Broad-spectrum adenovirus monoclonal antibody and application thereof

Abstract

The application discloses a broad-spectrum adenovirus monoclonal antibody and application thereof, and relates to the technical field of medicines.A kind of adenovirus monoclonal antibody, comprising: heavy chain variable region complementarity determining region and light chain variable region complementarity determining region selected from A1) or A2); A1) the amino acid sequences of heavy chain variable region CDR1-3 shown in SEQ ID NO.2-4 respectively, and the amino acid sequences of light chain variable region CDR1'-3' shown in SEQ ID NO.5-7 respectively; A2) the amino acid sequences of heavy chain variable region CDR1-3 shown in SEQ ID NO.10-12 respectively, and the amino acid sequences of light chain variable region CDR1'-3' shown in SEQ ID NO.13-15 respectively.The monoclonal antibody can specifically recognize human adenovirus 3, 4, 5, 7 and 55 true viruses, and also has good sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a broad-spectrum adenovirus monoclonal antibody and its applications. Background Technology

[0002] Human adenovirus (HAdV) is a non-enveloped, linear, double-stranded DNA virus consisting of a viral capsid and internal nucleic acid. HAdV is prevalent globally, with no clear seasonal pattern of transmission. The population is generally susceptible, but this susceptibility is strongly correlated with geographical location. High-density activity groups, children, and immunocompromised individuals are particularly vulnerable. This means that even healthy children and adults can easily become infected in enclosed or crowded environments. Numerous serological types of adenovirus exist; to date, 113 types of human adenovirus have been identified. Types 1-51 are classified based on serotypes, while types 52-113 are novel types obtained through whole-genome sequencing. The numerous serotypes can be divided into 7 subgenera, A to G. The adenovirus types associated with respiratory infections mainly include types 3, 7, 11, 14, 21 and 55 of subgenera B, types 1, 2, 5 and 6 of subgenera C, and type 4 of subgenera E. The adenovirus types that can cause outbreaks and widespread transmission are mainly types 3, 4, 5, 7, 11, 14 and 55.

[0003] Virus isolation and culture, along with nucleic acid detection, have long been considered the "gold standard" for detecting human respiratory adenoviruses. However, these methods are time-consuming, require highly qualified laboratories and specialized personnel, increasing the difficulty of detection and potentially delaying optimal treatment. Furthermore, there are numerous adenovirus types, with more than ten capable of causing severe respiratory infections in humans. Therefore, developing a monoclonal antibody specifically targeting human respiratory adenovirus proteins that can identify and detect multiple serotypes of human respiratory adenoviruses is of great significance for achieving rapid, early diagnosis of human adenovirus infections, early case detection, and prevention and treatment of adenovirus infection, particularly severe adenovirus pneumonia. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes an adenovirus monoclonal antibody that has excellent broad-spectrum activity, can specifically recognize human adenovirus types 3, 4, 5, 7 and 55, and also has excellent sensitivity.

[0005] The present invention also provides biological materials related to the above-mentioned monoclonal antibodies.

[0006] The present invention also provides a product for detecting human adenovirus.

[0007] The present invention also provides the use of the above-mentioned monoclonal antibody or the above-mentioned biological material in the preparation of products for detecting human adenovirus.

[0008] The present invention also provides the above-mentioned monoclonal antibody or the above-mentioned biological material or the above-mentioned product for use in immune detection and other experiments or operations involving specific binding of antigen and antibody.

[0009] An adenovirus monoclonal antibody according to a first aspect of the present invention includes: a heavy chain variable region complementarity-determining region (CDR) and a light chain variable region complementarity-determining region (CDR) selected from A1 or A2. A1) The amino acid sequences are the heavy chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.2~4, and the amino acid sequences are the light chain variable regions CDR1', CDR2' and CDR3' shown in SEQ ID NO.5~7; A2) The amino acid sequences are the heavy chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.10~12, and the amino acid sequences are the light chain variable regions CDR1', CDR2' and CDR3' shown in SEQ ID NO.13~15.

[0010] According to some embodiments of the present invention, the monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO.9.

[0011] According to some embodiments of the present invention, the monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.16, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO.17.

[0012] According to some embodiments of the present invention, the monoclonal antibody is any one of B1) to B4): B1) A single-chain antibody obtained by linking the above-mentioned heavy chain variable region and the above-mentioned light chain variable region; B2) A fusion antibody containing the single-chain antibody described in B1); B3) Fab containing the above-mentioned heavy chain variable region and the above-mentioned light chain variable region; B4) is a complete antibody containing the above-mentioned heavy chain variable region and the above-mentioned light chain variable region.

[0013] In this invention, the production of monoclonal antibodies is not limited to any specific method for producing monoclonal antibodies. For example, the antibodies can be obtained by culturing hybridoma cells as described in this invention and purifying them; or by using recombinant DNA technology (see Journal of virological methods, 2009, 158(1-2): 171-179).

[0014] According to a second aspect of the present invention, the biological material related to the above-described monoclonal antibody is any one of C1) to C4); C1) The nucleic acid molecule encoding the above-mentioned monoclonal antibody; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) A recombinant biological cell containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3).

[0015] According to some embodiments of the present invention, C1) the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

[0016] According to some embodiments of the present invention, C1) the nucleic acid molecule may be a gene encoding the monoclonal antibody.

[0017] According to some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule described in C1) is as shown in any one of D1) to D3); D1) The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.18, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.19; D2) The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.20, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.21; D3) is a nucleic acid molecule that has more than 90% identity with the nucleic acid molecules shown in D1) or D2) and encodes the monoclonal antibody.

[0018] According to some embodiments of the present invention, C2) the expression cassette refers to DNA capable of expressing the monoclonal antibody in a host cell. This DNA may include not only a promoter for initiating transcription of the coding gene of the monoclonal antibody, but also a terminator for terminating transcription of the coding gene of the monoclonal antibody. Further, the expression cassette may also include a replication start site, a transcription start sequence, an enhancer sequence, a selection element, or a reporter gene.

[0019] According to some embodiments of the present invention, the vector described in C3) may be a plasmid, a granule, a bacteriophage, or a viral vector. The vector may be a cloning vector or an expression vector.

[0020] According to some embodiments of the present invention, the biological cells described in C4) can be bacteria (such as Escherichia coli or Bacillus subtilis), algae, fungi (such as yeast or Aspergillus), insect cells (such as S2 Drosophila cells or Sf9 cells), or animal cells (such as CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells). The biological cells do not include reproductive materials.

[0021] According to some embodiments of the present invention, the biological cell described in C4) may specifically be a hybridoma cell. The hybridoma cell is obtained by fusing spleen cells with SP2 / 0 cells.

[0022] A product for detecting human adenovirus according to a third aspect of the present invention comprises the above-described monoclonal antibody.

[0023] According to some embodiments of the present invention, the product further includes one of enzyme-linked immunosorbent assay (ELISA) reagents, immunohistochemical reagents, immunofluorescence reagents, immunoblotting reagents, and immunoaffinity chromatography reagents. It is understood that the product of the present invention is not limited to the reagents exemplified above. Reagents for other methods commonly used in the art for detecting target viral or adenovirus DNA-binding proteins can also be used in the present invention.

[0024] According to some embodiments of the present invention, the product further includes a polyclonal antibody obtained by immunizing animals with a recombinant Hexon protein with an amino acid sequence as shown in SEQ ID NO.1.

[0025] According to some embodiments of the present invention, the method of using the product includes the following steps: Prepare a viral lysate containing the sample to be tested, and use the product to detect human adenovirus.

[0026] According to some embodiments of the present invention, the viral lysis buffer is used to lyse the virus. Each 100 mL of the viral lysis buffer comprises: 450 mmol–550 mmol Tris-HCl, 80 mmol–120 mmol EDTA, 3 mol–7 mol NaCl, and 0.8 g–1.2 g SDS. The pH of the viral lysis buffer is 7.0–8.0.

[0027] According to some embodiments of the present invention, the method for preparing the virus lysis buffer includes: mixing 15%~20% Tris-HCl solution, 20%~30% EDTA solution, 5%~15% NaCl solution, SDS, and 40%~50% water by volume fraction. The pH of the Tris-HCl solution is 8.0.

[0028] The use of the monoclonal antibody or the biological material described above according to the fourth aspect of the present invention in the preparation of products for detecting human adenovirus.

[0029] According to some embodiments of the present invention, the human adenovirus includes at least one of human adenovirus type 3, 4, 5, 7 and 55.

[0030] According to the fifth aspect of the present invention, the monoclonal antibody, the biological material, or the product described above are used for immunoassay and other experiments or procedures involving specific binding of antigen and antibody.

[0031] According to some embodiments of the present invention, the antigen includes at least one of human adenovirus type 3, 4, 5, 7, 55, human adenovirus DNA-binding protein, and human adenovirus Hexon protein.

[0032] The present invention has the following beneficial effects: The monoclonal antibodies 1F10 and 8D3 in the examples can specifically bind to multiple types of human adenoviruses (including human adenovirus types 3, 4, 5, 7 and 55) with high affinity, and have high sensitivity and low detection limits. They have clinical application value for broad-spectrum detection of human adenovirus infection and can be used to diagnose human adenovirus infection or diseases related to adenovirus infection (e.g., human adenovirus pneumonia).

[0033] Furthermore, this invention also found that pretreatment of the test sample with viral lysis buffer can further improve the sensitivity of monoclonal antibodies 1F10 and 8D3 to human adenovirus. This invention further found that combining monoclonal antibodies 1F10 and 8D3, or combining monoclonal antibody 1F10 with a polyclonal antibody obtained by immunization with Adv-Hexon antigen, for detection using a double-antibody sandwich ELISA method can further improve the detection sensitivity for human adenovirus.

[0034] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The results of homology analysis of amino acid sequences 520-975 of the human adenovirus Hexon protein are shown; where A is the molecular phylogenetic tree and B is the homology statistics. Figure 2 Results of hydrophilicity analysis of amino acid sequences 520-975 of the human adenovirus Hexon protein; Figure 3Results of B-cell linear epitope analysis of amino acid sequence 520-975 of human adenovirus Hexon protein; Figure 4 The results of SDS-PAGE analysis of recombinant Hexon protein expression; Figure 5 SDS-PAGE results for 1F10 and 8D3 monoclonal antibodies; Figure 6 The results show the binding activity of monoclonal antibody 1F10 (Example 1) against recombinant Hexon protein (A), inactivated human adenovirus type 3 (B), and inactivated human adenovirus type 55 (C), and monoclonal antibody 8D3 against recombinant Hexon protein (D), inactivated human adenovirus type 3 (E), and inactivated human adenovirus type 55 (F). Figure 7 The results show the binding activity of monoclonal antibody 1F10 in Example 1 against inactivated human adenovirus type 3 (A) and inactivated human adenovirus type 55 (B) after dilution with lysis buffer, and the binding activity of monoclonal antibody 8D3 against inactivated human adenovirus type 3 (C) and inactivated human adenovirus type 55 (D) after dilution with lysis buffer. Figure 8 The results of Western blot analysis of monoclonal antibodies 1F10 (A) and 8D3 (B) from Example 1 against different human adenovirus serotypes are shown; C represents the results of positive control and internal control. Figure 9 The results of double-antibody sandwich ELISA detection of different antigens using different antibody combinations in Example 4 are shown. Among them, A, B, and C are the detection results of Hexon antigen, human respiratory adenovirus type 3, and human respiratory adenovirus type 55, respectively, when monoclonal antibody 1F10 is used as the capture antibody and enzyme-labeled antibody 8D3 is used as the detection antibody; D, E, and F are the detection results of Hexon antigen, human respiratory adenovirus type 3, and human respiratory adenovirus type 55, respectively, when polyclonal antibody Hexon-421 is used as the capture antibody and enzyme-labeled antibody 1F10 is used as the detection antibody. Detailed Implementation

[0036] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0037] In the description of this invention, the terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion, for example, a process, method, system, product, or device that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or device.

[0038] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are generally performed in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Where specific conditions are not specified in the examples, they are performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0039] Unless otherwise specified, "room temperature" in this invention means 25℃±5℃.

[0040] The term "single-chain antibody" refers to an antibody fragment containing both a heavy chain variable region and a light chain variable region. Typically, single-chain antibodies also include a linker polypeptide between the heavy chain and light chain variable regions, which enables the single-chain antibody to form a desired structure for antigen binding.

[0041] The term "fusion antibody" refers to a product obtained by fusing a single-chain antibody with a biologically active functional protein molecule using genetic engineering techniques. Fusion antibodies do not affect the binding ability of the single-chain antibody to the antigen, nor do they affect the biological activity of the functional protein molecule to which it is fused.

[0042] The term "Fab" refers to an antibody fragment formed by an intact light chain (the variable region (VL) and the constant region (CL) of the light chain) through interchain disulfide bonds with an Fd fragment (the variable region (VH) of the heavy chain and the first constant region (CH1) of the heavy chain).

[0043] The term "intact antibody" consists of two identical heavy and light chains, each containing a variable region and one or more constant regions (C regions). The variable region is responsible for binding to the antigen, while the constant region is primarily responsible for binding to effector molecules. It can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0044] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. In the aforementioned biological materials, a greater than 90% identity can be defined as a nucleic acid molecule with at least 91%, 92%, 95%, 96%, 98%, or 99% identity.

[0045] The terms "human adenovirus" and "HAdV" refer to non-enveloped linear double-stranded DNA viruses, consisting of a viral capsid and internal nucleic acid. Each adenovirus particle primarily contains three capsid proteins: 240 hexagonal trimers, 12 pentameric trimers, and 12 ciliated trimers. The capsid also contains four minor proteins: IIIa, VI, VIII, and IX, while the core proteins include V, VII, Mu, TP, IVa2, and Protease. The two terms have the same meaning and are used interchangeably.

[0046] The term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules; that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen.

[0047] The term "myeloma cell" refers to a cell formed by the fusion of myeloma cells and B lymphocytes. Hybridoma cells are generally prepared through tumor cell culture. The technique for preparing hybridoma cells refers to the phenomenon of merging two or more cells to form a single cell; it allows nuclei from two different cell sources to express function within the same cell. By fusing two cell types while simultaneously maintaining the main characteristics of both, namely, mouse spleen cells immunized with an antigen and mouse myeloma cells, hybridoma cells are created. The main characteristics of the B lymphocytes are their antibody secretion function and ability to grow in selective culture media; while the mouse myeloma cells can divide and proliferate indefinitely under culture conditions, exhibiting so-called immortality. Under the influence of selective culture media, only hybrid cells formed by the fusion of B lymphocytes and myeloma cells possess the ability to proliferate continuously, forming cells that simultaneously possess both antibody secretion function and the characteristic of cell immortality.

[0048] The term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a rate of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10An antibody with an affinity of M or less (KD) binds to the antigen. The term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.

[0049] As used in this article, the term "Da" is a commonly used unit of molecular weight. Da stands for Dalton, and it is the algebraic sum of the atomic masses of all atoms in a molecule. Proteins are large molecules, so they are often represented by kDa (kilodaltons). The Dalton is a unit of atomic mass, and it is frequently used as D or KD in biochemistry, molecular biology, and proteomics.

[0050] In this invention, amino acids are typically represented using single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0051] Analysis of the conservation of human adenovirus antigens To obtain antibodies with broad-spectrum binding ability, the amino acid sequences of all proteins of human adenovirus type 2 and 7 were obtained from the Genebank database. The sequences were then aligned and analyzed for homology using ClustalX in BioEdit software, and the polypeptide sequences with the highest homology (amino acids 520 to 975 of the Hexon protein) were selected.

[0052] Peptide sequences of human adenoviruses (types 2, 4, 5, 7, 11, and 21) were obtained from the Genebank database. After alignment and homology analysis, amino acids 520-975 of the Hexon protein were identified as the most conserved peptide sequence for these types of human adenoviruses. Phylogenetic analysis of amino acid positions 520-975 of the human adenovirus Hexon protein was performed using the Neighbor-Joining method and the JTT model to construct a molecular phylogenetic tree for the peptide sequences. Subsequently, the hydrophilicity of the peptide sequences was analyzed using the ProtScale tool from the Expasy website. Finally, B-cell linear epitope prediction was performed using IEDB tools to obtain highly conserved peptides that are easily expressed and possess antigenicity.

[0053] Analysis results as follows Figures 1 to 3 As shown.

[0054] The amino acids 520-975 of the hexon protein of human adenovirus have the lowest homology (90.09%) among human adenovirus types 2, 4, 5, 7, 11, and 21; it is generally hydrophilic; and it contains a large number of antigenic determinant regions, thus exhibiting immunogenicity.

[0055] Prokaryotic expression, purification and refolding of recombinant Hexon protein A recombinant plasmid (Human adenovirus 7EHexon plasmid) with the pET-28α(+) prokaryotic expression vector as its backbone was constructed, which can express recombinant Hexon protein (a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, where amino acids 520-975 of the Hexon protein are represented). The relative molecular mass of this recombinant Hexon protein is approximately 51905 Da.

[0056] NTYEYMNGRVVSPSLVDSYINIGARWSLDPMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAVKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRTDGATISFTS INLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNIPISIPSRNWAAFRGWSFTRLKTKETPSLGSGFDPYFVYSGSIPYLDGTFYLNHTFKKVSIMFDS SVSWPGNDRLLSPNEFEIKRTVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPEGYKDRMYSFFRNFQPMSRQVVDEVNYTDYKAVTLPYQHNNSGFVGYLAPTMRQGEP YPANYPYPLIGTTAVKSVTQKKFLCDRTMWRIPFSSNFMSMGALTDLGQNMLYANSAHALDMTFEVDPMDEPTLLYLLFEVFDVVRVHQPHRGVIEAVYLRTPFSAGNATT (SEQ ID NO.1).

[0057] Transform the recombinant plasmid into E. coli E. coli In BL21(DE3), the cells were cultured overnight in LB agar dishes containing the corresponding antibiotics. Single colonies were then inoculated into LB liquid medium (containing 50 mg / L kanamycin). After large-scale activation culture, the OD of the bacterial culture was measured. 600When the protein concentration was 0.4–0.6, expression was induced with 0.5 mM IPTG. After centrifugation, the bacteria were harvested, resuspended in PBS buffer, and 1 mg / mL lysozyme was added. The mixture was then incubated on ice for 30 min, followed by sonication. After centrifugation, the supernatant and precipitate were collected for SDS-PAGE analysis. The analysis revealed that the recombinant protein was expressed as inclusion bodies in prokaryotic cells. Further purification was performed using His-tagged proteins via agarose gel permeation column purification. The protein was then refolded using a urea gradient dialysis method. SDS-PAGE electrophoresis was used to assess the expression, purification, and refolding effects.

[0058] The results are as follows Figure 4 As shown.

[0059] Depend on Figure 4 It can be seen that, through expression, purification and refolding, a recombinant Hexon protein with high purity (denoted as: Adv-Hexon antigen) with a size of 51905 Da was obtained.

[0060] It should be noted that the Adv-Hexon antigen is not limited to the method of this embodiment, but can also be prepared by conventional methods in the art. Of course, commercially available Adv-Hexon antigens can also be purchased.

[0061] Establishment of hybridoma cell lines and preparation of monoclonal antibodies (1) Purified and refolded Adv-Hexon antigen was used to repeatedly immunize 6-8 week old female BALB / c mice. The antigen dose of 50 μg / mouse was mixed with an equal volume of complete Freund's adjuvant and emulsified, and then injected subcutaneously at multiple sites (400 μL / mouse) to obtain mice for primary immunization. Subsequently, mice were immunized with an equal volume of incomplete Freund's adjuvant mixed with antigen (50 μg / mouse, 400 μL / mouse) for a total of 4 immunizations (including the primary immunization), with an interval of 2 weeks between each immunization. On the 12th day after the third immunization, blood was collected from the orbital cavity of the mice, and the serum titer was detected by indirect ELISA. The serum obtained by collecting blood from the orbital cavity of the mice before the first immunization was used as a negative control. When the immunotiter reached 100,000 or higher, the mice with the highest titer were selected 3 days before cell fusion. The mice were injected intraperitoneally with an antigen dose of 50 μg / mouse mixed with an equal volume of incomplete Freund's adjuvant (400 μL / mouse) to obtain mice for shock immunization. Three days after the shock immunization, spleen cells were collected from the shock immunized mice and fused with SP2 / 0 cells to obtain fused cells (hybridoma cells).

[0062] The specific steps of the indirect ELISA method are as follows: ① Coat a 96-well microplate with 100 μL / well of 1 μg / mL Adv-Hexon antigen solution (solvent: 1×PBS) and incubate at 37°C for 2 h. After washing, block the 96-well microplate with 200 μL / well of 5% skim milk powder solution and incubate at 37°C for 2 h or at 4°C overnight.

[0063] ② Mouse serum treated in a 56℃ metal bath for 30 min was used as the antibody sample to be tested, and serially diluted. The diluted antibody sample was added to each well and incubated at 37℃ for 30 min. After washing, 100 μL of HRP-goat anti-mouse IgG (diluted 1:5000) was added to each well and incubated at 37℃ for 30 min. After thorough washing, 100 μL of LELISA reagent was added to each well and incubated at 37℃ in the dark for 10-30 min. Then, 50 μL of H2SO4 solution was added to terminate the reaction, and OD was detected within 15 min. 450 Sample well OD 450 OD of negative control well 450 A ratio > 2.1 is considered positive.

[0064] (2) Take 50 μL of cell supernatant obtained from the culture of hybridoma cells as the test sample, and screen hybridoma cells according to the indirect ELISA method established above. Use the limiting dilution method to perform subcloning of the identified positive hybridoma cell lines 2-3 times, and finally select strong positive single cell lines against human adenovirus Hexon protein for expansion culture.

[0065] (3) One week in advance, inject 500 μL / mouse with incomplete Freund's adjuvant (or paraffin oil), and each mouse can be injected with 5 × 10 5 ~1×10 6 After injecting hybridoma cells (injection volume not exceeding 500 μL), ascites fluid was collected and diluted to remove impurities. The ascites fluid was then purified using affinity chromatography (Protein A agarose purification resin). The monoclonal antibodies secreted by the hybridoma cells were labeled 1F10 and 8D3, respectively. 20 μL of each antibody was added to 5 μL of SDS-PAGE Loading Buffer (5×) (catalog number: G3422-2), and the mixture was incubated in a metal bath at 100℃ for 10 min. The antibodies unwind at high temperature, and the heavy and light chains dissociate. The gel was prepared using the AmyKD-PAGE gel rapid preparation kit (catalog number: ATG0046), with a sample loading volume of 20 μL, and run at 90V for 120 min.

[0066] SDS-PAGE test results are as follows: Figure 5 As shown.

[0067] The heavy and light chains of the 1F10 and 8D3 monoclonal antibodies dissociated, showing two bands, with the heavy chain at approximately 55 kD and the light chain at approximately 25 kD.

[0068] Hybridoma cells prepared according to the above method can continuously and efficiently secrete monoclonal antibodies 1F10 and 8D3, and the method for preparing hybridoma cells is simple and easy to perform. Compared with in vitro culture, in vivo culture of hybridoma cells in mice can effectively reduce the impact of adverse external environmental factors on hybridoma cell proliferation and monoclonal antibody expression, providing a relatively stable survival environment for hybridoma cells, making them less susceptible to contamination and resulting in high production efficiency.

[0069] Example 1 This embodiment provides two monoclonal antibodies, namely monoclonal antibody 1F10 and monoclonal antibody 8D3.

[0070] The hybridoma cells prepared above were cultured in T75 cell culture flasks to a density of 70%–90%, collected by centrifugation, washed once with 10 mL of 1×PBS, centrifuged again, and the washing and centrifugation were repeated once more, reserving the hybridoma cells for later use. RNA was extracted from the hybridoma cells using the AGSteadyPure Universal RNA Extraction Kit II (catalog number AG21022) (which can be stored at -80℃ for later use). The extracted RNA was then reverse transcribed into cDNA using the Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit (catalog number 6210A) (which can be stored at -20℃ or -80℃ for later use). Subsequently, PCR amplification of the light and heavy chains of the 1F10 and 8D3 monoclonal antibodies was performed using the Takara Taq™ Version 2.0 plus dye premixed enzyme (catalog number RR901A). The amplified PCR products were analyzed by 1%–2% agarose gel electrophoresis, and bands of 300 bp–500 bp were sent for sequencing. The sequencing results were analyzed on the VBASE2 website to obtain the heavy and light chain variable region sequences of monoclonal antibody 1F10 and monoclonal antibody 8D3.

[0071] The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 58℃ annealing for 40 s, 72℃ extension for 1 min, for a total of 35 cycles; and 72℃ extension for 10 min.

[0072] The primers used for PCR amplification are shown in Table 1. Heavy chain downstream primers were combined with 15 heavy chain upstream primers for sequencing; light chain downstream primers were combined with 16 light chain upstream primers for sequencing.

[0073] Table 1

[0074] The monoclonal antibody 1F10 consists of a light chain and a heavy chain. The variable region of the heavy chain contains three complementarity-determining regions (CDR1, CDR2, and CDR3). The amino acid sequence of CDR1 is GFTFSSYG (SEQ ID NO.2), the amino acid sequence of CDR2 is VSSGGSYT (SEQ ID NO.3), and the amino acid sequence of CDR3 is AGGLSFDY (SEQ ID NO.4). The variable region of the light chain contains three complementarity-determining regions (CDR1', CDR2', and CDR3'). The amino acid sequence of CDR1' is QNILHSNGNTY (SEQ ID NO.5), the amino acid sequence of CDR2' is KVS (SEQ ID NO.6), and the amino acid sequence of CDR3' is FQGSHVPLT (SEQ ID NO.7).

[0075] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1F10: EVQLVESGGDLVKPGGSLKLSCAASGFTFSSYGMSWIRQTPDKRLEWVATVSSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCAGGLSFDYWGQGTTLTVSS (SEQ ID NO. 8).

[0076] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1F10: DVLMTQTPLSLPVSLGDQASISCRSSQNILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEL (SEQ ID NO. 9).

[0077] The monoclonal antibody 8D3 consists of a light chain and a heavy chain. The variable region of the heavy chain contains three complementarity-determining regions (CDR1, CDR2, and CDR3). The amino acid sequence of CDR1 is GYTFTDYS (SEQ ID NO.10), the amino acid sequence of CDR2 is INTTTGEP (SEQ ID NO.11), and the amino acid sequence of CDR3 is TRWWLLRRWFAY (SEQ ID NO.12). The variable region of the light chain contains three complementarity-determining regions (CDR1', CDR2', and CDR3'). The amino acid sequence of CDR1' is QDVSTA (SEQ ID NO.13), the amino acid sequence of CDR2' is SAS (SEQ ID NO.14), and the amino acid sequence of CDR3' is QQHYSTPLT (SEQ ID NO.15).

[0078] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 8D3: QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWMKQTPGKGLKWMGWINTDTGEPTYADDFRGRFAFSLETSASTAFLQINNLKNEDTATFFCTRWWLLRRWFAYWGQGTLVTVSA (SEQ ID NO. 16).

[0079] The amino acid sequence of the variable region of the light chain of monoclonal antibody 8D3: DIVMTPSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTRVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK (SEQ ID NO. 17).

[0080] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.18: GAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGATTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCGTTAGTAGTGGTGGTAGTTACA CCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCGGGAGGGCTAAGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO. 18).

[0081] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F10 is shown in SEQ ID NO.19: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTTTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAG TTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTG (SEQ ID NO. 19).

[0082] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 8D3 is shown in SEQ ID NO.20: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCACTGGATGAAGCAGACTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGACACTGGTGAGCCAACATATGCAGATGACTTCAGGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTTTTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATTTTTCTGTACTAGATGGTGGTTACTACGTCGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO.20).

[0083] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 8D3 is shown in SEQ ID NO.21: GACATTGTGATGACCCCGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGATACACTAGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO.21).

[0084] Example 2 This embodiment refers to the indirect ELISA method established in "Establishment of Hybridoma Cell Lines and Preparation of Monoclonal Antibodies" above. Recombinant Hexon protein (Hexon-7), inactivated human adenovirus type 3, and inactivated human adenovirus type 55 were used as coating antigens, respectively. Monoclonal antibody 1F10 and monoclonal antibody 8D3 from Example 1 were used as antibody samples to be tested. The binding activity of monoclonal antibody 1F10 and monoclonal antibody 8D3 from Example 1 with recombinant Hexon protein, inactivated human adenovirus type 3, and inactivated human adenovirus type 55 was detected by enzyme-linked immunosorbent assay (ELISA). NC-Ab (monkeypox monoclonal antibody) was used as a negative control antibody.

[0085] Test results as follows Figure 6 As shown.

[0086] The minimum reactive limits of monoclonal antibodies 1F10 and 8D3 against recombinant Hexon protein are 305.176 pg / mL and 305.176 pg / mL, respectively. Figure 6 As shown in A and D.

[0087] Monoclonal antibodies 1F10 and 8D3 both exhibited good binding activity against inactivated human adenovirus type 3 and inactivated human adenovirus type 55. The minimum reactive limits of monoclonal antibody 1F10 against inactivated human adenovirus type 3 and inactivated human adenovirus type 55 were 625 ng / mL and 625 ng / mL, respectively. Figure 6 As shown in Figures B and C. The minimum reactive limits of monoclonal antibody 8D3 against inactivated human adenovirus type 3 and inactivated human adenovirus type 55 are 1250 ng / mL and 2500 ng / mL, respectively. Figure 6 As shown in E and F.

[0088] Example 3 Considering that antibody recognition epitopes may not be exposed on the antigen surface, this embodiment refers to the indirect ELISA method established in "Establishment of Hybridoma Cell Lines and Preparation of Monoclonal Antibodies" above. Inactivated human adenovirus type 3 and inactivated human adenovirus type 55 diluted with lysis buffer were used as coating antigens, respectively. Monoclonal antibody 1F10 and monoclonal antibody 8D3 from Example 1 were used as antibody samples to be tested, and verification was performed by enzyme-linked immunosorbent assay (ELISA). NC-Ab (monkeypox monoclonal antibody) was used as a negative control antibody.

[0089] The lysis buffer was prepared as follows: 20 mL of Tris-HCl (pH 8.0, 500 mmol), 25 mL of 100 mmol EDTA solution, 10 mL of 5 mol NaCl solution and 1 g of SDS powder were mixed and then diluted to 100 mL with deionized water.

[0090] Test results as follows Figure 7As shown.

[0091] Monoclonal antibodies 1F10 and 8D3 showed improved binding ability to adenovirus antigen diluted with lysis buffer. Taking human adenovirus type 3 and 55 as examples, the minimum reaction limit of monoclonal antibody 1F10 against human adenovirus type 3 was reduced from 625 ng / mL. Figure 6 The concentration of B in the blood decreased to 312.5 ng / mL. Figure 7 (A), the minimum response limit of monoclonal antibody 8D3 to it is 1250 ng / mL ( Figure 6 The concentration of E in the middle reaches of the medium decreased to 625 ng / mL. Figure 7 (C); while for human adenovirus type 55, the minimum response limit of monoclonal antibody 1F10 is 625 ng / mL ( Figure 6 The concentration of C dropped to 312.5 ng / mL. Figure 7 (B), the minimum response limit of monoclonal antibody 8D3 to it is 2500 ng / mL ( Figure 6 The concentration of nitric acid (NF) decreased to 625 ng / mL. Figure 7 (D); the negative control antibody did not show specific binding activity.

[0092] Example 4 This embodiment uses Western blot to detect the reactivity of monoclonal antibodies 1F10 and 8D3 from Example 1 against different human adenoviruses. The specific detection method is as follows: Gel preparation was performed using the AmyKD-PAGE rapid gel preparation kit (catalog number: ATG0046). Human respiratory adenovirus types 3, 4, 5, 7, and 55 were used as the antigens to be detected. ADV DBP (the product of adenovirus E2 region gene expression, E2A, i.e., DNA-binding protein) was used as a positive control, and GADPH was used as a standardized internal control. After gel running, the gel was transferred to a polyvinylidene fluoride (PVDF) membrane using a semi-dry blot apparatus. The membrane was washed three times with 0.1% Tween 20 solution (solvent: 1×PBS) on a shaker for 5 min each time, and then blocked slowly on a shaker at room temperature for 2 h with 5% skim milk powder solution (solvent: PBST solution). After washing the membrane three times on a shaker with 0.1% Tween 20 solution (solvent: 1×PBS), it was slowly incubated on a shaker at 4°C for 6–8 h with monoclonal antibody 1F10 and monoclonal antibody 8D3 (diluted with 2% skim milk powder solution (solvent: PBST solution)) at a concentration of 2 μg / mL. Then, HRP-labeled goat anti-mouse IgG diluted 1:5000 was added and the membrane was slowly incubated on a shaker at room temperature for 1 h. Finally, it was developed with ECL chemiluminescent solution.

[0093] Test results as follows Figure 8 As shown in the figure. The Mock lane serves as a blank control.

[0094] Monoclonal antibodies 1F10 and 8D3 specifically recognize human adenovirus types 3, 4, 5, 7, and 55, all showing a band at approximately 120 kD. Based on the relative molecular mass of the structural proteins of human adenoviruses, the protein at 120 kD is inferred to be a Hexon subunit.

[0095] Example 5 This embodiment uses a double-antibody sandwich ELISA method to detect the reactivity of monoclonal antibodies 1F10 and 8D3 from Example 1 against different human adenoviruses. The specific detection method is as follows: ① Coat a 96-well microplate with 10 μL / well of 10 μg / mL capture antibody solution (solvent: 1×PBS) and incubate at 37℃ for 2 h. After washing with 1×PBST solution, block the 96-well microplate overnight at 4℃ with 200 μL / well of 5% skim milk powder solution.

[0096] The capture antibody is either monoclonal antibody 1F10 or polyclonal antibody Hexon-421; the preparation method of polyclonal antibody Hexon-421 is as follows: Mice were immunized with Adv-Hexon antigen (the immunization method is described in "Establishment of Hybridoma Cell Lines and Preparation of Monoclonal Antibodies" above). The serum of mice with the highest titer (1:102400) was collected. Cell debris and small particulate matter in the serum were removed by high-speed centrifugation, and the supernatant was collected. An equal volume of saturated ammonium sulfate solution was added to the supernatant, and the mixture was stirred at 4°C for 4-6 h to allow for sufficient precipitation. The mixture was then centrifuged at 12000 rpm for 15 minutes, and the supernatant was discarded. The precipitate was reconstituted with an appropriate amount of 1×PBS, and dialyzed with 1×PBS for 24 h to remove ammonium sulfate, thus obtaining polyclonal antibody Hexon-421.

[0097] ② Use 2 μg / mL Adv-Hexon antigen solution (solvent: 1×PBS) as the antigen to be tested and perform serial dilutions; use lysis buffer (same as in Example 3) to lyse human respiratory adenovirus type 3 (1.127×10⁻⁶) 4 PFU / mL) and human respiratory adenovirus (4.0999×10) 3 The sample was diluted 10-fold (PFU / mL) as the antigen to be tested, and serial dilutions were performed. NC-Ag (monkeypox A29L antigen) was used as the negative control antigen, and NC-Virus (Taterapox virus) was used as the negative control virus. The above samples were added to the ELISA plate and incubated at 37°C for 30 min.

[0098] ③ After washing, add 100 μL of enzyme-labeled antibody (detection antibody) solution (solvent is 5% skim milk powder) to each well and incubate at 37℃ for 30 min; The enzyme-labeled antibodies were obtained by labeling monoclonal antibody 1F10 and monoclonal antibody 8D3 with Glue-activated horseradish peroxidase (catalog number: MD010A) according to the instructions.

[0099] ④ After thorough washing, add 100 μL of ELISA reagent to each well and incubate at 37°C in the dark for 10-30 minutes. Then, add 50 μL of H2SO4 solution to terminate the reaction. Detect OD within 15 minutes. 450 Sample well OD 450 OD of negative control well 450 A ratio > 2.1 is considered positive.

[0100] Test results as follows Figure 9 As shown.

[0101] When tested using a sandwich ELISA method with monoclonal antibody 1F10 as the capture antibody and enzyme-labeled antibody 8D3 as the detection antibody, this paired antibody pair can detect low concentrations of Hexon antigen (limit of detection is 312.5 ng / mL). Figure 9 As shown in Figure A), human respiratory adenovirus type 3 (lowest detection limit is 36 PFU; as shown in Figure A). Figure 9 (as shown in B) and human respiratory adenovirus type 55 (lowest detection limit is 26 PFU; as shown in B) Figure 9 (As shown in C).

[0102] Using polyclonal antibody Hexon-421 as the capture antibody and enzyme-labeled antibody 1F10 as the detection antibody results in higher sensitivity. The limit of detection for Hexon antigen is 3.906 ng / mL; Figure 9 As shown in D. The limit of detection for human respiratory adenovirus type 3 is 141 PFU; as Figure 9 As shown in Figure E. The limit of detection for human respiratory adenovirus type 55 is 205 PFU; Figure 9 As shown in F.

[0103] In summary, monoclonal antibodies 1F10 and 8D3 not only have excellent broad-spectrum activity, specifically recognizing human adenovirus types 3, 4, 5, 7, and 55, but also exhibit high sensitivity. Therefore, they are of great significance for the clinical development of detection reagents or devices for human adenovirus.

[0104] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An adenovirus monoclonal antibody, characterized in that, It includes a heavy chain variable region and a light chain variable region, which includes: a heavy chain variable region complementary determination region and a light chain variable region complementary determination region selected from A1) or A2); A1) The amino acid sequences are the heavy chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.2~4 and the light chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.5~7, respectively. A2) The amino acid sequences are the heavy chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.10~12, and the amino acid sequences are the light chain variable regions CDR1, CDR2 and CDR3 shown in SEQ ID NO.13~15.

2. The adenovirus monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.8, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

9.

3. The adenovirus monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.16, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

17.

4. The adenovirus monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is any one of B1), B3), or B4. B1) A single-chain antibody obtained by linking the heavy chain variable region of claim 1 and the light chain variable region of claim 1; B3) Fab containing the heavy chain variable region as described in claim 1 and the light chain variable region as described in claim 1; B4) A complete antibody containing the heavy chain variable region and the light chain variable region as described in claim 1.

5. A biomaterial relating to the monoclonal antibody as described in any one of claims 1 to 4, characterized in that, The biomaterial is any one of C1) to C4); C1) A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4; C2) An expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) or the expression cassette described in C2); C4) A recombinant biological cell containing the nucleic acid molecule described in C1), the expression cassette described in C2), or the recombinant vector described in C3).

6. The biomaterial according to claim 5, characterized in that, C1) The nucleotide sequence of the nucleic acid molecule is more than 90% identical to that of the nucleic acid molecule shown in D1) or D2) and encodes the monoclonal antibody; D1) The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.18, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.19; D2) The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.20, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

21.

7. A product for detecting human adenovirus, characterized in that, Including the monoclonal antibody as described in any one of claims 1 to 4.

8. The product according to claim 7, characterized in that, The product is selected from one of the following: enzyme-linked immunosorbent assay (ELISA) reagents, immunohistochemical assay reagents, immunofluorescence assay reagents, and immunoblotting assay reagents.

9. The use of the monoclonal antibody according to any one of claims 1 to 4, or the biological material according to any one of claims 5 to 6, in the preparation of products for detecting human adenovirus.

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