A panel of recombinant mouse antibodies against adenovirus DBP protein and their application in detection of multiple types of adenovirus

Abstract

The present application provides a set of monoclonal antibody pairs targeting adenovirus DBP protein, which consists of a capture antibody and a detection antibody, both of which comprise a heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: the capture antibody is monoclonal antibody 19E3; the detection antibody is selected from monoclonal antibody 96-10D8, 95-2B7, 96-2C10, 96-8E10, 96-13D5 or 96-2B10. The monoclonal antibody pairs of the present application can be used for efficient detection of multiple types of HAdV DBP.

Description

Technical Field

[0001] This invention relates to the fields of immunology and molecular biology, and more particularly to monoclonal antibody pairs targeting adenovirus DBP protein and their uses. Background Technology

[0002] Human adenovirus (HAdV) belongs to the family Adenoviridae and the genus Mastadenovirus, encompassing seven species, from HADV-A to HADV-G. According to data from the Human Adenovirus Working Group, as of March 2024, 116 types of HADV have been identified. Human adenovirus is a non-enveloped, double-stranded DNA virus with a genome length of approximately 35 kb. The virus is spherical, and under negative-stain electron microscopy, the viral particle diameter is approximately 80 nm. The viral particle consists of a protein capsid and a DNA-containing core. The capsid is typically icosahedral in shape, composed of 252 capsomeres, containing 240 hexons and 12 pentons. Besides capsid proteins, human adenovirus structural proteins include small capsid proteins such as VI, IIIa, VIII, and IX, as well as core structural proteins, which are important antigenic components of the virus. Non-structural proteins mainly consist of preterminal proteins (pTP), viral DNA polymerase (Ad-Pol), viral single-stranded DNA-binding protein (DBP), and other early proteins, playing crucial roles in various stages of the viral life cycle.

[0003] Adenoviruses exhibit broad tissue tropism, infecting various cell types, including epithelial cells of the respiratory tract, gastrointestinal tract, urethra, and conjunctiva, causing related diseases. HAdV infection easily leads to outbreaks in kindergartens, schools, and among new recruits in military camps. In immunocompetent individuals, adenovirus infection is usually self-limiting and resolves spontaneously. However, in children, the elderly, and immunocompromised individuals (such as organ transplant recipients and HIV patients), adenovirus infection can lead to severe, fatal infections, such as fulminant pneumonia, hepatitis, encephalitis, and / or systemic infection. The virus's structural proteins are immunogenic and effectively elicit a host immune response, often serving as targets for diagnosis and treatment. The adenovirus nonstructural protein DBP is encoded by the early gene EA2. Studies have shown that DBP is expressed in large quantities in the early stages of viral infection and exhibits strong immunogenicity. It can induce a strong antibody response in the early stages of infection and shows strong conservation among different types of adenovirus (see Guo L, Wu C, Zhou H, et al. Identification of a nonstructural DNA-binding protein (DBP) as an antigen with diagnostic potential for human adenovirus. PLoS One. 2013;8(3):e56708.). Therefore, it has potential application value as a diagnostic biomarker.

[0004] Hybridoma cells are formed by fusing B cells capable of producing specific antibodies with myeloma cells that can proliferate indefinitely in vitro. They possess both the ability to continuously secrete monoclonal antibodies and the characteristic of long-term passage. Monoclonal antibodies, due to their high specificity, high homogeneity, strong targeting, low toxicity, and mild side effects, have been widely used in the diagnosis and treatment of various diseases. Therefore, specific hybridoma monoclonal antibodies against human adenovirus DBP can be used in the development of reagents for adenovirus infection detection, etiology, and immunology, and have significant application value. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a set of specific monoclonal antibody pairs against human adenovirus DBP, which can be used for efficient detection of multiple types of HAdV DBP.

[0006] A first aspect of the present invention provides a monoclonal antibody pair targeting adenovirus DBP protein, comprising a capture antibody and a detection antibody, both antibodies comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0007] The capture antibody is a monoclonal antibody 19E3, and the amino acid sequences of its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 1-3, respectively, and the amino acid sequences of its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively.

[0008] The detection antibody is selected from one of the following antibodies:

[0009] (1) Monoclonal antibody 96-10D8, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:7-9, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:10-12, respectively;

[0010] (2) Monoclonal antibody 95-2B7, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:13-15, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:16-18, respectively;

[0011] (3) Monoclonal antibody 96-2C10, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:19-21, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:22-24, respectively;

[0012] (4) Monoclonal antibody 96-8E10, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:25-27, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:28-30, respectively;

[0013] (5) Monoclonal antibody 96-13D5, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:31-33, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:34-36, respectively;

[0014] (6) Monoclonal antibody 96-2B10, the amino acid sequences of its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs:37-39, and the amino acid sequences of its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs:40-42.

[0015] In a preferred embodiment of the present invention, the heavy chain variable region further includes heavy chain frame regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region further includes light chain frame regions LFR1, LFR2, LFR3, and LFR4, wherein:

[0016] The capture antibody is a monoclonal antibody 19E3, whose HFR1, HFR2, HFR3, and HFR4 amino acid sequences are shown in SEQ ID NOs: 43-46, and whose LFR1, LFR2, LFR3, and LFR4 amino acid sequences are shown in SEQ ID NOs: 47-50, respectively.

[0017] The detection antibody is selected from one of the following antibodies:

[0018] (1) Monoclonal antibody 96-10D8, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 51-54, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 55-58, respectively;

[0019] (2) Monoclonal antibody 95-2B7, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 59-62, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 63-66, respectively;

[0020] (3) Monoclonal antibody 96-2C10, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 67-70, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 71-74, respectively;

[0021] (4) Monoclonal antibody 96-8E10, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 75-78, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 79-82, respectively;

[0022] (5) Monoclonal antibody 96-13D5, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 83-86, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 87-90, respectively;

[0023] (6) Monoclonal antibody 96-2B10, the amino acid sequences of its HFR1, HFR2, HFR3 and HFR4 are shown in SEQ ID NOs: 91-94, and the amino acid sequences of its LFR1, LFR2, LFR3 and LFR4 are shown in SEQ ID NOs: 95-98.

[0024] In a preferred embodiment of the present invention

[0025] The capture antibody is a monoclonal antibody 19E3, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 99, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 100;

[0026] The detection antibody is selected from one of the following antibodies:

[0027] (1) Monoclonal antibody 96-10D8, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 101, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 102;

[0028] (2) Monoclonal antibody 95-2B7, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 103, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 104;

[0029] (3) Monoclonal antibody 96-2C10, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 105, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 106;

[0030] (4) Monoclonal antibody 96-8E10, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 107, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 108;

[0031] (5) Monoclonal antibody 96-13D5, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 109, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 110;

[0032] (6) Monoclonal antibody 96-2B10, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 111, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 112.

[0033] In a preferred embodiment of the present invention, all monoclonal antibodies are recombinant mouse antibodies.

[0034] A second aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding any one of the above-described monoclonal antibody pairs, either a capture antibody or a detection antibody.

[0035] In a preferred embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding the capture antibody;

[0036] Preferably, it comprises a nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 19E3, as shown in SEQ ID NO: 113; and a nucleotide sequence encoding the light chain variable region of monoclonal antibody 19E3, as shown in SEQ ID NO: 114.

[0037] In a preferred embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding the detection antibody;

[0038] Preferably, it comprises:

[0039] (1) The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 96-10D8, as shown in SEQ ID NO: 115; and the nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 96-10D8, as shown in SEQ ID NO: 116; or

[0040] (2) The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 95-2B7, as shown in SEQ ID NO: 117; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 95-2B7, as shown in SEQ ID NO: 118; or

[0041] (3) The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 96-2C10, as shown in SEQ ID NO: 119; and the nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 96-2C10, as shown in SEQ ID NO: 120; or

[0042] (4) The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 96-8E10, as shown in SEQ ID NO: 121; and the nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 96-8E10, as shown in SEQ ID NO: 122; or

[0043] (5) The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 96-13D5, as shown in SEQ ID NO: 123; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 96-13D5, as shown in SEQ ID NO: 124; or

[0044] (6) The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 96-2B10, as shown in SEQ ID NO: 125; and the nucleotide sequence encoding the light chain variable region of monoclonal antibody 96-2B10, as shown in SEQ ID NO: 126.

[0045] A third aspect of the present invention provides a carrier comprising the above-described nucleic acid molecules.

[0046] A fourth aspect of the present invention provides a cell comprising the above-described nucleic acid molecule or the above-described carrier.

[0047] A fifth aspect of the present invention provides a kit for detecting human adenovirus, comprising the above-described monoclonal antibody pair.

[0048] In a preferred embodiment of the present invention, the kit further comprises: HRP marker, blocking solution, ELISA chromogenic solution, ELISA stop solution, and washing buffer.

[0049] In a more preferred embodiment of the present invention, the blocking solution is a 3% BSA / PBS solution; and the washing buffer is a 0.03% Tween-20 / PBS solution.

[0050] A sixth aspect of the present invention provides a method for preparing the above-mentioned monoclonal antibody pair, comprising culturing the above-mentioned cells, separating and recovering antibodies therefrom, and forming antibody pairs.

[0051] The seventh aspect of the present invention provides the use of the above-described monoclonal antibody pair, the above-described nucleic acid molecule, the above-described vector, or the above-described cell for preparing a reagent for detecting human adenovirus in a sample.

[0052] An eighth aspect of the present invention provides a method for detecting human adenovirus in a sample, comprising: firstly binding the sample to a capture antibody in the above-mentioned monoclonal antibody pair, then adding a labeled detection antibody, and obtaining a detection result of human adenovirus in the sample by detecting a reaction signal; wherein the capture antibody is monoclonal antibody 19E3, and the detection antibody is selected from one or more monoclonal antibodies 96-10D8, 95-2B7, 96-2C10, 96-8E10, 96-13D5, and 96-2B10.

[0053] In a preferred embodiment of the present invention, the detection antibody includes all of the monoclonal antibodies 96-10D8, 95-2B7, 96-2C10, 96-8E10, 96-13D5 and 96-2B10.

[0054] In a preferred embodiment of the present invention, the human adenovirus is selected from HAdV-B3, HAdV-E4, HAdV-C5, HAdV-D8, HAdV-A12, HAdV-F41, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21.

[0055] In a preferred embodiment of the present invention, wherein:

[0056] Antibodies 19E3 and 96-10D8 were used to detect HADV-B3, HADV-B7, HADV-B11, HADV-B14, and HADV-B21.

[0057] Antibodies 19E3 and 95-2B7 were used to detect HADV-E4;

[0058] Antibodies 19E3 and 96-2C10 were used to detect HADV-C5;

[0059] Antibodies 19E3 and 96-8E10 were used to detect HADV-D8;

[0060] Antibodies 19E3 and 96-13D5 were used to detect HADV-A12;

[0061] Antibodies 19E3 and 96-2B10 were used to detect HADV-F41. Attached Figure Description

[0062] Figure 1 The results of HAdV DBP protein gel electrophoresis identification in Example 1.1 are as follows: (A) HAdV-A12; (B) HAdV-B3; (C) HAdV-C5; (D) HAdV-D8; (E) HAdV-E4; (F) HAdV-F41.

[0063] Figure 2 The results show the serum titers of HAdV DBP protein in mice immunized in Example 1.2. (A) HAdV-B3 DBP; (B) HAdV-E4 DBP; (C) HAdV-C5 DBP; (D) HAdV-D8 DBP; (E) HAdV-A12 DBP; (F) HAdV-F41 DBP.

[0064] Figure 3 The results of detecting different types of HAdV DBP protein with the mouse recombinant monoclonal antibody in Example 3.2 are shown. (A) HAdV-B3 DBP; (B) HAdV-E4 DBP; (C) HAdV-C5 DBP; (D) HAdV-D8 DBP; (E) HAdV-A12 DBP; (F) HAdV-F41 DBP.

[0065] Figure 4 The results of using the mouse recombinant monoclonal antibody pair in Example 3.3 for sandwich ELISA to detect different types of HADV infected samples. Detailed Implementation

[0066] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0068] Example 1: Screening of murine monoclonal antibodies against adenovirus DBP

[0069] 1.1 Preparation of Adv DBP protein

[0070] (1) Representative adenovirus strains infecting humans were selected, specifically HAdV-A12, HAdV-B3, HAdV-C5, HAdV-D8, HAdV-E4, and HAdV-F41. The codons encoding the DBP gene of each strain were optimized and the gene sequence was synthesized. Using conventional molecular cloning techniques, the strains were cloned into the pFastBacHT A (Invitrogen) baculovirus expression vector using the Xho I-Hind III double restriction site to obtain plasmids. The recombinant baculovirus plasmid containing the target gene was transposed into DH10Bac competent cells (Invitrogen). Positive clones were selected, and plasmids containing recombinant baculovirus DNA (Bacmid) were prepared. The Bacmids were transfected into insect Sf9 cells. Five days after transfection, obvious cytopathic effects appeared. The cell culture supernatant was harvested to obtain recombinant baculovirus, which was then frozen at -80℃.

[0071] (2) High 5 cells with good growth were infected with recombinant baculovirus. After 5 days, the cell pellet was collected, treated with cell lysis buffer, purified by Ni-NTA column, concentrated by 30 kDa ultrafiltration tube and replaced with 0.01 MPBS (pH 7.4). After BCA protein quantification, the pellet was aliquoted and stored at -80℃.

[0072] Taking the preparation of HAdV-A12 DBP protein as an example, in short, the recombinant baculovirus containing the HAdV-A12 DBP gene was prepared and infected with High 5 insect cells in good growth condition at moi=0.1. The cells were then placed in an incubator and cultured by shaking at 28℃ and 90 rpm / min. After 5 days of culture, cells were centrifuged at 3500 g for 10 min, the culture supernatant was discarded, and the cell pellet was collected into a centrifuge tube. Three times the cell volume of cell lysis buffer (containing 20 mM Tris-HCl, 300 mM NaCl, RNase I, DNase, and protease inhibitor, pH 8.0) was added, and the cells were sonicated on ice for 30 min. After centrifugation at 12000 g for 20 min, the supernatant was transferred, and the cells were loaded into a Ni-NTA column by gravity at a flow rate of 0.5–1 mL / min. The flow-through was collected, and the loading was repeated once. Washing buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) was added at a flow rate of 1 mL / min, followed by 15 column volumes of elution buffer (20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0) at a flow rate of 0.5 mL / min. Elute at 3 column volumes (mL / min), collect the eluted liquid and add it to the inner tube of a 30 kDa ultrafiltration tube. Centrifuge at 4000 g for 20 min, discard the filtrate, and replenish the inner tube with 0.01 M PBS (pH 7.4). Repeat centrifugation once more, collect the liquid in the inner tube, and quantify the protein using a BCA quantitative kit (Pierce). Aliquot and store at -80℃. Prepare other viral strains of DBP using the same method.

[0073] (3) The prepared DBP proteins of each viral strain were thawed on ice. 1 μg of each strain was added to protein loading buffer (containing DTT (dithiothreitol), reducing, R, and without DTT, non-reducing, NR), respectively. The buffer was boiled at 100℃ for 10 min, then added to 4%–20% SDS-PAGE protein gels. The gels were run at 200 V for 35 min, and Coomassie staining was performed using an eBlot staining instrument for identification. The protein gel electrophoresis results are as follows: Figure 1 As shown, the molecular weight of the prepared DBP proteins of various types is 70-80 kDa, and the purity is greater than 85%, which can be used for subsequent animal immunization and immunoassay.

[0074] 1.2 Mouse immunization and serum antibody titer detection

[0075] (1) Mouse immunization

[0076] Ten 6-8 week old female Balb / C mice were randomly divided into two groups. Group A consisted of five mice that received an initial immunization of 50 μg each of HAdV-B3, HAdV-E4, and HAdV-C5 DBP proteins. Booster immunizations were administered on days 14 and 28 at doses of 25 μg each of the same proteins. Group B consisted of five mice that received an initial immunization of 50 μg each of HAdV-D8, HAdV-A12, and HAdV-F41 DBP proteins. Booster immunizations were administered on days 14 and 28 at doses of 25 μg each of the same proteins. After the immunization program was completed, peripheral blood of mice was collected by orbital blood sampling into centrifuge tubes, centrifuged at 4000 g for 10 min, and the serum was aliquoted and stored at -80℃.

[0077] (2) Detection of antibody titers in mouse serum

[0078] Different types of HADV DBP proteins, including HADV-B3, HADV-E4, HADV-C5 DBP, HADV-D8, HADV-A12, and HADV-F41DBP, were coated into 96-well ELISA plates (Coring) at a dose of 50 ng / 100 μL / well using coating buffer (an aqueous solution containing 1.59 g / L Na2CO3 and 2.94 g / L NaHCO3, pH 9.6). The plates were incubated overnight at 4°C. The coating buffer was discarded the next day, and 300 μL of blocking buffer (3% BSA / PBS) was added, with the plates blocked at 37°C for 2 h. Mouse immune sera were serially diluted 1:2000 to 1:12800 using sample dilution buffer (0.5% BSA / PBS), and 100 μL / well was added to the corresponding antigen-coated ELISA plates, incubating at 37°C for 1 h. Wash three times with washing buffer (0.03% Tween-20 / PBS). Add 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody diluted 1:10000 to each well. Incubate at 37°C for 1 h. Wash three times with washing buffer. Add 100 μL of ELISA development solution (Solepro) to each well and incubate at 37°C for 10 min. Add 50 μL of ELISA stop solution (Solepro). Use an Ensight microplate reader to read the absorbance at 450 nm. Results are as follows. Figure 2 As shown in the ELISA results, all immunized mice in each group were able to produce anti-DBP antibodies with titers greater than 128,000, indicating that the immunization of mice was successful.

[0079] Example 2: Hybridoma cell preparation and hybridoma sequencing

[0080] 2.1 Hybridoma cell preparation

[0081] (1) After the last immunization, the spleen of the mouse was removed in a sterile environment and a spleen cell suspension was prepared using a 100 μm cell filter. SP2 / 0 mouse myeloma cells in the logarithmic growth phase and in good growth condition were prepared. The spleen cells and myeloma cells were mixed at a ratio of 10:1. After centrifugation at 400 g, the supernatant was discarded. Preheated 50% polyethylene glycol solution was slowly added to the cell pellet and stirred to promote cell membrane fusion. The cells were diluted with RPMI-1640 medium to terminate the PEG effect. The fused cells were collected by centrifugation at 110 g.

[0082] (2) The fused cells were resuspended in HAT (hypoxanthine-aminopterin-thymine) selective medium and seeded into 96-well cell culture plates. They were cultured in a constant temperature incubator at 37°C and 5% CO2. After observing obvious cell clones, monoclonal cells were obtained by limiting dilution. After culturing for 7-14 days, when the cloned cells filled approximately 1 / 3 of the wells, the culture supernatant was aspirated, and the antibody titer was detected using the ELISA method described in section 1.2. The cell wells corresponding to antibody-positive cells were labeled, and after two subclonal selections, a single hybridoma cell line stably secreting highly specific monoclonal antibodies was obtained and transferred to 24-well cell culture plates for scale-up culture.

[0083] 2.2 Antibody purification from hybridoma supernatant

[0084] Hybridoma cell lines in good growth condition were digested and centrifuged, and then the concentration was adjusted to 5×10⁻⁶ using serum-free hybridoma cell culture medium (Beijing Yiqiao Shenzhou Technology Co., Ltd.). 5 Cells / mL were transferred to cell shake flasks and cultured on a cell shaker at 125 rpm, 37°C, and 5% CO2 for 3-5 days. AmgMagbeads Protein A (Genescript) was added and incubated for 2 hours. Purification was then performed using an AmgMag S1 (Genescript) automated antibody purification system. The procedure was as follows: wash buffer (0.01M PBS, pH 7.4; 5 volumes, 3 times); elution buffer (0.1M Glycine-HCl, pH 3.0; 10 volumes, 1 time, incubation time 10 minutes). The eluent was transferred to centrifuge tubes, and 0.1 volumes of neutralization buffer (1 M Tris-HCl, pH 8.0) were added. The mixture was then concentrated by centrifugation using a 50 kDa ultrafiltration tube, and the solution was replaced with 0.01 M PBS (pH 7.4). Antibody concentration was quantified by OD280 value and stored at -80°C.

[0085] 2.3 ELISA detection of hybridoma antibody reactivity

[0086] The titer of purified hybridoma antibodies was detected using the ELISA method described in 1.2. The antibody to be tested was diluted to 1 μg / mL using sample diluent (0.5% BSA / PBS), and the remaining steps were the same as in 1.2 (2). The concentration-effect curves of each antibody were plotted using GraphPad Prism software, and the median effect concentration (EC50) was calculated. The results are shown in Table 1. The antibody titer results showed that 19E3 was a high-titer broad-spectrum antibody, 96-10D8 was a HADV-B3 specific antibody, 95-2B7 was a HADV-E4 specific antibody, 96-2C10 was a HADV-C5 specific antibody, 96-8E10 was a HADV-D8 specific antibody, 96-13D5 was a HADV-A12 specific antibody, and 96-2B10 was a HADV-F41 specific antibody.

[0087]

[0088] 2.4 Hybridoma antibody variable region gene sequencing

[0089] (1) Collect hybridoma cell pellets (approximately 1 × 10⁻⁶) of clones 19E3, 96-10D8, 95-2B7, 96-2C10, 96-8E10, 96-13D5, and 96-2B10 after amplification in step 2.1. 6Cells were centrifuged at 400 g for 5 min, and the supernatant was discarded. RNA was extracted using the SuperFastPure Cell RNA Isolation Kit (Vazyme, Cat:RC102). 500 μL of Buffer CRL was added to the cell pellet, and the mixture was thoroughly vortexed until no obvious cell clumps remained. Subsequent procedures were performed according to the kit instructions. Finally, RNA was washed away with 50 μL of RNase-free water. The extracted RNA was quantified using a Nanodrop microspectrophotometer. Reverse transcription and antibody variable region (V region) amplification (including light and heavy chains) were performed using the Mouse Monoclonal Antibody Hybridoma Cell Gene Variable Region Fragment Amplification (Sequencing) Kit (Frdbio Bioscience & Technology, Cat:MAC0080K), following the kit instructions. The specific amplification product (~450 bp) was recovered from agarose gel and purified using a FastPure gel DNA Extraction kit (Vazyme, Cat: DC301-01). The product was eluted with 50 μL of RNase-free water, and 2 μL of the nucleic acid product was used to construct a cloning vector using the Ultra-Universal TOPO cloning kit (Thermo Scientific). The recombinant plasmid was transformed into DH5α competent cells, and 10 single colonies were picked the following day. The hybridoma antibody was sequenced using M13F / R universal primers. After obtaining the antibody sequence, the gene sequence of the variable region of the antibody was analyzed using the IgBlast online analysis tool.

[0090] 2.5 Preparation and HRP labeling of recombinant mouse monoclonal antibodies

[0091] (1) The V gene of the positive clone antibody was synthesized by Beijing Ruiboxing Biotechnology Co., Ltd. and cloned into mouse antibody expression vectors. The heavy chain V gene was ligated into the IgVec-mIgG2 vector by AgeI-SalI double digestion, and the light chain V gene was inserted into the IgVec-mIgK vector by AgeI-BswiI double digestion.

[0092] (2) Take Expi293 cells in good growth condition and adjust the concentration to 2.5 × 10⁻⁶. 6The heavy chain and light chain antibody plasmids were mixed at a ratio of 2:1. The PEI-MAX transfection reagent and plasmid were mixed at a ratio of 3:1 in Opti-MEM medium and slowly added dropwise to the cell suspension. The mixture was then placed in a cell shaker and cultured at 37°C, 8% CO2, and 125 rpm for 5-7 days. The antibody was purified according to the mouse antibody purification process in section 2.2 to obtain recombinant mouse monoclonal antibody. The purified antibody was quantified using the OD280 value and the BCA protein quantification kit.

[0093] (3) Adjust the concentration of the antibody to be labeled to 2 mg / mL, take 50 μL, add 5 μL of 250 mM sodium bicarbonate (pH=8.5), add the solution to ReadiUse Preactivated HRP HNS ester (AAT Bioquest, Cat: 11025), and react at room temperature in the dark for 2 h. Add an equal volume of 100 mM Tris-HCl buffer (pH=7.5) containing 0.5% BSA, and store at 4℃ in the dark for a long time.

[0094] Example 3: Mouse recombinant monoclonal antibody pair used for sandwich ELISA detection of human adenovirus.

[0095] 3.1 Pairing of recombinant mouse anti-AdV DBP antibodies

[0096] Unlabeled antibody 19E3 was used as the capture antibody (Cap Ab), diluted to 1 μg / mL with coating buffer, and added to a 96-well ELISA plate at 100 μL / well. The plate was then incubated at 4°C overnight. The following day, after blocking with blocking buffer (3% BSA / PBS) at 37°C for 2 h, 100 μL of different types of HADV DBP antigen at a concentration of 100 ng / mL was added, and the plate was incubated at 37°C for 1 h. After washing with 0.3% PBST, 100 μL of various HRP-conjugated antibodies (detection antibodies, Det Ab) at a concentration of 100 ng / mL were added, and the plate was incubated at 37°C for 1 h. After washing with 0.3% PBST, 100 μL of ELISA chromogenic solution was added, and the plate was incubated at room temperature for 15 minutes. 50 μL of ELISA stop solution was added, and the absorbance at 450 nm was measured using an Ensight microplate reader. A positive pairing reaction was defined as a signal higher than that of the blank and self-control. As shown in Table 2, the results indicate that the 19E3 (Cap Ab) and 96-10D8 (Det Ab) antibody pair can be used for the detection of HAdV-B3 DBP protein, the 19E3 (Cap Ab) and 95-2B7 (Det Ab) antibody pair can be used for the detection of HAdV-E4 DBP protein, the 19E3 (Cap Ab) and 96-2C10 (Det Ab) antibody pair can be used for the detection of HAdV-C5 DBP protein, the 19E3 (Cap Ab) and 96-8E10 (Det Ab) antibody pair can be used for the detection of HAdV-D8 DBP protein, the 19E3 (Cap Ab) and 96-13D5 (Det Ab) antibody pair can be used for the detection of HAdV-A12 DBP protein, and the 19E3 (Cap Ab) and 96-2B10 (Det Ab) antibody pair can be used for the detection of HAdV-F41 DBP protein.

[0097]

[0098] 3.2 Sandwich ELISA method for detecting different types and concentrations of HADV DBP antigen

[0099] Dilute Cap Ab antibody (19E3) with coating buffer and coat 96-well ELISA plates with 100 ng / 100 μL / well. Follow the procedure described in 3.1. After blocking the plate the next day, add HADV DBP antigen of different types and gradient concentrations. For antibody detection, use 100 ng / mL Det Ab and HRP-conjugated mouse anti-human IgG Fc monoclonal antibody (isotype control, Isotype, Abcam, cat: ab99759), 100 μL / well. After colorimetric reaction and termination with stop solution, measure the absorbance at 450 nm and plot the dose-response curve using GraphPad Prism software. Figure 3 As shown, the detection efficacy of 96-10D8 against HADV-B3, 95-2B7 against HADV-E4, 96-2C10 against HADV-C5, 96-8E10 against HADV-D8, 96-13D5 against HADV-A12, and 96-2B10 against HADV-F41 was further verified, and the dose-response curves of each antibody were all S-shaped.

[0100] 3.3 Sandwich ELISA method for detecting different types of HADV virus culture supernatant

[0101] (1) HeLa cells in good growth condition were digested with trypsin and then resuspended in DMEM medium containing 10% fetal bovine serum (FBS) to adjust the cell concentration to 5 × 10⁻⁶ cells / year. 5 Cells / mL, take 3 mL of cell suspension and add it to a 6-well cell culture plate, and incubate overnight at 37°C in a 5% CO2 incubator.

[0102] (2) The next day, the culture medium was replaced with DMEM containing 2% FBS, and different types of adenovirus with an infection multiplicity (MOI) of 5, including HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14, HAdV-B21, HAdV-E4, HAdV-C5 and HAdV-D8, were added. The cells were incubated at 37°C with 5% CO2 for 1 h. After 1 h, the culture supernatant was discarded, the cells were washed once with Versen buffer, and 3 mL of DMEM containing 2% FBS was added to each well. The cells were then incubated at 37°C with 5% CO2 for 24 h.

[0103] (3) The next day, the culture supernatant of different types of adenovirus was collected and transferred to centrifuge tubes. The tubes were centrifuged at 3000 g and 4℃ for 15 min. The supernatant was then transferred to a new centrifuge tube and stored at -80℃.

[0104] (4) Dilute Cap Ab antibody with coating buffer and coat 96-well ELISA plates with 100 ng / 100 μL / well. Follow the procedure described in 3.1. After blocking the next day, add supernatant of different types of HADV virus culture that has been inactivated. Dilute the virus culture supernatant with sample diluent (0.5% BSA / PBS). After incubation at 37°C for 1 h, wash with PBST. Then add 100 μL of a 100 ng / mL Det Ab mixture (a mixture of 96-10D8, 95-2B7, 96-2C10, 96-8E10, 96-13D5, and 96-2B10 antibodies in equal proportions) to each well, and add 100 ng / mL of 96-10D8, 95-2B7, 96-2C10, and 96-8E10 respectively. Incubate at 37°C for 1 h. After colorimetric reaction and termination with stop solution, measure 450. A positive result is defined as an absorbance value at a wavelength of nm that is higher than that of the culture medium control well. For example... Figure 4 As shown, the results indicate that Det Ab 96-10D8 can be used for the detection of HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21; Det Ab 95-2B7 can be used for the detection of HAdV-E4; Det Ab 96-2C10 can be used for the detection of HAdV-C5; Det Ab 96-8E10 can be used for the detection of HAdV-D8; and mixtures of Det Abs can be used for the detection of HAdV infection samples of different types.

[0105] Sequence information

[0106] SEQ ID NO: 1-19E3 HCDR1 amino acid sequence

[0107] GFSLSTSGMG

[0108] SEQ ID NO: 2——19E3 HCDR2 amino acid sequence

[0109] IWWDDVR

[0110] SEQ ID NO: 3——19E3 HCDR3 amino acid sequence

[0111] ARTWFAY

[0112] SEQ ID NO: 4——19E3 LCDR1 amino acid sequence

[0113] SQHSTYT

[0114] SEQ ID NO: 5——19E3 LCDR2 amino acid sequence

[0115] LKKDGSH

[0116] SEQ ID NO: 6—19E3 LCDR3 amino acid sequence

[0117] GVGDTIKEQFVYV

[0118] SEQ ID NO: 7——96-10D8 HCDR1 amino acid sequence

[0119] GFTFNTYA

[0120] SEQ ID NO: 8——96-10D8 HCDR2 amino acid sequence

[0121] IRTKSNNYAT

[0122] SEQ ID NO: 9——96-10D8 HCDR3 amino acid sequence

[0123] VRFAY

[0124] SEQ ID NO: 10——96-10D8 LCDR1 amino acid sequence

[0125] QSIVHSNGNTY

[0126] SEQ ID NO: 11——96-10D8 LCDR2 amino acid sequence

[0127] KVS

[0128] SEQ ID NO: 12——96-10D8 LCDR3 amino acid sequence

[0129] FQGSHVPYT

[0130] SEQ ID NO: 13——95-2B7 HCDR1 amino acid sequence

[0131] GYSITSGYS

[0132] SEQ ID NO: 14——95-2B7 HCDR2 amino acid sequence

[0133] IHYSGST

[0134] SEQ ID NO: 15——95-2B7 HCDR3 amino acid sequence

[0135] TNFDY

[0136] SEQ ID NO: 16——95-2B7 LCDR1 amino acid sequence

[0137] QSLLDSDGKTY

[0138] SEQ ID NO: 17——95-2B7 LCDR2 amino acid sequence

[0139] LVS

[0140] SEQ ID NO: 18——95-2B7 LCDR3 amino acid sequence

[0141] WQGTHFPRT

[0142] SEQ ID NO: 19——96-2C10 HCDR1 amino acid sequence

[0143] GYAFSSYW

[0144] SEQ ID NO: 20——96-2C10 HCDR2 amino acid sequence

[0145] IYPGDGDT

[0146] SEQ ID NO: 21——96-2C10 HCDR3 amino acid sequence

[0147] ANFDS

[0148] SEQ ID NO: 22——96-2C10 LCDR1 amino acid sequence

[0149] QSIVHSNGNTY

[0150] SEQ ID NO: 23——96-2C10 LCDR2 amino acid sequence

[0151] KVS

[0152] SEQ ID NO: 24——96-2C10 LCDR3 amino acid sequence

[0153] FQGSHVPWT

[0154] SEQ ID NO: 25——96-8E10 HCDR1 amino acid sequence

[0155] GFTFSSYA

[0156] SEQ ID NO: 26——96-8E10 HCDR2 amino acid sequence

[0157] ISSGGRT

[0158] SEQ ID NO: 27——96-8E10 HCDR3 amino acid sequence

[0159] ARDY

[0160] SEQ ID NO: 28——96-8E10 LCDR1 amino acid sequence

[0161] QSVDYDGESY

[0162] SEQ ID NO: 29——96-8E10 LCDR2 amino acid sequence

[0163] SAS

[0164] SEQ ID NO: 30——96-8E10 LCDR3 amino acid sequence

[0165] QQSHEDPWT

[0166] SEQ ID NO: 31——96-13D5 HCDR1 amino acid sequence

[0167] GFTFSSYA

[0168] SEQ ID NO: 32——96-13D5 HCDR2 amino acid sequence

[0169] ISSGGST

[0170] SEQ ID NO: 33——96-13D5 HCDR3 amino acid sequence

[0171] ARAY

[0172] SEQ ID NO: 34——96-13D5 LCDR1 amino acid sequence

[0173] QDISNY

[0174] SEQ ID NO: 35——96-13D5 LCDR2 amino acid sequence

[0175] YTS

[0176] SEQ ID NO: 36——96-13D5 LCDR3 amino acid sequence

[0177] QQGNTLPLT

[0178] SEQ ID NO: 37——96-2B10 HCDR1 amino acid sequence

[0179] GFTFSSYT

[0180] SEQ ID NO: 38——96-2B10 HCDR2 amino acid sequence

[0181] ISSGGSYT

[0182] SEQ ID NO: 39——96-2B10 HCDR3 amino acid sequence

[0183] TRFAY

[0184] SEQ ID NO: 40——96-2B10 LCDR1 amino acid sequence

[0185] ESVDSYGNSF

[0186] SEQ ID NO: 41——96-2B10 LCDR2 amino acid sequence

[0187] RAS

[0188] SEQ ID NO: 42——96-2B10 LCDR3 amino acid sequence

[0189] QQSNEDPPT

[0190] SEQ ID NO: 43——19E3 HFR1 amino acid sequence

[0191] QVTLKESGPGILQPSQTLSLTCSFS

[0192] SEQ ID NO: 44——19E3 HFR2 amino acid sequence

[0193] VGWLRQPSGKALEWLAH

[0194] SEQ ID NO: 45——19E3 HFR3 amino acid sequence

[0195] YYNPALKSRLTISKDTSSSQVFLKIASVDTADSATYYC

[0196] SEQ ID NO: 46——19E3 HFR4 amino acid sequence

[0197] WGQGTLVTVSA

[0198] SEQ ID NO: 47——19E3 LFR1 amino acid sequence

[0199] QLVLTQSSSASFSLGASAKLTCTLS

[0200] SEQ ID NO: 48——19E3 LFR2 amino acid sequence

[0201] IEWYQQQPLKPPKYVME

[0202] SEQ ID NO: 49——19E3 LFR3 amino acid sequence

[0203] STGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYIC

[0204] SEQ ID NO: 50-19E3 LFR4 amino acid sequence

[0205] FGGGTKVTVL

[0206] SEQ ID NO: 51——96-10D8 HFR1 amino acid sequence

[0207] EVQLVESGGGLVQPKGSLKLSCAAS

[0208] SEQ ID NO: 52——96-10D8 HFR2 amino acid sequence

[0209] MHWVCQAPGKGLEWVAR

[0210] SEQ ID NO: 53——96-10D8 HFR3 amino acid sequence

[0211] YYADSVKDRFTISRDDSQNILYLQMNNLKTEDTAIYYC

[0212] SEQ ID NO: 54——96-10D8 HFR4 amino acid sequence

[0213] WGQGTLVTVSA

[0214] SEQ ID NO: 55——96-10D8 LFR1 amino acid sequence

[0215] DVLMTQTPLSLPVSLGDQASISCRSS

[0216] SEQ ID NO: 56——96-10D8 LFR2 amino acid sequence

[0217] LEWYLQKPGQSPKLLIY

[0218] SEQ ID NO: 57——96-10D8 LFR3 amino acid sequence

[0219] NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC

[0220] SEQ ID NO: 58——96-10D8 LFR4 amino acid sequence

[0221] FGGGTKLEIK

[0222] SEQ ID NO: 59——95-2B7 HFR1 amino acid sequence

[0223] DVQLQESGPDLVKPSQSLSLTCTVT

[0224] SEQ ID NO: 60——95-2B7 HFR2 amino acid sequence

[0225] WHWIRQFPGNKLEWMGY

[0226] SEQ ID NO: 61——95-2B7 HFR3 amino acid sequence

[0227] NYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYC

[0228] SEQ ID NO: 62——95-2B7 HFR4 amino acid sequence

[0229] WGQGTTLTVSS

[0230] SEQ ID NO: 63——95-2B7 LFR1 amino acid sequence

[0231] DVVMTQTPLTLSVTIGQPASISCKSS

[0232] SEQ ID NO: 64——95-2B7 LFR2 amino acid sequence

[0233] LNWLLQRPGQSPKRLIY

[0234] SEQ ID NO: 65——95-2B7 LFR3 amino acid sequence

[0235] KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC

[0236] SEQ ID NO: 66——95-2B7 LFR4 amino acid sequence

[0237] FGGGTKLEI

[0238] SEQ ID NO: 67——96-2C10 HFR1 amino acid sequence

[0239] QVQLQQSGAELVRPGSSVKISCKAS

[0240] SEQ ID NO: 68——96-2C10 HFR2 amino acid sequence

[0241] MHWVKQRPGQGLEWIGQ

[0242] SEQ ID NO: 69——96-2C10 HFR3 amino acid sequence

[0243] DYIEKFKGKVTLTADKSSSTAYMQLSSLTSEDSAVYFC

[0244] SEQ ID NO: 70——96-2C10 HFR4 amino acid sequence

[0245] WGQGTTLTVSS

[0246] SEQ ID NO: 71——96-2C10 LFR1 amino acid sequence

[0247] DVLMTQTPLSLPVSLGDQASISCRSS

[0248] SEQ ID NO: 72——96-2C10 LFR2 amino acid sequence

[0249] LEWYLQKPGQSPKLLIY

[0250] SEQ ID NO: 73——96-2C10 LFR3 amino acid sequence

[0251] NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC

[0252] SEQ ID NO: 74——96-2C10 LFR4 amino acid sequence

[0253] FGGGTKLEIK

[0254] SEQ ID NO: 75——96-8E10 HFR1 amino acid sequence

[0255] EVKLVESGGGLVKPGGSLKLSCAVS

[0256] SEQ ID NO: 76——96-8E10 HFR2 amino acid sequence

[0257] MSWVRQTPEKRLEWVAS

[0258] SEQ ID NO: 77——96-8E10 HFR3 amino acid sequence

[0259] YYLDILKGRFTISRDNARNTLYLQMSSLRSEDTAIYYC

[0260] SEQ ID NO: 78——96-8E10 HFR4 amino acid sequence

[0261] WGQGTTLTVSS

[0262] SEQ ID NO: 79——96-8E10 LFR1 amino acid sequence

[0263] GTSPAGLNEFALKQWYQRRVRACKAS

[0264] SEQ ID NO: 80-96-8E10 LFR2 amino acid sequence

[0265] LNWHQQKPGQPPRLLIY

[0266] SEQ ID NO: 81——96-8E10 LFR3 amino acid sequence

[0267] TLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC

[0268] SEQ ID NO: 82——96-8E10 LFR4 amino acid sequence

[0269] FGGGTMLEIK

[0270] SEQ ID NO: 83——96-13D5 HFR1 amino acid sequence

[0271] EVKLVESGGGLVKPGGSLKLSCAAS

[0272] SEQ ID NO: 84——96-13D5 HFR2 amino acid sequence

[0273] MSWVRQTPEKRLEWVAS

[0274] SEQ ID NO: 85-96-13D5 HFR3 amino acid sequence

[0275] YYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYC

[0276] SEQ ID NO: 86-96-13D5 HFR4 amino acid sequence

[0277] WGQGTLVTVS

[0278] SEQ ID NO: 87——96-13D5 LFR1 amino acid sequence

[0279] DIQMTQTTSSLSASLGDRVTISCRAS

[0280] SEQ ID NO: 88——96-13D5 LFR2 amino acid sequence

[0281] LNWYQQKPDGTVKLLIY

[0282] SEQ ID NO: 89——96-13D5 LFR3 amino acid sequence

[0283] RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC

[0284] SEQ ID NO: 90——96-13D5 LFR4 amino acid sequence

[0285] FGAGTKLE

[0286] SEQ ID NO: 91-96-2B10 HFR1 amino acid sequence

[0287] DVKLVESGGGLVKPGGSLKLSCAAS

[0288] SEQ ID NO: 92——96-2B10 HFR2 amino acid sequence

[0289] MSWVRQTPEKRLEWVAT

[0290] SEQ ID NO: 93-96-2B10 HFR3 amino acid sequence

[0291] YYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYC

[0292] SEQ ID NO: 94——96-2B10 HFR4 amino acid sequence

[0293] WGQGTLVTVSA

[0294] SEQ ID NO: 95-96-2B10 LFR1 amino acid sequence

[0295] DIVLTQSPASLAVSLGQRATISCRAS

[0296] SEQ ID NO: 96——96-2B10 LFR2 amino acid sequence

[0297] MHWYQQKPGQPPKLLIY

[0298] SEQ ID NO: 97——96-2B10 LFR3 amino acid sequence

[0299] NLESGIPARFSGSGSRTDFTLTINPVEADDVATYYC

[0300] SEQ ID NO: 98——96-2B10 LFR4 amino acid sequence

[0301] FGGGTKLEIK

[0302] SEQ ID NO: 99——19E3 VH amino acid sequence

[0303] QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWLRQPSGKALEWLAHIWWDDVRYYNPALKSRLTISKDTSSSQVFLKIASVDTADSATYYCARTWFAYWGQGTLVTVSA

[0304] SEQ ID NO: 100-19E3 VL amino acid sequence

[0305] QLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKPPKYVMELKKDGHSTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTIKEQFVYVFGGGTKVTVL

[0306] SEQ ID NO: 101——96-10D8 VH amino acid sequence

[0307] EVQLVESGGGLVQPKGSLKLSCAASGFTFNTYAMHWVCQAPGKGLEWVARIRTKSNNYATYYADSVKDRFTISRDDSQNILYLQMNNLKTEDTAIYYCVRFAYWGQGTLVTVSA

[0308] SEQ ID NO: 102——96-10D8 VL amino acid sequence

[0309] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0310] SEQ ID NO: 103 - Amino acid sequence of 95 - 2B7 VH

[0311] DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHYSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTNFDYWGQGTTLTVSS

[0312] SEQ ID NO: 104 - Amino acid sequence of 95 - 2B7 VL

[0313] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEI

[0314] SEQ ID NO: 105 - Amino acid sequence of 96 - 2C10 VH

[0315] QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMHWVKQRPGQGLEWIGQIYPGDGDTDYIEKFKGKVTLTADKSSSTAYMQLSSLTSEDSAVYFCANFDSWGQGTTLTVSS

[0316] SEQ ID NO: 106 - Amino acid sequence of 96 - 2C10 VL

[0317] DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK

[0318] SEQ ID NO: 107 - Amino acid sequence of 96 - 8E10 VH

[0319] EVKLVESGGGLVKPGGSLKLSCAVSGFTFSSYAMSWVRQTPEKRLEWVASISSGGRTYYLDILKGRFTISRDNARNTLYLQMSSLRSEDTAIYYCARDYWGQGTTLTVSS

[0320] SEQ ID NO: 108 - Amino acid sequence of 96 - 8E10 VL

[0321] GTSPAGLNEFALKQWYQRRVRACKASQSVDYDGESYLNWHQQKPGQPPRLLIYSASTLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSHEDPWTFGGGTMLEIK

[0322] SEQ ID NO: 109 - Amino acid sequence of 96 - 13D5 VH

[0323] EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARAYWGQGTLVTVS

[0324] SEQ ID NO: 110 - Amino acid sequence of 96 - 13D5 VL

[0325] DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPLTFGAGTKLE

[0326] SEQ ID NO: 111 - Amino acid sequence of 96 - 2B10 VH

[0327] DVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCTRFAYWGQGTLVTVSA

[0328] SEQ ID NO: 112 - Amino acid sequence of 96 - 2B10 VL

[0329] DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASNLESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPPTFGGGTKLEIK

[0330] SEQ ID NO: 113 - 19E3 VH nucleotide sequence

[0331] caggttactctgaaagagtctggccctgggatattgcagccctcccagaccctcagtctgacttgttctttctctgggttttcactgagcacttctggtatgggtgtaggctggcttcgtcagccatcaggtaaggctctggagtggctggcacacatttggtgggatgatgtcaggtactataacccagccctgaagagccgactgactatctccaaggatacctccagtagccaggttttcctcaagatcgccagtgtggacactgcagattctgccacatactactgtgctcgaacctggtttgcttactggggccaagggactctggtcactgtctctgca

[0332] SEQ ID NO: 114 - 19E3 VL nucleotide sequence

[0333] caacttgtgctcactcagtcatcttcagcctctttctccctgggagcctcagcaaaactcacgtgcaccttgagtagtcagcacagtacgtacaccattgaatggtatcagcaacagccactcaagcctcctaagtatgtgatggagcttaagaaagatggaagccacagcacaggtgatgggattcctgatcgcttctctggatccagctctggtgctgatcgctaccttagcatttccaacatccagcctgaagatgaagcaatatacatctgtggtgtgggtgatacaattaaggaacaatttgtgtatgttttcggcggtggaaccaaggtcactgtccta

[0334] SEQ ID NO: 115 - VH nucleotide sequence of 96 - 10D8

[0335] gaggtgcagcttgttgagtctggtggaggattggtgcagcctaaaggttcattgaaactctcatgtgccgcctctggtttcaccttcaatacctatgccatgcactgggtctgccaggctccaggaaagggtttggaatgggttgctcgcataagaactaaaagtaataattatgcaacatattatgccgattcagtgaaagacagattcaccatctccagagatgattcacagaacattctctatctgcaaatgaacaacctgaaaactgaggacacagccatatattactgtgtgaggtttgcttactggggccaagggactctggtcactgtctctgcag

[0336] SEQ ID NO: 116 - VL nucleotide sequence of 96 - 10D8

[0337] gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaaggttcacatgttccgtacacgttcggaggggggaccaagctggaaataaaac

[0338] SEQ ID NO: 117 - VH nucleotide sequence of 95 - 2B7

[0339] gatgtgcagcttcaggagtcaggacctgacctggtgaaaccttctcagtcactttcactcacctgcactgtcactggctactccatcaccagtggttatagctggcactggatccggcagtttccaggaaacaaactggaatggatgggctacatacactacagtggtagcactaactacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattactgtacaaactttgactactggggccaaggcaccactctcacagtctcctcag

[0340] SEQ ID NO: 118 - Nucleotide sequence of 95 - 2B7 VL

[0341] gatgttgtgatgacccagactccactcactttgtcggttaccattggacaaccagcctccatctcttgcaagtcaagtcagagcctcttagatagtgatggaaagacatatttgaattggttgttacagaggccaggccagtctccaaagcgcctaatctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcagggacagatttcacactgaaaatcagcagagtggaggctgaggatttgggagtttattattgctggcaaggtacacattttcctcggacgttcggtggaggcaccaagctggaaatca

[0342] SEQ ID NO: 119 - Nucleotide sequence of 96 - 2C10 VH

[0343] caggttcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagttactggatgcactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttatcctggagatggtgatactgactacattgaaaaatttaagggtaaagtcacactgactgcagacaaatcctccagcacagcctacatgcaactcagcagcctaacatctgaggactctgcggtctatttctgtgcaaactttgactcctggggccaaggcaccactctcacagtctcctcag

[0344] SEQ ID NO: 120 - 96 - 2C10 VL nucleotide sequence

[0345] gatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagagcattgtacatagtaatggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaagctcctgatctacaaagtttccaaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgttttcaaggttcacatgttccgtggacgttcggtggaggcaccaagctggaaatcaaac

[0346] SEQ ID NO: 121 - 96 - 8E10 VH nucleotide sequence

[0347] gaagtgaagctggtggagtccgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagtctctggattcactttcagtagttatgccatgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatccattagtagtggtggtaggacctactatctagacattttgaagggccgattcaccatctccagagataatgccaggaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatctattactgtgcaagagactactggggccaaggcaccactctcacagtctcctcag

[0348] SEQ ID NO: 122 - Nucleotide sequence of 96 - 8E10 VL

[0349] ggtactagtcctgcaggtttaaatgaattcgcccttaagcagtggtatcaacgcagagtacgggcctgcaaggccagccaaagtgttgattatgatggtgaaagttatctgaactggcaccaacagaaaccaggacagccacccagactcctcatctattctgcatccactctagaatctgggatcccagccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcaaagtcatgaggatccgtggacgttcggtggaggcaccatgttggaaatcaaac

[0350] SEQ ID NO: 123 - Nucleotide sequence of 96 - 13D5 VH

[0351] gaagtgaagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctatgccatgtcttgggttcgccagactccagagaagaggctggagtgggtcgcatccattagtagtggtggtagcacctactatccagacagtgtgaagggccgattcaccatctccagagataatgccaggaacatcctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagggcttactggggccaagggactctggtcactgtctct

[0352] SEQ ID NO: 124 - Nucleotide sequence of 96 - 13D5 VL

[0353] gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgctcacgttcggtgctgggaccaagctggagct

[0354] SEQ ID NO: 125 - Nucleotide sequence of 96 - 2B10 VH

[0355] gacgtgaagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagctataccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtagtggtggtagttacacctactatccagacagtgtgaagggccgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgtacaagatttgcttactggggccaagggactctggtcactgtctctgcag

[0356] SEQ ID NO: 126 - Nucleotide sequence of 96 - 2B10 VL

[0357] Gacattgtgctgacccaatctccagcttctttggctgtgtctctagggcagagggccaccatatcctgcagagccagtgaaagtgttgatagttatggcaatagttttatgcactggtaccagcagaaaccaggacagccacccaaactcctcatctatcgtgcatccaacctagaatctgggatccctgccaggttcagtggcagtgggtctaggacagacttcaccctcaccattaatcctgtggaggctgatgatgttgcaacctattactgtcagcaaagtaatgaggatcctccgacgttcggtggaggcaccaagctggaaatcaaac。

Claims

1. A monoclonal antibody pair targeting human adenovirus DBP protein, comprising a capture antibody and a detection antibody, both antibodies containing a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region contains light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein: The capture antibody is a monoclonal antibody 19E3, whose amino acid sequences of HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 1-3, and whose amino acid sequences of LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 4-6, respectively. The detection antibody is monoclonal antibody 96-10D8, and the amino acid sequences of its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NOs: 7-9, respectively, and the amino acid sequences of its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NOs: 10-12, respectively.

2. The monoclonal antibody pair according to claim 1, wherein the heavy chain variable region further comprises heavy chain framework regions HFR1, HFR2, HFR3, and HFR4, and the light chain variable region further comprises light chain framework regions LFR1, LFR2, LFR3, and LFR4, wherein: The capture antibody is a monoclonal antibody 19E3, whose HFR1, HFR2, HFR3, and HFR4 amino acid sequences are shown in SEQ ID NOs: 43-46, and whose LFR1, LFR2, LFR3, and LFR4 amino acid sequences are shown in SEQ ID NOs: 47-50, respectively. The detection antibody is monoclonal antibody 96-10D8, and the amino acid sequences of its HFR1, HFR2, HFR3, and HFR4 are shown in SEQ ID NOs: 51-54, respectively, and the amino acid sequences of its LFR1, LFR2, LFR3, and LFR4 are shown in SEQ ID NOs: 55-58, respectively.

3. The monoclonal antibody pair according to claim 2, wherein: The capture antibody is a monoclonal antibody 19E3, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 99, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 100; The detection antibody is monoclonal antibody 96-10D8, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 101, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

102.

4. The monoclonal antibody pair according to any one of claims 1-3, wherein the monoclonal antibodies are all recombinant mouse antibodies.

5. A nucleic acid molecule comprising a nucleotide sequence encoding a detection antibody against a monoclonal antibody pair as described in any one of claims 1-4.

6. The nucleic acid molecule according to claim 5, comprising a nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 96-10D8, as shown in SEQ ID NO: 115; and a nucleotide sequence encoding the light chain variable region of monoclonal antibody 96-10D8, as shown in SEQ ID NO:

116.

7. A vector comprising the nucleic acid molecule of claim 5 or 6.

8. A cell comprising the nucleic acid molecule of claim 5 or 6 or the vector of claim 7.

9. A kit for detecting human adenovirus, comprising a monoclonal antibody pair according to any one of claims 1-4.

10. The kit according to claim 9, further comprising: HRP marker, blocking solution, ELISA chromogenic solution, ELISA stop solution, and washing buffer.

11. The kit according to claim 10, wherein the blocking solution is a PBS solution containing 3% BSA; and the washing buffer is a PBS solution containing 0.03% Tween-20.

12. Use of the monoclonal antibody pair of any one of claims 1-4, the nucleic acid molecule of claim 5 or 6, the vector of claim 7, or the cell of claim 8 for the preparation of a reagent for detecting human adenovirus in a sample; The human adenovirus mentioned therein is selected from HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 or HAdV-B21.

13. A non-diagnostic method for detecting human adenovirus in an isolated sample, comprising: First, the in vitro sample is bound to the capture antibody in the monoclonal antibody pair according to any one of claims 1-4, and then the labeled detection antibody is added. The detection result of human adenovirus in the sample is obtained by detecting the reaction signal. The human adenovirus mentioned therein is selected from HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 or HAdV-B21.

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