent to each well (the concentration of the detection antibody in the antibody diluent is 8 μg / mL-0.000512 μg / mL, with a 5-fold gradient), incubate at 37°C for 1.5 h, and wash. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 1 h, and wash. Then, add TMB substrate solution for color development, and measure OD 450 >0.2 as the cut-off value for judging positive, the lowest concentration of antibody that can be detected as positive is compared, and the lower the concentration of antibody that can be detected as positive, the higher the binding activity of the antibody.

[0134] The results are shown in Figure 3 , Figure 4 , Figure 5 and Figure 6 . Using NP antigens of H1, H3, H5 and H7 subtypes of influenza virus for detection, both VHH108 antibody and VHH108-Fc antibody as detection antibodies can bind, indicating that the antibody can detect multiple subtypes of influenza virus. Taking OD 450 >0.2 as the cut-off value for judging ELISA results. The results show that the lowest concentration of biotin-labeled VHH108-Fc antibody for detecting NP antigen is 0.00256 μg / ml, while the lowest concentration of biotin-labeled VHH108 antibody for detecting NP antigen is 0.0128 μg / ml, indicating that biotin-labeled VHH108-Fc antibody exhibits higher binding activity.

[0135] III. Affinity Comparison

[0136] The tested antibodies are VHH108 antibody (provided by the VHH108 antibody solution prepared in Example 1) or VHH108-Fc antibody (provided by the VHH108-Fc antibody solution prepared in Example 1).

[0137] The Gator protein characterization analysis system was used to detect the affinity of the test antibody to the NP antigen of influenza A virus (H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein, respectively). NP antigen diluted with Q Buffer (NP antigen concentration of 2 μg / mL) was added to the test hole of the low adsorption special plate, and only Q Buffer was added to the blank hole. Then 2-fold serially diluted test antibody (test antibody concentration of 10 μg / mL-0.3125 μg / mL) was added, and the prepared plate was placed in the Gator protein characterization analysis system instrument for affinity determination and calculation of the affinity dissociation constant. The affinity of the antibody to the antigen was determined by comparing the size of the affinity dissociation constant KD value, and the smaller the KD value, the greater the affinity of the antibody.

[0138] Q Buffer: containing 0.02% Tween 20, 0.2% BSA and 0.05% NaN3, and the rest is PBS buffer (pH 7.4).

[0139] The results of VHH108 antibody are shown in Figure 7 The results of VHH108-Fc antibody are shown in Figure 8 The affinity is shown in Table 1.

[0140] Compared with VHH108 antibody, the affinity of VHH108-Fc antibody to H1N1-NP protein was increased by about 3 orders of magnitude. Compared with VHH108 antibody, the affinity of VHH108-Fc antibody to H3N2-NP protein was increased by about 37 times. Compared with VHH108 antibody, the affinity of VHH108-Fc antibody to H5N1-NP protein was increased by about 2 orders of magnitude. Compared with VHH108 antibody, the affinity of VHH108-Fc antibody to H7N9-NP protein was increased by about 2 orders of magnitude.

[0141] Based on the ELISA detection and affinity analysis results of VHH108 antibody and VHH108-Fc antibody to NP antigen, VHH108-Fc antibody has higher detection sensitivity to NP antigen of multiple subtypes of influenza A virus, and has stronger affinity to NP antigen of multiple subtypes of influenza A virus in affinity analysis. It is shown that the affinity of the antibody is significantly improved by bivalent modification of the nanobody. It is speculated that the reason is that after the nanobody is recombined with the Fc segment, the cysteine residues on the two peptide segments can form disulfide bonds, thereby forming two VHH-Fc bivalent nanobodies connected by disulfide bonds (natural SDS-PAGE results confirm that VHH108-Fc antibody exists in the form of bivalent antibody), and the bivalent antibody has two identical antigenic determinants, which is more stable in structure, thereby showing stronger binding activity

[0142] Table 1 Affinity determination of antibodies to different subtypes of NP antigens

[0143]

[0144] Example 3, Specificity detection of VHH108 antibody and VHH108-Fc antibody

[0145] The test viruses are respectively: influenza virus subtype H9N2 (H9N2), adenovirus (ADV), influenza B virus (IBV), enterovirus EV71 (EV71), SARS-CoV-2 (SCoV), coronavirus 229E (229E), vesicular stomatitis virus (VSV), herpes simplex virus 2 (HSV-2), rubella virus (RV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), respiratory syncytial virus RSV-A or respiratory syncytial virus RSV-B. MDCK cells are used as cell controls for the test viruses.

[0146] ADV (adenovirus), IBV (influenza B virus), EV71 (Enteroviruses 71), SCoV (SARS-CoV-2), 229E (coronaviruses 229E) are described in document 1. The ADV strain is ADV55 / Shenyang2018-1815-2 strain. The IBV strain is FluB / Xinjiang2018-1801-03 / YAM strain. The EV71 strain is AH / 08 / 06 (EV 71) strain. The SCoV strain is BetaCoV / Beijing / IME-BJ05 / 2020 strain. The 229E strain is CoV229E / Xinjiang2018-1750-2 strain. Document 1: Ying Tang, An integrated rapid nucleic acid detection assay based on recombinant polymerase amplification for SARS-CoV-2, Virologica Sinica 37 (2022) 138-141.

[0147] H9N2 (H9N2 influenza virus) is described in document 2. The H9N2 strain is NJ01 strain. Document 2: Zhao Haiming et al., Isolation, screening and identification of epidemic strain of avian influenza virus (H9 subtype), China Animal Health Care, 2024, 26(08).

[0148] VSV (vesicular stomatitis virus) is described in document 3. Document 3: Li G, et al. Pathophysiological characteristics of vesicular stomatitis virus infection in rabbits. Medical Animal Prevention and Treatment; 2022, 38(09).

[0149] HSV-2 (human herpes virus 2), RV (rubella virus), JEV (Japanese encephalitis virus) are described in document 4. Document 4: Development of rapid nucleic acid assays based on the recombinant polymerase amplification for monkeypox virus, Yuchang Li a, 1, Yanhong Gao b, 1, Ying Tang a, Jing Li a, Sen Zhang a, Tao Jiang, Xiaoping Kang, Virologica Sinica 38 (2023) 165-170.

[0150] TBEV (tick-borne encephalitis virus) is described in document 5. Document 5: Kang X, Li Y, Fan L, Lin F, Wei J, Zhu X, Hu Y, Li J, Chang G, Zhu Q, Liu H, Yang Y. Development of an ELISA-array for simultaneous detection of five encephalitis viruses. Virol J. 2012 Feb 27;9:56. doi: 10.1186 / 1743-422X-9-56. PMID: 22369052; PMCID: PMC3305475.

[0151] RSV-A (respiratory syncytial virus A) and RSV-B (respiratory syncytial virus B) are described in document 6. Document 6: Respiratory syncytial virus surge in 2022 caused by lineages already present before the COVID-19 pandemic; J Med Virol. 2023;95:e28830.

[0152] The test antibodies are respectively biotin-labeled VHH108 antibody prepared in Example 1 or biotin-labeled VHH108-Fc antibody prepared in Example 1.

[0153] Take 96-well ELISA microplates, add 100 μL of test virus solution (virus concentration is 2.5 x 10 4 PFU / mL) to each well, and coat overnight at 4°C. Then block. Then, add 100 μL of test antibody solution (antibody concentration is 0.5 μg / mL) to each well, incubate at 37°C for 1.5 h, and wash. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 1 h, and wash. Then, develop color by adding TMB substrate solution, and measure OD 450 value.

[0154] The average value of the blank control plus 3 times the standard deviation is used as the cut off value for judging positive, to determine the cross-reactivity specificity of the test antibody with other irrelevant viruses.

[0155] The results are shown in Figure 9 . Both VHH108 antibody and VHH108-Fc antibody can specifically detect H9N2, and have no cross-reactivity with other respiratory viruses.

[0156] Example 4, Performance detection of VHH57 antibody

[0157] The binding activity, specificity and affinity of VHH57 antibody are detected.

[0158] I. Capture effect of capture antibody

[0159] The test capture antibody is VHH57 antibody (provided by VHH57 antibody solution prepared in Example 1).

[0160] The antigens are respectively H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein.

[0161] Take 96-well ELISA microplates, add 100 μL of coating solution containing 2 μg / mL of test capture antibody to each well, and coat overnight at 4°C. Then block. Then, add 100 μL of antigen diluent (in the antigen diluent, the concentration of the antigen is 2 μg / mL-0.000128 μg / mL, with 5-fold gradient) to each well, incubate at 37°C for 1.5 h, and wash. Then, add 100 μL of HRP-labeled anti-His antibody to each well, incubate at 37°C for 1.5 h, and wash. Then, develop color by adding TMB substrate solution, and measure OD 450 value.

[0162] The results of the binding activity are shown in Figure 10 .

[0163] II. Specificity

[0164] The test antibody was biotin-labeled VHH57 antibody prepared in Example 1.

[0165] The method was the same as in Example 3.

[0166] The results of specificity are shown in Figure 11 .

[0167] III. Affinity

[0168] The test antibody was VHH57 antibody (provided by VHH57 antibody solution prepared in Example 1).

[0169] The influenza A virus NP antigen was H1N1-NP protein, H3N2-NP protein or H7N9-NP protein, respectively.

[0170] The results of affinity are shown in Figure 12 and Table 2.

[0171] Table 2 Affinity determination of antibodies to different subtypes of NP antigens

[0172]

[0173] The results of this example show that the VHH57 antibody has good binding activity and specificity to influenza virus antigens.

[0174] Example 5, Double antibody sandwich ELISA

[0175] The capture antibody was VHH57 antibody (provided by VHH57 antibody solution prepared in Example 1).

[0176] The detection antibody was biotin-labeled VHH108 antibody prepared in Example 1 or biotin-labeled VHH108-Fc antibody prepared in Example 1, respectively.

[0177] H9N2 (H9N2 influenza virus) is described in document 2. The H9N2 strain is NJ01 strain. Document 2: Zhao Haiming et al., Isolation, screening and identification of epidemic strain of avian influenza virus (H9 subtype), China Animal Health, 2024, 26(08).

[0178] Take 96-well ELISA microplates, add 100 μL of coating solution containing 2 μg / mL of capture antibody to each well, and coat overnight at 4°C. Then, block. Then, add 100 μL of virus dilution solution (H9N2 content: 2.5 x 10 4 PFU / mL-2.44 x 10 1PFU / mL, 2-fold gradient), incubate at 37°C for 1.5 hours, and wash. Then, add 100 μL of detection antibody solution (antibody concentration is 0.5 μg / mL) to each well, incubate at 37°C for 1.5 hours, and wash. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 1 hour, and wash. Then, add TMB substrate solution for color development and measure OD 450 value.

[0179] OD 450 The cut-off value of >0.2 was used as the positive value to determine the detection sensitivity of the double-antibody sandwich ELISA for H9N2 influenza virus.

[0180] See the results Figure 13 The combination of VHH57 antibody and VHH108-Fc antibody has a minimum detection limit of 195 PFU / mL.

[0181] Example 6. Double Antibody Sandwich ELISA (Nluc Luciferase-Labeled VHH108-Fc Antibody)

[0182] Capture antibody: VHH57 antibody (provided by the VHH57 antibody solution prepared in Example 1).

[0183] Detection antibody: VHH108-Fc-Nluc antibody (provided by the VHH108-Fc-Nluc antibody solution prepared in Example 1).

[0184] Substrate working solution: Luciferase was first diluted to 50 times the volume with buffer, and then diluted to 2000 times the volume with PBS buffer. Luciferase: Promega, product catalog number N1120. Buffer: Promega, product catalog number N112B.

[0185] H9N2 (H9N2 influenza virus) is described in Reference 2. The H9N2 strain is the NJ01 strain. Reference 2: Zhao Haiming et al., Isolation, screening and identification of epidemic strains of avian influenza virus (H9 subtype), Chinese Journal of Animal Health, 2024, 26(08).

[0186] Take a 96-well ELISA microplate and add 100 μL of coating solution containing 2 μg / mL capture antibody to each well. Incubate overnight at 4°C. Then, block the plate. Then, add 100 μL of virus dilution solution (H9N2 content of 2.5×10 4 PFU / mL-7.6×10 - 1PFU / mL, 2-fold gradient), 37°C incubation for 1.5h, washing. Then, 100μL detection antibody solution (antibody concentration is 0.5μg / mL) was added to each well, 37°C incubation for 1.5h, washing. Then, 100μl substrate working solution was added to each well, and the fluorescence value was detected on the promaga fluorescence detector.

[0187] The results are shown in Figure 14 The detection sensitivity of VHH108-Fc-Nluc antibody ELISA was further improved to 1.5 PFU / mL. It is shown that the Nluc labeled VHH108-Fc antibody can significantly improve the detection sensitivity compared with the original structure of the nanobody.

[0188] Example 7, Comparison with the detection effect of commercial kit

[0189] The tested viruses were H1N1, H3N2, H7N9 or H9N2.

[0190] The H1N1 strain was A / California / 7 / 2009(H1N1) strain. The H3N2 strain was A / Wisconsin / 67 / 2005(H3N2) strain. The H7N9 strain was A / Anhui / 01 / 2013(H7N9) strain. All are recorded in document 1. Document 1: Ying Tang, An integrated rapid nucleic acid detection assay based on recombinant polymerase amplification for SARS-CoV-2, Virologica Sinica 37(2022) 138-141.

[0191] H9N2(H9N2 influenza virus) is recorded in document 2. The H9N2 strain is NJ01 strain. Document 2: Zhao Haiming et al., Isolation, screening and identification of epidemic strain of avian influenza virus (H9 subtype), China Animal Health, 2024, 26(08).

[0192] I. Detection by commercial kit

[0193] Commercial kit: Influenza A / B virus antigen detection kit (colloidal gold method, Guangzhou Wanfu Company product), according to the instruction. Add 400μL sample extraction solution in the sample extraction tube, dilute the tested virus by 2-fold gradient (1×10 5 PFU / mL-1.95×10 2PFU / mL), then 80 μL of the diluted virus sample was taken and added into the sample extraction tube, and after several times of blowing, the dropper was covered, and 4 drops of the sample were dropped into the sample well of the detection card, and the results were observed within 15 min.

[0194] The results are shown in Table 3.

[0195] II. Detection by double antibody sandwich ELISA

[0196] Capture antibody: VHH57 antibody (provided by the VHH57 antibody solution prepared in Example 1).

[0197] Detection antibody: VHH108-Fc-Nluc antibody (provided by the VHH108-Fc-Nluc antibody solution prepared in Example 1).

[0198] Substrate working solution: Luciferase was first diluted with Buffer to 50 times the volume, and then diluted with PBS buffer to 2000 times the volume. Luciferase: Promega Company, product catalog number N1120. Buffer: Promega Company, product catalog number N112B.

[0199] Take a 96-well ELISA microplate, add 100 μL of coating solution containing 2 μg / mL of capture antibody to each well, and coat overnight at 4°C. Then, block. Then, add 100 μL of virus diluent (the content of the test virus is 2.5 x 10 4 PFU / mL-7.6 x 10 -1 PFU / mL, 2-fold gradient), incubate at 37°C for 1.5 h, and wash. Then, add 100 μL of detection antibody solution (antibody concentration is 0.5 μg / mL) to each well, incubate at 37°C for 1.5 h, and wash. Then, add 100 μl of substrate working solution to each well, and detect the fluorescence value on the promaga fluorescence detector.

[0200] The results are shown in Table 3.

[0201] The results show that compared with the commercial kit, the sensitivity of VHH108-Fc-Nluc antibody in detecting each influenza A virus subtype is extremely significantly improved.

[0202] Table 3

[0203]

[0204] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. An antibody that binds to influenza virus, named VHH108-Fc-Nluc antibody, whose amino acid sequence is shown in SEQ ID NO:

7.

2. An antibody that binds to influenza virus, named VHH108-Fc antibody, whose amino acid sequence is shown in SEQ ID NO:

5.

3. Use of the antibody according to claim 1 or 2 in the preparation of a kit for detecting influenza virus; the influenza virus is an H1N1 subtype influenza virus, an H3N2 subtype influenza virus, an H5N1 subtype influenza virus, an H7N9 subtype influenza virus, or an H9N2 subtype influenza virus.

4. Use of the VHH108-Fc-Nluc antibody and the VHH57 antibody in preparing a kit for detecting influenza virus; the VHH108-Fc-Nluc antibody is as described in claim 1; the VHH57 antibody is an antibody that binds to influenza virus, and its amino acid sequence is shown in SEQ ID NO: 3; the influenza virus is an H1N1 subtype influenza virus, an H3N2 subtype influenza virus, an H5N1 subtype influenza virus, an H7N9 subtype influenza virus, or an H9N2 subtype influenza virus.

5. Use of the VHH108-Fc antibody and the VHH57 antibody in preparing a kit for detecting influenza virus; the VHH108-Fc antibody is as described in claim 2; the VHH57 antibody is as described in claim 4; the influenza virus is an H1N1 subtype influenza virus, an H3N2 subtype influenza virus, an H5N1 subtype influenza virus, an H7N9 subtype influenza virus, or an H9N2 subtype influenza virus.

6. Antibody-related biological materials, which are (a) or (b) or (c) or (d) below: (a) VHH108-Fc-Nluc antibody-related biomaterials; (b) VHH108-Fc antibody-related biomaterials; (c) VHH108-Fc-Nluc antibody-related biomaterials and VHH57 antibody-related biomaterials; (d) VHH108-Fc antibody-related biomaterials and VHH57 antibody-related biomaterials; The VHH108-Fc-Nluc antibody-related biological material is a nucleic acid molecule expressing the VHH108-Fc-Nluc antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule; the VHH108-Fc-Nluc antibody is as described in claim 1; The VHH108-Fc antibody-related biological material is a nucleic acid molecule expressing the VHH108-Fc antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule; the VHH108-Fc antibody is as described in claim 2; The VHH57 antibody-related biological material is a nucleic acid molecule expressing the VHH57 antibody, an expression cassette having the nucleic acid molecule, a recombinant vector having the nucleic acid molecule, a recombinant microorganism having the nucleic acid molecule, or a recombinant cell having the nucleic acid molecule; the VHH57 antibody is as described in claim 4.

7. Use of the antibody-related biomaterial according to claim 6 in the preparation of a kit for detecting influenza virus; the influenza virus is an H1N1 subtype influenza virus, an H3N2 subtype influenza virus, an H5N1 subtype influenza virus, an H7N9 subtype influenza virus, or an H9N2 subtype influenza virus.

8. A kit for detecting influenza virus, comprising VHH108-Fc-Nluc antibody or VHH108-Fc antibody; the VHH108-Fc-Nluc antibody is as described in claim 1; the VHH108-Fc antibody is as described in claim 2.

9. A kit for detecting influenza virus, comprising a VHH108-Fc-Nluc antibody and a VHH57 antibody; the VHH108-Fc-Nluc antibody is as described in claim 1; the VHH57 antibody is as described in claim 4.

10. A kit for detecting influenza virus, comprising a VHH108-Fc antibody and a VHH57 antibody; the VHH108-Fc antibody is as described in claim 2; the VHH57 antibody is as described in claim 4.

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