Anti-human adenovirus antibody, and preparation method therefor and use thereof

Abstract

An anti-human adenovirus (AdV) antibody, a preparation method, a kit comprising the antibody, and the related use thereof. The anti-human adenovirus antibody can specifically bind hexon proteins of various types of adenoviruses, shows high sensitivity and high specificity for adenoviruses, can be used for detecting various types of adenoviruses, greatly increases the detection rate of adenoviruses, reduces the detection cost, and provides a feasible scheme for rapid detection of adenoviruses in the respiratory tract and the digestive tract.

Description

Anti-human adenovirus antibody and its preparation method and application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an anti-human adenovirus (AdV) antibody, a preparation method, a kit comprising the antibody, and related applications. Background Art

[0002] Adenovirus particles are non-enveloped spherical structures with a diameter of 70 to 90 nm. Each virus particle contains a 36 kb linear double-stranded DNA. The adenovirus capsid has icosahedral symmetry and is composed of 252 capsomers with a diameter of 8 to 10 nm. The 12 vertebral capsomers of the icosahedron are pentons. In addition to the pentons, there are 240 non-vertebral capsomers, called hexons. Hexons carry primary genus- and subgenus-specific antigenic determinants and secondary species-specific antigenic determinants, which can serve as diagnostic criteria for different serotypes.

[0003] Adenoviruses are widely distributed in nature. There are seven groups of adenoviruses, AG, that can infect humans. Currently, there are 55 different serotypes known, of which the most common pathogenic types are types 1-8. Adenovirus type 55 is a new virus produced by the recombination of human adenovirus types 11 and 14.

[0004] Adenoviruses are primarily transmitted through airborne droplets, though most types can also spread through the digestive tract. Adenovirus infection in humans can cause a variety of illnesses, including acute upper respiratory tract infection (ARI), adenovirus pneumonia, fulminant conjunctivitis, acute hemorrhagic cystitis, and infant gastroenteritis. Adenovirus pneumonia accounts for approximately 10% of childhood pneumonia cases, most commonly in infants aged 6 months to 2 years. It is a serious disease and a leading cause of death and disability from pneumonia in infants and children. Types 3 and 7 are the most common types of adenovirus pneumonia in infants and children, while type 55 is the most common type causing community-acquired adenovirus pneumonia in adults.

[0005] Currently, the main methods for diagnosing adenovirus infection include virus isolation and serological identification, viral nucleic acid detection, and antigen detection. Traditional virus isolation and serotyping methods are the gold standard for adenovirus diagnosis, but are not suitable for early clinical diagnosis. Viral nucleic acid detection has high sensitivity but is expensive and requires strict laboratory conditions. Antigen detection targeting the adenovirus capsid hexon, using nasopharyngeal swabs, sputum, or stool as specimens, can be highly effective within 3-5 days of onset.

[0006] my country is a vast country, and adenovirus respiratory infections can occur year-round. Adenovirus infection patterns exhibit complex and differentiated epidemiological characteristics across time and space. The dominant adenovirus strains prevalent in different regions vary in type and time of year: they are more common in winter and spring in northern my country, and more common in spring and summer in southern China. The dominant strains in the north, northwest, and south belong to the HAdV-B group, primarily types 3 and 7; while the dominant strains in the east belong to the HAdV-C group, primarily types 1 and 2. Furthermore, in recent years, localized outbreaks of the novel adenovirus type 55 have occurred in various locations in both southern and northern my country. Enteric adenoviruses, primarily types 40 and 41 of the F subgroup, are the second leading cause of diarrhea in children, after rotavirus.

[0007] The regional and temporal variations in the prevalence of adenovirus types have hindered the rapid diagnosis of adenovirus infection. Currently, a single antibody can typically detect only one type of adenovirus, necessitating the use of multiple antibodies to improve detection rates when testing samples such as throat swabs or stool. This increases testing costs and still carries a certain risk of false negatives.

[0008] Therefore, there is a need for an adenovirus detection product and method that can simultaneously detect multiple types with high specificity and sensitivity.

[0009] Summary of the Invention

[0010] As mentioned above, there is a need in the art for an anti-adenovirus antibody that has high specificity and high sensitivity and is applicable to multiple types of adenoviruses.

[0011] Considering that the hexon proteins of different adenovirus types are highly conserved (78% to 95%), and that inter-type and tissue-specific epitopes are located in these conserved regions and may have certain antigenicity, the inventors used the hexon protein as a target site for antigenic diagnosis of adenovirus infection. They immunized mice with the hexon protein (amino acid sequence: SEQ ID NO: 62) of adenovirus type 3 (HAdV3), fused the mouse spleen cells with myeloma cells, and screened for hybridoma cell lines that specifically bound to the hexon proteins of multiple adenovirus types using ELISA. Recombinant antibodies were then constructed to obtain recombinant anti-adenovirus antibodies targeting the hexon protein of adenovirus. This led to the present invention.

[0012] Therefore, in a first aspect, the present invention provides an anti-adenovirus monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0013] a) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, respectively; H CDR1, V H CDR2 and VH CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6, respectively. L CDR1, V L CDR2 and V L CDR3;

[0014] b) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 5 and SEQ ID NO: 10, respectively. L CDR1, V L CDR2 and V L CDR3;

[0015] c) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3;

[0016] d) the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 12-14, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 15-17, respectively. L CDR1, V L CDR2 and V L CDR3;

[0017] e) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 18-20, respectively H CDR1, V HCDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 21, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0018] f) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 25-27, respectively. L CDR1, V L CDR2 and V L CDR3;

[0019] g) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 28, and SEQ ID NO: 29, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3;

[0020] h) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 32-34, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 35-37, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0021] i) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 38-40, respectively H CDR1, V HCDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 41-43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0022] In a second aspect, a nucleic acid molecule is provided, which encodes the anti-adenovirus monoclonal antibody according to the first aspect.

[0023] In a third aspect, a vector is provided, comprising the nucleic acid molecule described in the second aspect.

[0024] In a fourth aspect, an expression cell is provided, comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0025] In a fifth aspect, a method for detecting adenovirus is provided, comprising the step of using the anti-adenovirus monoclonal antibody described in the first aspect.

[0026] In a sixth aspect, a method for diagnosing adenovirus infection is provided, comprising the step of using the anti-adenovirus monoclonal antibody described in the first aspect.

[0027] In a seventh aspect, provided is a use of the anti-adenovirus monoclonal antibody of the first aspect in preparing a reagent for detecting adenovirus.

[0028] In an eighth aspect, a kit for detecting adenovirus is provided, comprising the anti-adenovirus monoclonal antibody described in the first aspect and instructions for use.

[0029] In summary, the present invention provides a new anti-adenovirus monoclonal antibody that can specifically bind to the hexon protein of multiple types of adenovirus, including types 1, 2, 3, 4, 7, 40, 41, and 55. It can be used to detect multiple types of adenovirus, showing high sensitivity and high specificity for adenovirus, and can achieve more sensitive detection at picogram concentrations, greatly improving the detection rate of adenovirus and reducing the detection cost, providing a feasible solution for the rapid detection of adenovirus in the respiratory and digestive tracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific implementation or the description of the prior art.

[0031] Figure 1 shows the binding reaction results of the monoclonal antibody A18 of the present invention with the hexon proteins of eight types of adenovirus and the new coronavirus Np protein (control).

[0032] Figure 2 shows the binding reaction results of the monoclonal recombinant antibody A18 of the present invention with the hexon proteins of eight types of adenovirus and the new coronavirus Np protein (control).

[0033] Figure 3 shows the binding reaction results of monoclonal antibodies A54, A15, A5, A7, A13, A1, A27 and A8 prepared according to one embodiment of the present invention with hexon proteins of eight types of adenovirus and SARS-CoV-2 Np protein (control). DETAILED DESCRIPTION

[0034] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the following description is merely illustrative of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. Furthermore, those skilled in the art will understand that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0036] Where a numerical range is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of the range and any other stated or intervening values ​​in the stated range are encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the subject matter.

[0037] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." can generally be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." can also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0038] For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood as being modified in all cases by the term "about", unless otherwise indicated. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that may vary depending on the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0039] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the antibody's isotype can be defined accordingly as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C LComposition. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). For each heavy chain or light chain, its variable region each contains three CDRs, namely CDR1, CDR2, and CDR3. Therefore, each V H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L ) respectively form the antigen binding site.

[0040] The rules for assigning amino acids to regions or domains are defined in several publications: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)); Chothia & Lesk J. Mol. Biol. 1987; 196: 901-917; Chothia et al., Nature 1989; 342: 878-883; Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1): D301-D307.

[0041] The exact boundaries of CDRs have been defined differently according to different systems. The Kabat system not only provides a clear residue numbering system that can be applied to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs are called Kabat CDRs. Chothia discovered that certain sub-portions within the Kabat system CDRs, despite having great diversity at the amino acid sequence level, have almost identical peptide backbone conformations. These sub-portions are called Chothia CDRs, and Chothia CDRs have overlapping boundaries with Kabat CDRs. The above-mentioned overlapping boundaries are further described by Padlan and MacCallum. CDR boundary definitions may not strictly adhere to the above system, such as the AbM definition. In this article, CDRs defined according to any of these systems can be used, although the preferred embodiment uses the antibody numbering system of Chothia et al. to define CDRs.

[0042] As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule can be an immunoglobulin, whether it is a natural immunoglobulin or an immunoglobulin obtained partially or entirely by a synthetic method. The antibody molecule can also include all polypeptides or proteins with an antibody structural binding domain, and antibody fragments with antibody structural domains are molecules such as Fab, scFv, Fv, dAb, Fd, and bifunctional antibodies. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on the antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256 (5517): 495), but can also be obtained using recombinant DNA technology (e.g., see US Patent 4,816,567). As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0043] As used herein, the term "recombinant antibody" refers to an antibody obtained by cloning an antibody gene into an expression vector and then transfecting the expression vector into a suitable host cell line for expression by molecular biology techniques. The recombinant antibody encoding gene may be identical or non-identical to the antibody encoding gene of natural origin. For example, the complete encoding gene of the antibody obtained by immunizing an animal may be cloned into an expression vector for expression, thereby obtaining an antibody identical to the antibody obtained by the immunizing animal. Alternatively, the gene encoding the variable region (including heavy chain variable region and light chain variable region) of the antibody obtained by immunizing an animal may be cloned into an expression vector together with the gene encoding the constant region of the antibody in another species (such as human) source, thereby obtaining an antibody comprising heavy chain and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody with mouse heavy chain and light chain variable region connected to a human constant region. This antibody is commonly referred to as a "chimeric antibody" in the art.

[0044] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen.

[0045] In the present invention, primers are also used to perform PCR amplification of the nucleotide sequence encoding the antibody. In the primer sequence, some sites only involve a single base, such as any one of adenine (A), guanine (G), cytosine (C), and thymine (T), while some sites involve a combination of two, three, or four bases. In this case, these bases are referred to as degenerate bases of each other, which are mainly determined based on the degeneracy of the codons. Degenerate bases can be represented by the letters R, Y, M, K, S, W, H, B, V, D, and N, where R represents A / G, Y represents C / T, M represents A / C, K represents G / T, S represents C / G, W represents A / T, H represents A / T / C, B represents G / T / C, V represents G / A / C, D represents G / A / T, and N represents A / T / C / G.

[0046] As used herein, the terms sequence "identity," "identity," or "homology" have their art-recognized meanings, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art to apply conservative amino acid substitutions in peptides or proteins, and generally can be made without altering the biological activity of the resulting molecule. In general, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0047] As previously mentioned, the present invention aims to provide an anti-adenovirus monoclonal antibody with high sensitivity and specificity against multiple adenovirus types. As previously mentioned, the inventors used the hexon protein as a target site for antigenic diagnosis of adenovirus infection. Mice were immunized with the hexon protein of adenovirus type 3 (HAdV3) (amino acid sequence set forth in SEQ ID NO: 62). The mouse spleen cells were then fused with myeloma cells. Using ELISA, hybridoma cell lines were identified that specifically bound to the hexon proteins of multiple adenovirus types. These hybridoma cell lines were designated A54, A15, A5, A1, A27, A8, A18, A7, and A13, respectively.

[0048] Therefore, in a first aspect, the present invention provides an anti-adenovirus monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0049] a) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3, respectively; H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6, respectively. L CDR1, V L CDR2 and V L CDR3;

[0050] b) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 5 and SEQ ID NO: 10, respectively. L CDR1, V L CDR2 and V L CDR3;

[0051] c) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 8, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 6, respectively.L CDR1, V L CDR2 and V L CDR3;

[0052] d) the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 12-14, H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 15-17, respectively. L CDR1, V L CDR2 and V L CDR3;

[0053] e) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 18-20, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 21, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0054] f) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes light chain complementary determining regions VL CDR1, VL CDR2, and VL CDR3 represented by amino acid sequences of SEQ ID NOs: 25-27, respectively;

[0055] g) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 28, and SEQ ID NO: 29, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and VL CDR3;

[0056] h) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 32-34, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 35-37, respectively. L CDR1, V L CDR2 and V L CDR3; or

[0057] i) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 38-40, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 41-43, respectively. L CDR1, V L CDR2 and V L CDR3.

[0058] In a specific embodiment, the antibody is a complete antibody comprising a variable region and a constant region. For the antibodies of the present invention, any framework region (FR) and any constant region can be used. The amino acid sequence of the FR or constant region used in the antibodies of the present invention can be the amino acid sequence of the original FR or constant region as a source, or it can be a different amino acid sequence obtained by performing one or more amino acid substitutions, deletions, additions and / or insertions on the amino acid sequence of the original FR or constant region. The structure used to support the CDR or CDR group of the present invention generally belongs to an antibody heavy chain or light chain sequence or a major part thereof, wherein the CDR or CDR group is located in a region corresponding to a naturally occurring V sequence encoded by a rearranged immunoglobulin gene. H and V L The CDR or CDR group of the antibody variable domain corresponds to the position of the antibody variable domain.

[0059] As an example, each framework region (FR) may have the following sequence:

[0060] HFR1 (heavy chain framework region 1): QLQQSGPGLVAPSQSLSITCTVS

[0061] HFR2 (heavy chain framework region 2): WMHWVMQRPGQGLEWIGVI

[0062] HFR3 (heavy chain framework region 3): TNYNSALMSRLSISKDNSKSQVFLKMNSLQSEDSAVYYCAR

[0063] HFR4 (heavy chain framework region 4): WGQGTTLTVS

[0064] LFR1 (light chain framework region 1): VVMTQTPLTLSVTIGQPASISC

[0065] LFR2 (light chain framework region 2): WYLQKPGQSPKLLIY

[0066] LFR3 (light chain framework region 3): GVPSRFSSSGSGTDFVFKISRVEAEDLGVYYC

[0067] LFR4 (light chain framework region 4): FGGGTKLEI

[0068] In a specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:44, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:44, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:45, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:45.

[0069] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:46, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:46, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:47.

[0070] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:48, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:48, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:49, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:49.

[0071] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:50, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:51, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:51.

[0072] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:52, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:53, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:53.

[0073] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:54, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:55.

[0074] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:56, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:57, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:57.

[0075] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:58, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:59, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:59.

[0076] In another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence of SEQ ID NO:60, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:60, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:61, or a sequence having greater than 80%, greater than 85%, greater than 90%, or greater than 95%, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence of SEQ ID NO:61.

[0077] In a specific embodiment, the antibody further includes a constant region sequence, such as but not limited to a constant region sequence selected from any one of IgG, IgA, IgM, IgE and IgD. Those skilled in the art can make a selection based on needs, and this is not particularly limited herein.

[0078] In another specific embodiment, the species origin of the constant region sequence can be rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose or human, but is not limited thereto.

[0079] In a specific embodiment, the constant region of the antibody of the present invention is derived from mouse.

[0080] In some embodiments, the species origin of the constant region sequence is mouse.

[0081] In a specific embodiment, the monoclonal antibody targets the hexon protein of adenovirus. The hexon protein is highly conserved (78% to 95%) in adenovirus and carries major genus and subgenus specific antigenic determinants and minor species specific antigenic determinants, and can be used as a standard for diagnosing different serotypes.

[0082] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-adenovirus monoclonal antibody according to the first aspect.

[0083] For those skilled in the art, knowing the amino acid sequence of a protein, such as the anti-adenovirus monoclonal antibody of the present invention, it is well within their capabilities to determine its nucleic acid encoding sequence. Furthermore, to obtain a monoclonal antibody via recombinant means, the nucleic acid molecule can be cloned into a vector, and the vector can be further introduced into an expression cell, which can then express the antibody protein.

[0084] In a third aspect, the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the present invention.

[0085] In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

[0086] In a specific embodiment, the vector is a pTOPO vector.

[0087] In another specific embodiment, the vector is a eukaryotic expression vector.

[0088] In a preferred embodiment, the pcDNA vector may be a pCDNA3.1 vector.

[0089] In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.

[0090] The expression cell is prepared by introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into a host cell by molecular biological methods well known to those skilled in the art.

[0091] As previously mentioned, the present inventors used hexon protein as a target site for antigenic diagnosis of adenovirus infection, immunized mice with hexon protein (Hexon) of type 3 adenovirus (HAdV3), and fused the mouse spleen cells with myeloma cells. Hybridoma cell lines that specifically bind to hexon proteins of various types of adenoviruses were screened by ELISA and named A54, A15, A5, A1, A27, A8, A18, A7, and A13, respectively. After screening for monoclonal cell lines that secrete the target antibody, heavy and light chain variable region cDNAs can be recovered from the cell lines by reverse transcriptase-PCR, and appropriate immunoglobulin constant regions (e.g., human constant regions) can be selected. The heavy and light chain variable region cDNAs and constant region cDNAs are then transferred into host cells such as COS or CHO cells, thereby obtaining cells expressing the target antibody of the present invention. Other antibodies or chimeric molecules that retain the specificity of the original antibody can be produced using monoclonal and other antibody and recombinant DNA techniques. These techniques may include introducing DNA encoding the variable region or complementarity determining region (CDR) of an antibody immunoglobulin into the constant region or constant region plus framework regions of a different immunoglobulin.

[0092] In a specific embodiment, the expressing cells can be mammalian cells, such as Chinese hamster ovary cells, baby hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells. In a more specific embodiment, the expressing cells can be, for example, monkey kidney cells transformed with SV40 (COS-7, ATCC CRL1651), human embryonic kidney cells (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216; e.g., DG44), mouse thymoma cells (NSO), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (HepG2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.

[0093] In a fifth aspect, the present invention provides a method for detecting adenovirus, comprising the step of using the anti-adenovirus monoclonal antibody described in the first aspect.

[0094] In a sixth aspect, a method for diagnosing adenovirus infection is provided, comprising the step of using the anti-adenovirus monoclonal antibody described in the first aspect.

[0095] Those skilled in the art will appreciate that the method for detecting adenovirus can be used for non-diagnostic purposes or diagnostic purposes.

[0096] In a specific embodiment, the detection or diagnosis of adenovirus is achieved by the anti-adenovirus monoclonal antibody of the present invention specifically binding to the adenovirus hexon protein.

[0097] In another specific embodiment, the detection or diagnosis of adenovirus is performed by immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence, or electrochemiluminescence.

[0098] In a preferred embodiment, the detection can be a direct method, an indirect method, a sandwich method, or a competitive method.

[0099] In a preferred embodiment, the immunochromatography includes but is not limited to fluorescent microsphere immunochromatography, colloidal gold immunochromatography, colored latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography.

[0100] In the present invention, the anti-adenovirus monoclonal antibody can be used as a labeled antibody. For example, the anti-adenovirus monoclonal antibody can be combined with magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, etc.

[0101] Without wishing to be bound by theory, the anti-adenovirus monoclonal antibodies of the present invention may also be used as coating antibodies. For example, the anti-adenovirus monoclonal antibodies may be bound to a solid phase, such as a solid support. The solid support used in the detection method of the present invention is not particularly limited and may be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, and the like.

[0102] For example, when performing a colloidal gold immunochromatographic test, the anti-adenovirus monoclonal antibody of the present invention (such as A1, A8, etc., but not limited thereto) can be labeled with colloidal gold, and another anti-adenovirus monoclonal antibody of the present invention (such as antibody A18, but not limited thereto) or the prior art can be coated with a nitrocellulose membrane (NC membrane), and the membrane is drawn to obtain a detection line (T line). According to the preparation method of the immune test strip, the colloidal gold test strip is assembled. During the test, the analyte in the positive sample binds to the colloidal gold-labeled anti-adenovirus monoclonal antibody to form a complex, and the complex binds to the coated antibody at the T line to form a sandwich complex, where the colloidal gold aggregates and precipitates to show red, indicating that the sample is positive.

[0103] Those skilled in the art will appreciate that, in the context of the present invention, "another anti-adenovirus monoclonal antibody" refers to an antibody that binds to the same antigen as the anti-adenovirus monoclonal antibody of the present invention, preferably to a different epitope of the same antigen. Such an antibody may be one of the anti-adenovirus monoclonal antibodies of the present invention or another anti-adenovirus monoclonal antibody in the prior art, but is not limited thereto. It is noteworthy that the anti-adenovirus monoclonal antibody of the present invention, whether used as a coating antibody or a labeled antibody, when used in combination with another anti-adenovirus monoclonal antibody, can detect adenovirus with high sensitivity and specificity.

[0104] In a seventh aspect, the present invention provides use of the anti-adenovirus monoclonal antibody of the first aspect in preparing a reagent for detecting adenovirus.

[0105] In a specific embodiment, the detection of adenovirus is achieved by the anti-adenovirus monoclonal antibody of the present invention specifically binding to the adenovirus hexon protein.

[0106] In another specific embodiment, the detection is performed by immunochromatography, enzyme-linked antibody assay (ELISA), chemiluminescence, or electrochemiluminescence. The immunochromatography may include, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, immunochromatography based on colored latex microspheres, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography. Those skilled in the art can select the detection method according to their needs, and the present invention is not particularly limited thereto.

[0107] In yet another preferred embodiment, ELISA can be a direct method, an indirect method, a sandwich method, or a competitive method.

[0108] As previously mentioned, the anti-adenovirus monoclonal antibodies of the present invention can be used as either labeled antibodies or coated antibodies. For example, the anti-adenovirus monoclonal antibodies can be conjugated to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, and the like, and used as labeled antibodies. In another example, the anti-adenovirus monoclonal antibodies can be conjugated to a solid phase, such as a solid support, and used as a coated antibody. The solid support used in the detection method of the present invention is not particularly limited and can be a porous or non-porous material, such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, and the like.

[0109] In an eighth aspect, the present invention provides a kit for detecting adenovirus, comprising the anti-adenovirus monoclonal antibody described in the first aspect and instructions for use.

[0110] The kit of the present invention can be used in point-of-care testing (POCT) or electrochemical immunoassay systems.The kit according to the present invention and any exemplary form thereof can be used in automated and semi-automated systems and optimized.

[0111] Example

[0112] The following examples illustrate the preparation methods and characterization of the relevant properties of the antibodies of the present invention. Unless otherwise specified, the experimental methods used are all conventional methods, and unless otherwise specified, the experimental materials used in the following examples are all purchased from conventional reagent stores. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0113] It should be noted that the terms used in the description of the present invention are intended only to describe specific embodiments and are not intended to limit the present invention. The above summary of the invention and the detailed description below are intended only to illustrate the present invention and are not intended to limit the present invention in any way. Without departing from the spirit and purpose of the present invention, the scope of the present invention is determined by the appended claims.

[0114] Example 1: Preparation of anti-adenovirus monoclonal antibodies

[0115] Antigen conjugation and immunization: The purified type III adenovirus hexon protein (amino acid sequence: SEQ ID NO: 62: MATPSMMPQWAYMHIAGQDASGYLSPGLVQFARATDTYFSMGNKFRNPTVAPTHDVTTDRSQRLMLRFVPVDREDNTYSYKVRYTLAVGDNRVLDMASTFFDIRGVLDRGPSFKPYSGTAYNSLAPKGAPNTSQWIVTTNGDNAVTTTTNTFGIASMKGGNITKEGLQIGKDITTTEGEEKPIYADKTYQPEPQVGEESWTDTDGTNEKFGGRALKPATNMKPCYGSFARPTNIKGGQAKNRKVKPTTEGGVETEEPDIDMEFFDGRDAVAGALAPEIVLYTENVNLETPDSHVVYKPETSNNSHANLGQQAMPNRPNYIGFRDNFVGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSLGDRTRYFSMWNQAVDSYDPDVRIIENHGIEDELPNYCFPLNGIGPGHTYQGIKVKTDDTNGWEKDANVAPANEITIGNNLAMEINIQANLWRSFLYSNVALYLPDVYKYTPPNITLPTNTNTYEYMNGRVVSPSLVDSYINIGARWSLDPMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAVKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRTDGATISFTSINLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNIPISIPSRNWAAFRGWSFTRLKTKETPSLGSGFDPYFVYSGSIPYLDGTFYLNHTFKKVAIMFDSSVSWPGNDRLLSPNEFEIKRTVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPEGYKDRMYSFFRNFQPMSRQVVDEVNYTDYKAVTLPYQHNNSGFVGYLAPTMRQGEPYPANYPYPLIGTTAVKSVTQKKFLCDRTMWRIPFSSNFMSMGALTDLGQNMLYANSAHALDMTFEVDPMDEPTLLYLLFEVFDVVRVHQPHRGVIEAVYLRTPFSAGNATT) was used as an immunogen for immunizing mice.Female BALB / c mice aged 6-8 weeks were selected. The mice were immunized a total of 4 times, with an interval of 2 weeks between each immunization, and the immunization dose was 100 μg / mouse. For the first immunization, hexon protein was mixed with Freund's complete adjuvant (Sigma-Aldrich) in equal volumes and injected subcutaneously at multiple points on the back. For the next three immunizations, hexon protein was mixed with Freund's incomplete adjuvant (Sigma-Aldrich) in equal volumes and injected intraperitoneally. Seven days after the fourth immunization, the mice were tail-cut and bled, and the serum was separated. The antibody titer of the immunized mouse antiserum was detected by indirect ELISA to observe the immune response effect. Mice with serum antibody titers higher than 1:10,000 were selected for cell fusion experiments. Three days before the cell fusion experiment, the mice were boosted with hexon protein without adjuvant (100 μg / mouse) via intraperitoneal injection.

[0116] Establishment of hybridoma cells: On the day of fusion, the spleen of the immunized mouse was removed under sterile conditions and the organ was made into a single cell suspension. Mouse myeloma cells (SP2 / 0) were fused with the above-mentioned immunized BALB / c mouse spleen cells at a ratio of 1:5, mixed thoroughly, and washed twice before fusion with PEG. Preheated PEG1500 was added, gently shaken, the cells were washed with preheated serum-free RPMI-1640 medium, and then resuspended with HAT selective medium. The cell suspension was plated into a 96-well culture plate at 200 μL / well and the cells were cultured at 37°C and 5% CO2. After culturing for 4 to 7 days, HT medium was used for culture. When the fused cells grew to 1 / 10-1 / 5 of the bottom area of ​​the wells of the 96-well plate, the supernatant was taken for antibody detection.

[0117] Screening of positive hybridoma cells: dilute hexon protein with coating buffer (0.05 mol / L, pH 9.6, PBS) to a final concentration of 1 μg / ml, add 100 μL / well to a 96-well plate, and coat overnight at 4°C; discard the coating solution, wash three times with phosphate buffered saline (PBST), and pat dry; The plates were blocked with BSA in PBST (150 μL / well) and incubated at 37°C for 2 h, washed three times with PBST, and patted dry. The supernatant of the fused cells, 1:1000 diluted positive serum from immune mice (as a positive control), and 1:1000 diluted negative serum from mice (as a negative control) were added to the corresponding wells at 100 μL / well and incubated at 37°C for 1 h, washed three times with PBST, and patted dry. 100 μL / well of 1:4000 diluted horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) was added and incubated at 37°C for 1 h, washed three times with PBST, and patted dry. 100 μL / well of tetramethylbenzidine (3,3',5,5'-Tetramethylbenzidine, TMB) substrate was added and color was developed at room temperature in the dark for 10 min. 50 μL of 2 mol / L sulfuric acid was added to each well to terminate the reaction.

[0118] Detect the OD of all wells in the microplate at a wavelength of 450 nm on a microplate reader. 450nm When the OD of negative serum 450nm ≤0.1, to measure the absorbance value OD of the well 450nm The value is the OD of the negative well 450nm The positive hybridoma cells were selected for cloning.

[0119] Cloning of positive cell lines: After sampling and counting the positive cell wells that secrete antibodies, dilute them to 100 cells / 10mL culture medium, plate the diluted cell suspension at 100μL / well to a 96-well cell culture plate, and culture in a 37°C, 5% CO2 cell culture incubator. After 6-7 days, the formation of cloned cells can be observed under a microscope. Mark the single clone growth well, remove the cell supernatant, perform ELISA test (the same as the above fusion test), and select positive monoclonal cells. Perform limiting dilution on the positive well cells, measure the ELISA value 5-6 days after each limiting dilution, and pick the OD value obtained by ELISA test. 450nm Limiting dilutions were performed on wells containing monoclonal clones with high positive values ​​until the entire 96-well plate was positive by ELISA. Monoclonal clones with high positive values ​​were selected and cloned. Finally, nine cell lines that stably secreted anti-hexon antibodies were obtained and designated hybridoma cell lines A54, A15, A5, A1, A27, A8, A18, A7, and A13.

[0120] Preparation and purification of monoclonal antibodies on cells: Hybridoma cell lines were cultured in RPMI-1640 medium containing 15% serum in 10 cm culture dishes. When the culture was expanded to about 4×10 7 When the cells were 10 cells / dish, centrifuge at 800 rpm for 5 min, discard the supernatant and transfer the cells to a 2 L spinner flask, add serum-free medium, and make the cell density about 3 × 10 5 After 1-2 weeks of culture, when the cell death rate reaches 80%-90% (the cell density is about 1×10 6 -2×10 6 The cell suspension was collected and centrifuged at 6000 rpm for 20 min. The supernatant was purified by Protein A immunochromatography.

[0121] Monoclonal antibodies produced by hybridoma cells are also referred to as A54, A15, A5, A1, A27, A8, A18, A7, and A13 antibodies, respectively.

[0122] The concentrations of the above monoclonal antibodies were all greater than 4 mg / mL as determined by microspectrophotometry. The purified monoclonal antibodies were then aliquoted and stored at 4°C-8°C.

[0123] The monoclonal antibody was identified by SDS-PAGE electrophoresis. The antibody had an antibody heavy chain band of about 51 KD and an antibody light chain band of about 26 KD.

[0124] Purity testing: Monoclonal antibodies were analyzed by size exclusion chromatography (SEC-HPLC). When all components in the sample were detected, the purity percentage of the main peak was calculated using the peak area normalization method. The purity was greater than 98%.

[0125] Example 2: Indirect ELISA method for detecting antibody titer

[0126] The hexon protein was diluted with 0.05 mol / L carbonate buffer at pH 9.6 to a concentration of 1 μg / ml. 100 μL / well was added to a 96-well ELISA plate and coated overnight at 4°C. The plates were washed three times with PBST on an automatic plate washer and patted dry. Blocked with PBST containing 2% BSA, 150 μL / well was incubated at 37°C for 2 h, washed three times with PBST, and patted dry. The nine anti-adenovirus hexon protein monoclonal antibodies obtained in Example 1 were serially diluted with PBS buffer at pH 7.4, 0.02 M. The antibody starting concentration was 5 μg / ml, and serially diluted threefold to obtain a series of monoclonal antibody samples of different concentrations. The diluted monoclonal antibody samples were added to the above ELISA plate at 100 μL / well, incubated at 37°C for 1 h, washed three times, and patted dry. Add 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Sigma) at a dilution of 1:5000, incubate at 37°C for 1 hour, wash three times with PBST, and pat dry. Add 100 μL / well of TMB substrate and develop for 10 minutes at room temperature in the dark. Terminate the reaction by adding 50 μL / well of 2 mol / L sulfuric acid. Measure the OD value using a microplate reader. 450nm The results are shown in Figures 1 and 3. Figure 1 shows the results of the binding reaction of monoclonal antibody A18 with the hexon protein of eight types of adenovirus (Adv) and the Np protein of the new coronavirus (as a control). As can be seen from Figure 1, monoclonal antibody A18 can bind to the hexon protein of eight types of adenovirus, but not to the Np protein of the new coronavirus, and the antibody has good specificity. In addition to antibody A18, the other eight monoclonal antibodies can also bind to the hexon protein of eight types of adenovirus, but not to the Np protein of the new coronavirus (as shown in Figure 3).

[0127] In addition, the ELISA results were further analyzed using software to obtain the EC50 values ​​of each monoclonal antibody against the hexon proteins of eight types of adenovirus. The EC50 values ​​are shown in Table 1 below.

[0128] Table 1: EC50 (nM) values ​​of nine monoclonal antibodies against hexon proteins of eight adenovirus types

[0129] From the data given in Table 1, it can be seen that the affinities of the nine clones to the hexon proteins of various types of adenovirus are all in the nM level, among which A54, A27 and adenovirus type 55 hexon proteins have the highest affinity, A15, A1, A18 and adenovirus type 3 hexon proteins have the highest affinity, and A5 and adenovirus type 7 hexon proteins have the highest affinity.

[0130] Example 3: Cloning and sequencing of antibody variable region sequences

[0131] Total RNA was isolated from the nine hybridoma cell lines obtained in Example 1, and cDNA was prepared by reverse transcription to clone immunoglobulin sequences from the hybridoma cell lines. The variable region sequences of the antibodies of the hybridoma cell lines were determined as follows.

[0132] a. RNA extraction: Total RNA was extracted from the hybridoma cell lines according to the instructions of the M5 total RNA extraction kit (purchased from Beijing PolyBiotech Co., Ltd.);

[0133] b. RNA reverse transcription into cDNA: The total RNA extracted in the previous step was reverse transcribed using the M5 First Strand cDNA Synthesis Kit (purchased from Beijing Polymer Biotechnology Co., Ltd.) to produce cDNA, which was then frozen at -20°C until use;

[0134] c. PCR amplification and recovery of variable region sequences: Using the cDNA obtained in the previous step as a template, immunoglobulin heavy chain (IgH) cDNA was amplified by PCR using universal heavy chain primers Mu Ig VH 5′-A and Mu IgG VH 3′-2. Similarly, immunoglobulin light chain (IgK) cDNA was amplified by PCR using light chain primers Mu IgκVL 5′-A and Mu IgκVL 3′-1, and the PCR product was recovered. The thermostable Pfu DNA polymerase was used throughout the PCR reaction.

[0135] d. Cloning and sequencing of variable region sequences: According to the instructions of the cloning vector pTOPO-Blunt Cloning kit (purchased from Beijing Polymer Biotechnology Co., Ltd.), the heavy and light chain variable region genes were ligated to the pTOPO vector, transformed into Escherichia coli DH5α, and positive clones were selected and sequenced by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0136] The antibody heavy chain variable region gene sequences and light chain variable region gene sequences of the hybridoma cell lines obtained by sequencing were analyzed, and the heavy chain complementary determining region sequences and light chain complementary determining region sequences are shown in Table 2 below.

[0137] Table 2: Heavy chain complementary determining regions, heavy chain variable regions, light chain complementary determining regions, and light chain variable regions of nine monoclonal antibodies

[0138] Example 4: Preparation and purification of recombinant antibodies

[0139] Construct recombinant antibodies, prepare cell lines that stably express antibodies through eukaryotic expression, and culture and purify them on a large scale.

[0140] The PCR method was used to amplify the variable region genes of the heavy and light chains of mouse monoclonal antibodies using the cDNA obtained by reverse transcription as a template. H and V L ), and the sequence was confirmed by sequencing. L and V H Genes were obtained and recombinant antibody eukaryotic expression vectors were constructed using molecular cloning methods. The heavy and light chain gene expression plasmid pCDNA3.1 was electroporated into CHO host cells. After electroporation, the cells were cultured in pressure selection medium (50 μM MSX) for 20 days. The supernatant was then collected and analyzed by ELISA (using horseradish peroxidase (HRP)-labeled goat anti-mouse IgG as a secondary antibody, using the same method as above) to identify cell lines stably expressing the recombinant antibody.

[0141] The selected stable cell lines were cultured on a large scale using roller bottle culture technology for the preparation of recombinant antibodies. The cells were cultured in Vega CHO medium at a rate of (0.2-0.3) × 10 6 Cells are inoculated into roller bottles at a rate of 1 cell / ml, containing 300 ml of culture medium (Vega CHO). The number of inoculated bottles is determined based on production needs. The inoculated roller bottles are placed in a cell roller and cultured in a cell culture incubator. Culture conditions are 900 rpm, 37°C, and 5% CO2. After 7-9 days of culture, samples are taken for microscopic observation. When cell viability is less than 50%, the samples are collected by centrifugation. The samples are affinity purified using a protein A affinity chromatography column to obtain the recombinant monoclonal antibody.

[0142] According to the method described in Example 2, the binding ability of each recombinant antibody to the hexon protein of eight types of adenovirus was detected. Figure 2 shows the binding curve of recombinant antibody A18 to the hexon protein of eight types of adenovirus. As can be seen from the figure, the ability of recombinant antibody A18 to bind to the hexon protein of eight types of adenovirus is also very strong, while it does not bind to the control (new coronavirus Np protein), and the antibody specificity is good. In addition to antibody A18, the other eight monoclonal antibodies obtained similar results that were able to bind to the hexon protein of eight types of adenovirus, but not to the new coronavirus Np protein (results not shown).

[0143] Furthermore, the ELISA results were analyzed by software to obtain the EC50 (nM) values ​​of the nine recombinant antibodies, which are shown in Table 3 below.

[0144] Table 3: EC50 (nM) values ​​of recombinant antibodies against hexon proteins of eight types of adenovirus

[0145] From the data given in Table 3, it can be seen that the affinities of the nine recombinant antibodies to the hexon proteins of various types of adenovirus are all in the nM level, which is basically similar to the monoclonal antibodies from which they are derived.

[0146] Example 5: Double sandwich ELISA to verify the sensitivity of recombinant antibodies

[0147] In this example, one recombinant antibody of the present invention (the first antibody) was used as the labeled antibody, and another recombinant antibody of the present invention (the second antibody) was used as the coating antibody, and the following steps were performed:

[0148] a) Labeling the primary antibody with horseradish peroxidase (HRP) to obtain a primary antibody-HRP conjugate.

[0149] b) Use double antibody sandwich ELISA to verify the sensitivity of the primary antibody-HRP according to the following steps:

[0150] Coating: Dilute the coating antibody / secondary antibody to a concentration of 1 μg / mL in 0.05 mol / L carbonate buffer (pH 9.6). Add 100 μL / well to a 96-well ELISA plate. Coat overnight at 4°C. Wash three times with PBST in an automated plate washer and pat dry.

[0151] Blocking: Add 120 μL of blocking solution (PBST containing 2% BSA) to each well and block at 37°C for 2 hours. Shake dry and set aside for use.

[0152] Add sample: add 50 μL of the sample to be tested, positive control, and negative control, respectively, and incubate at 37°C for 60 min;

[0153] Add enzyme: Add the primary antibody-HRP conjugate to the enzyme diluent at a certain concentration and mix well. Add 50 μL to each well and incubate at 37°C for 30 minutes.

[0154] Washing: Tap off the liquid in the well plate and wash the plate 5 times;

[0155] Color development: Add 100 μL of TMB substrate to each well, shake gently to mix, and develop color at room temperature in the dark for 30 minutes;

[0156] Termination: Add 50 μL of 2 mol / L sulfuric acid to each well to terminate the reaction and gently shake to mix;

[0157] Reading value: Read the OD value at 450nm and 630nm on a microplate reader;

[0158] The recombinant hexon protein was diluted in several gradients starting from 100 ng / mL. Then, the recombinant antibodies of the present invention were used to detect different concentrations of hexon protein. At the same time, the same concentration of (COVID-19 Np protein) was diluted as a control. The detection value of the COVID-19 Np protein was used as the standard, and the lowest concentration of hexon protein with a detection value greater than or equal to 2.1 times the detection value of the same COVID-19 Np protein concentration was used as the sensitivity of this method for detecting the antigen. The detection results of the nine recombinant monoclonal antibodies are shown in Table 4 below.

[0159] Table 4: Results of the recombinant monoclonal antibodies of the present invention detecting different concentrations of recombinant hexon protein

[0160] As shown in Table 4, even when the concentration of recombinant adenovirus hexon protein was diluted to 10 pg / mL (i.e., 0.01 nm / mL), the ELISA test results obtained by the antibodies of the present invention were still approximately 3-5 times that of the corresponding concentration of SARS-CoV-2 Np (as a control). Therefore, according to the above judgment criteria, the lowest concentration of recombinant hexon protein detected by the antibodies of the present invention for eight types of adenoviruses can reach 10 pg / mL. This result also demonstrates that the recombinant monoclonal antibodies of the present invention have good affinity and extremely high sensitivity.

[0161] Example 6: Colloidal gold immunochromatographic test

[0162] In this example, a colloidal gold immunochromatographic test was performed using one recombinant antibody of the present invention (the first antibody) as a labeled antibody and another recombinant antibody of the present invention (the second antibody).

[0163] Preparation of colloidal gold: Add 200 ml of ultrapure water to a conical flask, heat to boiling, add 1 ml of 2% chloroauric acid solution (Sigma-Aldrich, Catalog No.: 16961-25-4), and immediately add 1 ml of 2% trisodium citrate solution (Sigma-Aldrich, Catalog No.: 6132-04-3). Continue stirring and boiling for 10 minutes, then cool naturally and set aside.

[0164] Labeling the colloidal gold conjugate: Take 10 ml of the above colloidal gold and place it in a beaker. Add 140 μL of 0.2M K2CO3 to adjust the pH to 7.4 while stirring, and continue stirring for 10 seconds; add 100 μg of the primary antibody and continue stirring for 5 minutes; add 0.1 ml of 10% BSA and continue stirring for 5 minutes; centrifuge at 12000g for 10 minutes, discard the supernatant, and dilute the precipitate to 1 ml with colloidal gold diluent (10 mM PB, 150 mM NaCl, 0.2% BSA, 0.1% TritonX-100, 3% Sucrose, 0.01% Proclin300) to prepare the anti-adenovirus hexon protein antibody colloidal gold complex.

[0165] Preparation of colloidal gold pads: The colloidal gold complexes were diluted 10 times with colloidal gold diluent, soaked in glass fiber (Shanghai Jinbiao Company), and freeze-dried to prepare gold label pads.

[0166] Nitrocellulose membrane (NC membrane) coating: dilute the secondary antibody to 1.2 mg / ml to prepare the detection line working solution, use a membrane spotter to draw lines on the corresponding positions of nitrocellulose membrane (Millipore, catalog number: HF135002), dry at 50℃ for 1 hour, and set aside.

[0167] Assembly of colloidal gold immunochromatographic test reagent strip: Assemble the above-mentioned gold label pad, nitrocellulose membrane coated with antibody, absorbent paper, polyester plate, and sample pad into a colloidal gold immunoassay reagent strip.

[0168] Sensitivity test: 8 adenovirus hexon protein antigen samples of varying concentrations were tested. Specifically, 80 μl of the sample to be tested was dripped onto the sample pad, allowed to stand at room temperature for 10-15 minutes, and the results were determined. The depth of the displayed band color indicated the binding activity of the antigen and antibody in the sample. The color of the T-bar band developed by the colloidal gold test paper was compared with a standard color chart. The closest color was selected, and the color number corresponding to that color was used to mark the product activity. The results are shown in Table 5 below.

[0169] Table 5: Detection results of the recombinant monoclonal antibodies of the present invention against hexon proteins of eight types of adenovirus

[0170] * A1 / A2, where A1 represents the primary antibody and A2 represents the secondary antibody.

[0171] From the above results, it can be seen that when the recombinant monoclonal antibody of the present invention is assembled into a colloidal gold chromatography reagent, the minimum detection limit for hexon proteins of eight types of adenovirus can reach 100 pg (C8).

[0172] Specificity test: 500 nasopharyngeal swab samples from healthy individuals were tested using a reagent strip prepared with the recombinant monoclonal antibody of the present invention. The results showed that the reagent strip prepared with the recombinant monoclonal antibody of the present invention had no false positive results and a specificity of 100%.

Claims

1. An anti-adenovirus monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein: a) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 1-3 respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 4-6 respectively L CDR1, V L CDR2 and V L CDR3; b) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 5 and SEQ ID NO: 10, respectively. L CDR1, V L CDR2 and V L CDR3; c) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO: 8, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 6, respectively. L CDR1, V L CDR2 and V L CDR3; d) the heavy chain variable region comprises a heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 12-14, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 15-17 respectively. L CDR1, V L CDR2 and V L CDR3; e) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 18-20, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 21, respectively. L CDR1, V L CDR2 and V L CDR3; f) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 22-24, respectively. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 25-27 respectively. L CDR1, V L CDR2 and V L CDR3; g) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 22, SEQ ID NO: 28 and SEQ ID NO: 29, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 30 and SEQ ID NO: 31, respectively. L CDR1, V L CDR2 and V L CDR3; h) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 32-34, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V shown in SEQ ID NO: 35-37 respectively. L CDR1, V L CDR2 and V L CDR3; or i) the heavy chain variable region comprises the heavy chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 38-40, respectively H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the light chain complementary determining region V represented by the amino acid sequences of SEQ ID NOs: 41-43, respectively. L CDR1, V L CDR2 and V L CDR3.

2. The anti-adenovirus monoclonal antibody according to claim 1, wherein a) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 44 or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity with the amino acid sequence shown in SEQ ID NO: 44, and the light chain The variable region includes the amino acid sequence shown in SEQ ID NO:45, or a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence shown in SEQ ID NO:45; b) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:46, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:46, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:47, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:47; c) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:48, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:48, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:49, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:49; d) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:50, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:51, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence shown in SEQ ID NO:51 e) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:52, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence of SEQ ID NO:52, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:53, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence of SEQ ID NO:53; f) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:54, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence of SEQ ID NO:54, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:55, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence of SEQ ID NO:55; g) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:56, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:56, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:57, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:57; h) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:58, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO:59, or a sequence having 80% or more, 85% or more, 90% or more, or 95% identity to the amino acid sequence of SEQ ID NO:59; or i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:60, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence shown in SEQ ID NO:60, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:61, or a sequence having 80% or more, 85% or more, 90% or more identity with the amino acid sequence shown in SEQ ID NO:

61.

3. A nucleic acid molecule encoding the anti-adenovirus monoclonal antibody according to claim 1.

4. The nucleic acid molecule according to claim 3, wherein The anti-adenovirus monoclonal antibody is the anti-adenovirus monoclonal antibody according to claim 2. A vector comprising the nucleic acid molecule according to claim 3 .

6. The carrier according to claim 5, wherein The vector is a plasmid vector.

7. The carrier according to claim 6, wherein The plasmid vector is any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT, pTT3, pEFBOS, pBV, pJV and pBJ. An expression cell comprising the nucleic acid molecule of claim 3.

9. An expression cell comprising the vector according to claim 5.

10. The expression cell according to claim 9, wherein The expression cells are mammalian cells.

11. The expression cell according to claim 10, wherein The mammalian cells are Chinese hamster ovary cells, miniature hamster kidney cells, monkey kidney cells, mouse thymoma cells, and human embryonic kidney cells.

12. A method for detecting adenovirus, especially for diagnosing adenovirus infection, comprising the step of using the anti-adenovirus monoclonal antibody according to claim 1.

13. The method according to claim 12, wherein: The anti-adenovirus monoclonal antibody is the anti-adenovirus monoclonal antibody according to claim 2.

14. The method according to claim 12, wherein: The detection is performed by immunochromatography, enzyme-labeled antibody method (ELISA), chemiluminescence method, and electrochemiluminescence method.

15. The method according to claim 14, wherein: The immunochromatography method includes fluorescent microsphere immunochromatography, colloidal gold immunochromatography, immunochromatography based on colored latex microspheres, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography and quantum dot immunochromatography.

16. The method according to claim 14, wherein: The ELISA methods include direct method, indirect method, sandwich method and competitive method.

17. Use of the anti-adenovirus monoclonal antibody according to claim 1 in preparing a reagent for detecting adenovirus.

18. The use according to claim 17, wherein The anti-adenovirus monoclonal antibody is the anti-adenovirus monoclonal antibody according to claim 2.

19. A kit for detecting adenovirus, comprising the anti-adenovirus monoclonal antibody of claim 1 and instructions for use.

20. The kit according to claim 19, wherein The anti-adenovirus monoclonal antibody is the anti-adenovirus monoclonal antibody according to claim 2.

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