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

Abstract

The invention discloses an anti-influenza virus high-sensitivity antibody as well as a preparation method and application thereof. The invention provides an antibody combined with influenza virus, which is named as a VHH108-Fc-Nluc antibody and comprises the following three sections: a VHH108 antibody, an Fc and NLuc luciferase, and the VHH108 antibody has a section as shown in the 3rd to 125th sites in SEQ ID NO: 1. The invention also provides a kit for detecting the influenza virus, and the kit comprises the VHH108-Fc-Nluc antibody and the VHH57 antibody. And the VHH57 antibody has segments as shown in the 230th site to the 350th site in SEQ ID NO: 3. The kit is suitable for detecting various subtypes of influenza A viruses, has the advantages of high sensitivity and high specificity, and has market value and popularization and application value.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to highly sensitive antibodies against influenza viruses and a preparation method and application thereof. Background Art

[0002] Influenza virus is an enveloped, single-stranded, negative-sense RNA virus belonging to the family Orthomyxoviridae. It is spherical in appearance with a diameter of about 80-120 nm. Its structure can be divided into three parts from the outside to the inside: the envelope, the matrix protein (M protein) and the core protein (NP protein). Its genetic composition consists of 7-8 RNA gene fragments encoding different proteins. In the classification of influenza viruses, influenza viruses can be divided into four types: influenza A virus (also known as influenza A virus), influenza B virus (also known as influenza B virus), influenza C virus (also known as influenza C virus) and influenza D virus due to the differences in the antigenicity of nucleoprotein and matrix protein.

[0003] Among the above influenza virus types, influenza A virus is the most harmful to humans, poultry and mammals. It is also the most common type of seasonal influenza virus and is prone to cause pandemics. Influenza A virus can be divided into different subtypes based on the different antigenicity of hemagglutinin (HA) and neuraminidase (NA) embedded on the surface of virus particles. To date, influenza A virus is divided into 18 HA subtypes and 11 NA subtypes. The most common influenza A virus subtypes include H1N1, H3N2, H5N1, H7N9 and H9N2.

[0004] Influenza A viruses are mostly transmitted in poultry, and some subtypes can be transmitted across species. Currently, seasonal influenza A viruses that spread in humans are mostly H1N1 and H3N2 subtypes. Highly pathogenic avian influenza viruses such as H7N9 and H5N1 also cause fatal infections in humans. Therefore, establishing a detection technology that can detect multiple subtypes of influenza A viruses is of great significance for infection control of the virus.

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

[0006] The traditional method of antibody preparation is mainly hybridoma antibody preparation technology, which uses specific antigens to immunize mice to stimulate B lymphocyte activation, proliferation, and differentiation into sensitized lymphocytes; collect mouse spleen cells, fuse them with myeloma cells and monoclonize them, and screen and isolate cell lines that can secrete specific monoclonal antibodies. However, during the subculture of hybridoma cell lines, there is often a risk of antibody genes being lost. Although the use of molecular biology technology can achieve hybridoma antibody gene expression and intracellular culture, which can overcome the problem of antibody gene loss, the in vitro expression of antibody genes needs to be carried out in eukaryotic cells, with high expression costs and low expression levels. Therefore, antibody preparation technology needs to be improved urgently.

[0007] Single-chain antibodies (VHH antibodies), also known as nanobodies, are a new type of antibody found in alpacas. Although nanobodies are smaller than traditional antibodies IgG, in terms of structure, the complementary determining regions CDR1 and CDR3 of nanobodies are longer than the CDR regions of traditional antibodies, and can penetrate deep into the antigen and bind to secret target binding sites that traditional antibodies cannot bind to; thereby increasing the antigen binding affinity of nanobodies. Nanobodies have a small molecular weight, a simple structure, and no modification sites such as glycosylation. They can be expressed in large quantities in prokaryotic systems and have low preparation costs. This solves the problem of high preparation costs for traditional antibodies. In short, compared with traditional antibodies, nanobodies have the advantages of small molecular weight (approximately 12-15kDa), high chemical tolerance, good stability, high affinity, and easy expression. The development of nanobodies as detection antibodies is expected to develop antigen detection methods with better sensitivity. Summary of the invention

[0008] The purpose of the present invention is to provide a highly sensitive anti-influenza virus antibody and a preparation method and application thereof.

[0009] The present invention 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 at positions 3-125 in SEQ ID NO:1.

[0010] Specifically, the VHH108-Fc-Nluc antibody includes the following three segments from the N-terminus to the C-terminus: VHH108 antibody, Fc and NLuc luciferase.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0045] The antibody-related biomaterial is a VHH108-Fc-Nluc antibody-related biomaterial and / or a VHH108-Fc antibody-related biomaterial and / or a VHH108 antibody-related biomaterial and / or a VHH57 antibody-related biomaterial.

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

[0047] The antibody-related biomaterial is a VHH108-Fc-Nluc antibody-related biomaterial and / or a VHH108-Fc antibody-related biomaterial and / or a VHH108 antibody-related biomaterial and / or a VHH57 antibody-related biomaterial.

[0048] The present invention also provides a kit for detecting influenza virus, which comprises 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 biological material related to the VHH108-Fc-Nluc antibody is a nucleic acid molecule, an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell expressing the VHH108-Fc-Nluc antibody. Specifically, the nucleic acid molecule is as shown in SEQ ID NO: 8, positions 7-1629. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing the small fragment between the AflII and EcoRI restriction recognition sites in the vector pcDNA3.1 (+) with the 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 biological material related to the VHH108-Fc antibody 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 positions 7-1074 of SEQ ID NO: 6. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing the small fragment between the AflII and EcoRI restriction recognition sites in the vector pcDNA3.1 (+) with the double-stranded DNA molecule shown in positions 7-1072 of SEQ ID NO: 6. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21 (DE3).

[0052] The biological material related to the VHH108 antibody 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 positions 1-1053 of SEQ ID NO: 4. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between the EcoRI and XhoI restriction recognition sites in the vector pGEX-4T-1 with a double-stranded DNA molecule shown in positions 688-1053 of SEQ ID NO: 4. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21 (DE3).

[0053] The biological material related to the VHH57 antibody 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 positions 3-404 of SEQ ID NO: 2. Specifically, the recombinant vector is a recombinant plasmid obtained by replacing a small fragment between the NcoI and XhoI restriction sites in the vector pET-28a (+) with a double-stranded DNA molecule shown in positions 7-404 of SEQ ID NO: 2. Specifically, the recombinant microorganism is a recombinant bacterium obtained by introducing the recombinant plasmid into Escherichia coli BL21 (DE3).

[0054] Fc, the antibody FC fragment, is also known as the antibody constant region.

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

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

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

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

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

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

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

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

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

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

[0065] Any of the above influenza viruses may be influenza A virus (influenza A virus).

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

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

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

[0069] In view of the current situation that there are insufficient detection reagents for influenza A antigens that can detect multiple subtypes, the present invention has developed and screened nano antibodies VHH108 and VHH57 that can bind to multiple subtypes of influenza viruses. Further, in the development of double antibody sandwich ELISA detection, it was found that when VHH nano antibodies were used as capture antibodies, they showed a more sensitive detection effect. However, the sensitivity of nano antibodies as detection antibodies is not high, significantly lower than that of traditional IgG antibodies. The main reason is that small molecule nano antibodies are not as stable as long fragment nano antibodies. Further, the present invention optimizes the structure of VHH108 nano antibodies, and by recombinantly expressing the Fc fragment at the end of the antibody, two VHH antibodies are expressed and spontaneously form disulfide bonds through the cysteine ​​residues on the Fc, thereby forming a 2-valent VHH-FC antibody. The affinity analysis results show that the affinity of the modified VHH108-FC antibody is 10 times higher than that of the original VHH108 nano antibody; in the double antibody sandwich ELISA experiment, the detection sensitivity is also significantly improved. Furthermore, the present invention recombined the sensitive detection marker Nluc at the C-terminus of the VHH-FC antibody and established a highly sensitive Nluc nanobody ELISA, the detection sensitivity of which was 100 times higher than that of the traditional ELISA.

[0070] The present invention is applicable to the detection of various 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 a diagram showing the results of electrophoresis detection in step six of Example 1.

[0072] Figure 2 Detection of 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 values ​​(NP antigen of H1 subtype influenza virus).

[0074] Figure 4 Detection of 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 This is the result diagram of affinity detection in step 3 of Example 2 (VHH108 antibody).

[0078] Figure 8 This is a graph showing the affinity detection results of step 3 of Example 2 (VHH108-Fc antibody).

[0079] Fig. 9 Detection of OD for Example 3 450 Result graph of the values.

[0080] Fig.10 Detection of OD for step 1 of Example 4 450 Result graph of the values.

[0081] Fig.11 Detect OD for step 2 of Example 4 450 Result graph of the values.

[0082] Fig.12 This is a graph showing the results of affinity testing in step 3 of Example 4.

[0083] Fig.13 Detection of OD for Example 5 450 Result graph of the values.

[0084] Fig.14 This is a graph showing the results of detecting fluorescence values ​​in Example 6. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0086] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. 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 with SV40 virus genes. TMSulfo-NHS biotinylation kit: Thermo Scientific, product catalog number 21425. Coating solution: Beijing Solebao Technology Co., Ltd., product catalog number C1055. HRP-labeled anti-Flag antibody: Beijing Sino Biological Technology Co., Ltd., product catalog number 100233-MM01-H. HRP-labeled anti-His antibody: Beijing Sino Biological Technology Co., Ltd., product catalog number 105327-MM02T-H. MDCK cells (canine kidney cells): ATCC number CCL-34. TMB substrate solution: Tiangen Biochemical Technology (Beijing) Co., Ltd., product catalog number PA107-01. Horseradish peroxidase-labeled streptavidin (product specifications: liquid, 1 mg / ml): Beijing Bioson Biotechnology Co., Ltd., product catalog number bs-0437p-HRP.

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

[0089] Example 1. Preparation of Antibodies

[0090] 1. Construction of recombinant plasmid

[0091] The double-stranded DNA molecule shown at positions 7-404 in SEQ ID NO: 2 is used to replace the small fragment between the NcoI and XhoI restriction recognition sites in the vector pET-28a (+) to obtain the recombinant plasmid pET28a-VHH108. The recombinant plasmid pET28a-VHH108 contains the DNA molecule shown in SEQ ID NO: 2. The nucleotides at positions 3-404 in SEQ ID NO: 2 constitute an open reading frame, encoding the protein shown in SEQ ID NO: 1 (named VHH108-Flag fusion protein, i.e., VHH108 antibody with a flag tag at the C-terminus, referred to as VHH108 antibody). In SEQ ID NO: 1, the amino acid residues at positions 3-125 constitute the VHH108 antibody, and the amino acid residues at positions 126-133 constitute the flag tag.

[0092] The double-stranded DNA molecule shown at positions 688-1053 in SEQ ID NO: 4 was used to replace the small fragment between the EcoRI and XhoI restriction sites in the vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-4T-1-VHH57. The recombinant plasmid pGEX-4T-1-VHH57 contains the DNA molecule shown in SEQ ID NO: 4. The nucleotides at positions 1-1053 in SEQ ID NO: 4 constitute an open reading frame, encoding the protein shown in SEQ ID NO: 3 (named GST-VHH57 fusion protein, i.e., VHH57 antibody with a GST tag at the N-terminus, referred to as VHH57 antibody). In SEQ ID NO: 3, the amino acid residues at positions 1-229 constitute the GST tag, and the amino acid residues at positions 230-350 constitute the VHH57 antibody.

[0093] The double-stranded DNA molecule shown at positions 7-1072 in SEQ ID NO: 6 was used to replace the small fragment between the AflII and EcoRI restriction enzyme recognition sites in the vector pcDNA3.1 (+) to obtain the recombinant plasmid pcDNA3.1-VHH108-Fc. The recombinant plasmid pcDNA3.1-VHH108-Fc contains the DNA molecule shown in SEQ ID NO: 6. In SEQ ID NO: 6, nucleotides at positions 7-1074 constitute an open reading frame, encoding the protein shown in SEQ ID NO: 5 (named VHH108-Fc fusion protein, also known as VHH108-Fc antibody). In SEQ ID NO: 5, amino acid residues at positions 2-124 constitute the VHH108 antibody, and amino acid residues at positions 125-355 constitute Fc.

[0094] The double-stranded DNA molecule shown at positions 7-1629 in SEQ ID NO: 8 was used to replace the small fragment between the AflII and EcoRI restriction enzyme recognition sites in the vector pcDNA3.1 (+), thereby obtaining the recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc. The recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc contains the DNA molecule shown in SEQ ID NO: 8. In SEQ ID NO: 8, nucleotides at positions 7-1629 constitute an open reading frame encoding 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 Fc, and amino acid residues at positions 371-540 constitute NLuc luciferase.

[0095] 2. Expression and purification of VHH108 antibody

[0096] 1. Introduce the recombinant plasmid pET28a-VHH108 into Escherichia coli BL21 (DE3) competent cells to obtain recombinant bacteria.

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

[0098] 3. After completing step 2, collect the bacteria, disrupt the bacteria, collect the supernatant, and use Anti-Flag affinity purification gel (Yisheng Biotechnology (Shanghai) Co., Ltd., product catalog number 20584ES08, follow the instructions) to perform protein purification operations, and then use ultrafiltration tubes to concentrate and exchange the liquid (make the buffer system PBS buffer) to obtain an antibody solution, named VHH108 antibody solution.

[0099] 3. Expression and purification of VHH57 antibody

[0100] 1. Introduce the recombinant plasmid pGEX-4T-1-VHH57 into Escherichia coli BL21 (DE3) competent cells to obtain recombinant bacteria.

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

[0102] 3. After completing step 2, collect the bacteria, disrupt the bacteria, collect the supernatant, and use Anti-GST affinity purification gel (Yisheng Biotechnology (Shanghai) Co., Ltd., product catalog number 20507ES10, follow the instructions) to purify the protein, then use an ultrafiltration tube to concentrate and exchange the liquid (make the buffer system PBS buffer) to obtain an antibody solution, named VHH57 antibody solution.

[0103] IV. Expression and purification of VHH108-Fc antibody

[0104] 1. COS7 cells were seeded into 24-well plates and cultured in DMEM medium containing 10% FBS until the confluence reached 80%-90%, and then transfected with the recombinant plasmid pcDNA3.1-VHH108-Fc (with the help of Lipofectamine TM 3000 μL transfection reagent and operate according to the instruction manual), culture for 3-4 days, and then collect the supernatant.

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

[0106] 3. After completing step 2, collect the magnetic beads, wash them with PBST solution, then add 2 mL of pH 3.0 citrate buffer to the magnetic beads, vortex mix for 5 minutes, then discard the magnetic beads and collect the supernatant.

[0107] 4. Take the supernatant obtained in step 3 and add 1M Tris buffer to a pH value of 7.0 to obtain an antibody solution named VHH108-Fc antibody solution.

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

[0109] 5. Expression and purification of VHH108-Fc-Nluc antibody

[0110] The recombinant plasmid pcDNA3.1-VHH108-Fc-Nluc was used to replace the recombinant plasmid pcDNA3.1-VHH108-Fc, and the rest was the same as step 4. An antibody solution was obtained, which was named VHH108-Fc-Nluc antibody solution.

[0111] 6. Electrophoresis detection

[0112] The test samples were: the VHH108 antibody solution prepared in step 2, the VHH57 antibody solution prepared in step 3, the VHH108-Fc antibody solution prepared in step 4, or the VHH108-Fc-Nluc antibody solution prepared in step 5.

[0113] 1. Reduction SDS-PAGE

[0114] Take 20 μL of sample, add 5 μL of 5× protein loading buffer (Beijing Jinpulai Biotechnology Co., Ltd., product catalog number P06M18), boil for 10 minutes, and load the sample after cooling. The sample and standard protein marker (molecular weight 10-170 kDa; Thermo Scientific, product catalog number 26616) are loaded in an amount of 10 μL. Use 15% separation gel (Shanghai Bioengineering Co., Ltd., product catalog number C651103-0001). During electrophoresis, set the concentration gel conditions to 80V, 30min, and the separation gel conditions to 120V, 40min. After the electrophoresis, use ProteinShow-G250 protein fast staining reagent (Kangwei reagent product, product catalog number CW0023S, operate according to the instructions) for operation, and then use a gel imager to collect information.

[0115] Reducing SDS-PAGE results can destroy the disulfide bonds between peptides, thereby accurately displaying the molecular weight of the protein. Figure 1 A. Figure 1 In A: 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 showed that the band size was consistent with the expected molecular weight of the antibody.

[0116] 2. Non-reducing SDS-PAGE

[0117] Take 10 μL of sample and add 10 μL of 2×Native protein loading buffer (Shanghai Bioengineering Co., Ltd., product catalog number C506025-0003). The sample loading volume is 20 μL, and the standard protein marker (molecular weight 10-170 kDa; Thermo Scientific, product catalog number 26616) loading volume is 10 μL. Use 12% separation gel (Shanghai Bioengineering Co., Ltd., product catalog number C651102-0001). During electrophoresis, set the concentration gel conditions to 80V, 30min, and the separation gel conditions to 120V, 40min. After the electrophoresis, use ProteinShow-G250 protein fast staining reagent (Kangwei reagent product, product catalog number CW0023S, operate according to the instructions) for operation, and then use a gel imager to collect information.

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

[0119] VII. Biotin labeling of VHH108 antibody and VHH108-Fc antibody

[0120] Take the VHH108 antibody solution prepared in step 2 and use EZ-Link TM The Sulfo-NHS biotinylation kit was operated according to the instructions to obtain the biotin-labeled VHH108 antibody.

[0121] Take the VHH57 antibody solution prepared in step 3 and use EZ-Link TMThe Sulfo-NHS biotinylation kit was operated according to the instructions to obtain the biotin-labeled VHH57 antibody.

[0122] Take the VHH108-Fc antibody solution prepared in step 4 and use EZ-Link 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 Binding Activity between VHH108 Antibody and VHH108-Fc Antibody

[0124] 1. Comparison of capture effects of capture antibodies

[0125] The capture antibodies are 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 antigens are: H1N1-NP protein or H3N2-NP protein.

[0127] Take a 96-well ELISA microplate, add 100 μL of coating solution containing 2 μg / mL capture antibody to each well, and coat overnight at 4°C. Then block. Then, add 100 μL of antigen diluent to each well (in the antigen diluent, the concentration of antigen is 8 μg / mL-0.000512 μg / mL, 5-fold gradient), incubate at 37°C for 1.5 hours, and wash. Then, add 100 μL of HRP-labeled anti-His antibody to each well, incubate at 37°C for 1.5 hours, and wash. Then add TMB substrate solution for color development and measure OD 450 value.

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

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

[0130] 2. Comparison of detection effects as detection antibodies

[0131] The detection antibodies are: the biotin-labeled VHH108 antibody prepared in Example 1 or the biotin-labeled VHH108-Fc antibody prepared in Example 1.

[0132] Take a 96-well ELISA microplate, add 100 μL of coating solution containing 1 μg / mL antigen to each well (the antigens are H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein), and coat overnight at 4°C. Then block. Then, add 100 μL of antibody diluent to each well (in the antibody diluent, the concentration of the detection antibody is 8 μg / mL-0.000512 μg / mL, 5-fold gradient), 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.

[0133] OD 450 The cut-off value for judging as positive was >0.2, and the lowest concentration of the antibody that could detect positive was compared. The lower the antibody concentration that could detect positive, the higher the antibody binding activity.

[0134] Results Figure 3 , Figure 4 , Figure 5 and Figure 6 . The NP antigens of four influenza virus subtypes H1\H3\H5\H7 were used for detection. Both VHH108 antibody and VHH108-Fc antibody were able to bind as detection antibodies, indicating that the antibody can detect multiple subtypes of influenza virus. 450 >0.2 is the cutoff value for ELISA result judgment. The results showed that the lowest concentration of biotin-labeled VHH108-Fc antibody for NP antigen detection was 0.00256μg / ml, while the lowest concentration of biotin-labeled VHH108 antibody for NP antigen detection was 0.0128μg / ml, indicating that biotin-labeled VHH108-Fc antibody showed higher binding activity.

[0135] 3. Affinity Comparison

[0136] The test antibodies were: 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 influenza A virus NP antigen (H1N1-NP protein, H3N2-NP protein, H5N1-NP protein or H7N9-NP protein). NP antigen diluted with Q Buffer was added to the test wells of the low adsorption special plate (NP antigen concentration was 2μg / mL), and only Q Buffer was added to the blank wells. Then, a 2-fold serial dilution of the test antibody (test antibody concentration was 10μg / mL-0.3125μg / mL) was added, and the configured plate was placed in the Gator protein characterization analysis system for affinity determination and calculation of the affinity dissociation constant. The affinity of the antibody and the antigen was determined by comparing the affinity dissociation constant KD value. The smaller the KD value, the greater the affinity of the antibody.

[0138] Q Buffer: contains 0.02% Tween 20, 0.2% BSA and 0.05% NaN3, and the balance 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 the VHH108 antibody, the affinity of the VHH108-Fc antibody to the H1N1-NP protein increased by about 3 orders of magnitude. Compared with the VHH108 antibody, the affinity of the VHH108-Fc antibody to the H3N2-NP protein increased by about 37 times. Compared with the VHH108 antibody, the affinity of the VHH108-Fc antibody to the H5N1-NP protein increased by about 2 orders of magnitude. Compared with the VHH108 antibody, the affinity of the VHH108-Fc antibody to the H7N9-NP protein increased by about 2 orders of magnitude.

[0141] Based on the results of ELISA detection and affinity analysis of VHH108 antibody and VHH108-Fc antibody for NP antigen, VHH108-Fc antibody has a higher detection sensitivity for multi-subtype influenza A virus NP antigen, and in affinity analysis, VHH108-Fc antibody has a stronger affinity for multi-subtype influenza A virus NP antigen. This shows that the nanobody significantly improves the antibody affinity through bivalent modification. The reason is speculated to be that after the nanobody and Fc segment are recombined, the cysteine ​​residues of the Fc segment on the two peptide segments can form a disulfide bond, thereby forming a bivalent nanobody in which two VHH-Fc are connected by a disulfide bond (the natural SDS-PAGE results confirmed that VHH108-Fc antibody exists in the form of a bivalent antibody). The bivalent antibody has two identical antigenic determinants, and the structure is more stable when binding to the antigen, thus showing a stronger binding activity.

[0142] Table 1 Determination of the affinity 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 were: influenza virus H9N2 subtype (H9N2), adenovirus (ADV), influenza B virus (IBV), enterovirus EV71 (EV71), SARS-CoV-2 (ScoV), coronavirus 229E (229E), vesicular stomatitis virus (VSV), herpes virus type 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 were used as cell controls for the test viruses.

[0146] ADV (adenovirus), IBV (influenza B virus), EV71 (Enteroviruses 71), SCoV (SARS-CoV-2), and 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 recorded in document 2. The H9N2 strain is the NJ01 strain. Document 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).

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

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

[0150] TBEV (tick-borne encephalitis virus) is described in Document 5. Literature 5: Kang X, Li Y, FanL, 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.VirolJ.2012Feb 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 reference 6. Reference 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 were: the biotin-labeled VHH108 antibody prepared in Example 1 or the biotin-labeled VHH108-Fc antibody prepared in Example 1.

[0153] Take a 96-well ELISA microplate and add 100 μL of the test virus solution (virus concentration is 2.5×10 4 PFU / mL), coated overnight at 4℃. Then blocked. Then, 100μL of test antibody solution (antibody concentration is 0.5μg / mL) was added to each well, incubated at 37℃ for 1.5h, and washed. Then, 100μL of horseradish peroxidase-labeled streptavidin was added to each well, incubated at 37℃ for 1h, and washed. Then, TMB substrate solution was added for color development, and OD was measured. 450 value.

[0154] The mean value of the blank control plus 3 times the standard deviation was used as the cut-off value for judging as positive to determine the cross-reaction specificity of the test antibody with other unrelated viruses.

[0155] Results Fig. 9 Both VHH108 antibody and VHH108-Fc antibody can specifically detect H9N2 and have no cross-reaction with other respiratory viruses.

[0156] Example 4. Performance testing of VHH57 antibody

[0157] The binding activity, specificity and affinity of the VHH57 antibody were tested.

[0158] 1. Capture effect of capture antibody

[0159] The capture antibody tested was: VHH57 antibody (provided by the VHH57 antibody solution prepared in Example 1).

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

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

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

[0163] Specificity

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

[0165] The method is the same as Example 3.

[0166] Specific results are shown in Fig.11 .

[0167] 3. Affinity

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

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

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

[0171] Table 2 Determination of the affinity 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 with influenza virus antigens.

[0174] Example 5: Double Antibody Sandwich ELISA

[0175] Capture antibody: VHH57 antibody (provided by the VHH57 antibody solution prepared in Example 1).

[0176] The detection antibodies are: the biotin-labeled VHH108 antibody prepared in Example 1 or the biotin-labeled VHH108-Fc antibody prepared in Example 1.

[0177] H9N2 (H9N2 influenza virus) is recorded in document 2. The H9N2 strain is the NJ01 strain. Document 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).

[0178] Take a 96-well ELISA microplate and add 100 μL of coating solution containing 2 μg / mL capture antibody to each well. Coat at 4°C overnight. Then, block. Then, add 100 μL of virus dilution solution (H9N2 content of 2.5×10 4 PFU / mL-2.44×10 1PFU / mL, 2-fold gradient), incubate at 37°C for 1.5h, wash. Then, add 100 μL of detection antibody solution (antibody concentration is 0.5 μg / mL) to each well, incubate at 37°C for 1.5h, wash. Then, add 100 μL of horseradish peroxidase-labeled streptavidin to each well, incubate at 37°C for 1h, 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] Results Fig.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 is N1120. Buffer: Promega, product catalog number is N112B.

[0185] H9N2 (H9N2 influenza virus) is recorded in document 2. The H9N2 strain is the NJ01 strain. Document 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. Coat at 4°C overnight. Then, block. 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), incubate at 37°C for 1.5h, 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 1.5h, and wash. Then, add 100 μL of substrate working solution to each well and place it on the promaga fluorescence detector to detect the fluorescence value.

[0187] Results Fig.14 The detection sensitivity of the VHH108-Fc-Nluc antibody ELISA was further improved to 1.5 PFU / mL. This shows 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 of detection results with commercial kits

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

[0190] The H1N1 strain is A / California / 7 / 2009 (H1N1) strain. The H3N2 strain is A / Wisconsin / 67 / 2005 (H3N2) strain. The H7N9 strain is 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 the NJ01 strain. Document 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).

[0192] 1. Detection using commercial kits

[0193] Commercial kit: Influenza A / B virus antigen detection kit (colloidal gold method, product of Guangzhou Wanfu Company), operate according to the instructions. Add 400 μL sample extract to the sample extraction tube, dilute the test virus 2 times (1×10 5 PFU / mL-1.95×10 2PFU / mL), then draw 80 μL of the diluted virus sample and add it to the sample extraction tube. After blowing several times, cover the dropper and drop 4 drops of the sample into the sample well of the test card. Observe the results within 15 minutes.

[0194] The results are shown in Table 3.

[0195] 2. Detection using 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 to 50 times the volume with Buffer, and then diluted to 2000 times the volume with PBS buffer. Luciferase: Promega, product catalog number is N1120. Buffer: Promega, product catalog number is N112B.

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

[0200] The results are shown in Table 3.

[0201] The results showed that compared with commercial kits, the sensitivity of VHH108-Fc-Nluc antibody in detecting various influenza A virus subtypes was 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. An antibody that binds to influenza virus, named VHH108-Fc-Nluc antibody, comprising the following three segments: VHH108 antibody, Fc and NLuc luciferase; the VHH108 antibody has the segments shown at positions 3-125 in SEQ ID NO:

1.

2. An antibody that binds to influenza virus, named VHH108-Fc antibody, comprising the following two segments: VHH108 antibody and Fc; the VHH108 antibody has the segment shown at positions 3-125 in SEQ ID NO:

1.

3. An antibody that binds to influenza virus, named VHH108 antibody, which has the segment shown at positions 3-125 in SEQ ID NO:

1.

4. An antibody that binds to influenza virus, named VHH57 antibody, which has the segment shown at positions 230-350 in SEQ ID NO:

3.

5. Use of the antibody according to any one of claims 1 to 4 in the preparation of a kit for detecting influenza virus.

6. Application of VHH108-Fc-Nluc antibody and VHH57 antibody in the preparation of a kit for detecting influenza virus; The VHH108-Fc-Nluc antibody is as described in claim 1; The VHH57 antibody is as described in claim 4.

7. Use of VHH108-Fc antibody and VHH57 antibody in the preparation of 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.

8. Use of VHH108 antibody and VHH57 antibody in the preparation of a kit for detecting influenza virus; The VHH108 antibody is as described in claim 3; The VHH57 antibody is as described in claim 4.

9. Antibody-related biomaterials or applications of antibody-related biomaterials; The antibody-related biomaterial is a VHH108-Fc-Nluc antibody-related biomaterial and / or a VHH108-Fc antibody-related biomaterial and / or a VHH108 antibody-related biomaterial and / or a VHH57 antibody-related biomaterial; The application of the antibody-related biological material is the application of the antibody-related biological material in preparing a kit for detecting influenza virus; The VHH108-Fc-Nluc antibody is as described in claim 1; The VHH108-Fc antibody is as described in claim 2; The VHH108 antibody is as described in claim 3; The VHH57 antibody is as described in claim 4.

10. A kit for detecting influenza virus, comprising antibodies or antibody-related biological materials; The antibody is the VHH108-Fc-Nluc antibody of claim 1 and / or the VHH108-Fc antibody of claim 2 and / or the VHH108 antibody of claim 3 and / or the VHH57 antibody of claim 4; The antibody-related biological material is as described in claim 9.

Images