Neutralizing monoclonal antibody 10g8 for recognizing CV-a6 and use thereof

Abstract

The present invention provides a neutralizing monoclonal antibody 10G8 for recognizing CV-A6. Six CDR regions of the monoclonal antibody 10G8 are specifically as follows: (1) the amino acid sequence of a heavy chain CDR1 is set forth in SEQ ID NO. 1; (2) the amino acid sequence of a heavy chain CDR2 is set forth in SEQ ID NO. 2; (3) the amino acid sequence of a heavy chain CDR3 is set forth in SEQ ID NO. 3; (4) the amino acid sequence of a light chain CDR1 is set forth in SEQ ID NO. 4; (5) the amino acid sequence of a light chain CDR2 is set forth in SEQ ID NO. 5; and (6) the amino acid sequence of a light chain CDR3 is set forth in SEQ ID NO. 6. The monoclonal antibody 10G8 provided in the present invention can recognize the CV-A6 virus, has neutralizing activity, has a 100% protection rate in mice, and is of great significance for the treatment of diseases caused by the CV-A6 pathogen and for vaccine research.

Description

A neutralizing monoclonal antibody 10G8 that recognizes CV-A6 and its applications Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a neutralizing monoclonal antibody 10G8 that can recognize Coxsackievirus A6 (CV-A6) and its applications. Background Technology

[0002] The viruses that cause hand, foot, and mouth disease (HFMD) belong to the genus Enterovirus of the family Picornavirus. They are single-stranded positive-sense RNA viruses, including Coxsackievirus A types 2, 4, 5, 6, 7, 9, 10, and 16, Coxsackievirus B types 1, 2, 3, 4, 5, 6, and 13, Human enterovirus 71 (EV-A71), and echoviruses. Early studies identified EV-A71 as the main causative agent of HFMD. In recent years, the enterovirus pathogen spectrum for HFMD in mainland China has changed. Epidemiological data from recent years show that HFMD cases caused by CV-A6 have been steadily increasing and have become the main pathogen causing HFMD outbreaks.

[0003] Neutralizing antibodies are better able to reflect the antigenicity, immunogenicity, and function of viruses. Therefore, developing neutralizing monoclonal antibodies that target and recognize the CV-A6 structural protein is of great significance for quality control in vaccine development, detection of clinical samples, and identification of viruses in the laboratory. Summary of the Invention

[0004] Therefore, this invention involves immunizing mice with purified, inactivated CV-A6 stock solution, followed by fusion of splenic lymphocytes and myeloma cells, and screening to obtain hybridoma cells 10G8, which in turn yields the monoclonal antibody 10G8. This monoclonal antibody differs from existing CV-A6 monoclonal antibodies; it binds to different neutralizing sites, enabling it to recognize CV-A6 and exhibit neutralizing activity. It can be used for CV-A6 virus quantification and reflects viral load and function, making it crucial for quality control in vaccine development.

[0005] One objective of this invention is to provide a neutralizing monoclonal antibody 10G8 that recognizes CV-A6, wherein the six CDR regions of the neutralizing monoclonal antibody 10G8 are as follows:

[0006] (1) The amino sequence of the heavy chain CDR1 is shown in SEQ ID NO.1: GFTLKNYA;

[0007] (2) The amino sequence of the heavy chain CDR2 is shown in SEQ ID NO.2: VSSGGST;

[0008] (3) The amino sequence of the heavy chain CDR3 is shown in SEQ ID NO.3: EREEGGYAEAWFAY;

[0009] (4) The amino sequence of the light chain CDR1 is shown in SEQ ID NO.4: QSIVQSNGNTY;

[0010] (5) The amino sequence of the light chain CDR2 is shown in SEQ ID NO.5: EVS;

[0011] (6) The amino sequence of the light chain CDR3 is shown in SEQ ID NO.6: FQGSHVPFT.

[0012] Furthermore, the full-length amino acid sequence of the heavy chain variable region of the monoclonal antibody 10G8 is as shown in SEQ ID NO.7, or is an amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.7.

[0013] The full-length amino acid sequence of the light chain variable region of the monoclonal antibody 10G8 is as shown in SEQ ID NO.8, or an amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.8.

[0014] In some specific embodiments, preferably, the SEQ ID NO.7 is: EVKLVESGGGLVKPGGSLKLSCAASGFTLKNYAMSWVRQTPEKRLEWVASVSSGGSTYYLDSVKGRFTVSRDNARNILYLQMSSLRSEDTAMYYCEREEGGYAEAWFAYWGQGTLVTVSA.

[0015] In some specific embodiments, preferably, the SEQ ID NO.8 is: AVLMTQTPLSLTVSLGDQASISCRSSQSIVQSNGNTYLEWYLQKSGQSPKLLLYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK.

[0016] Furthermore, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO.9; and the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.8 is shown in SEQ ID NO.10.

[0017] In some specific embodiments, preferably, the SEQ ID NO.9 is specifically: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTAAAAAAACTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCGTTAGTAGTGGTGGTAGCACCTACTA TTTAGACAGTGTGAAGGGCCGATTCACCGTCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGAAAGAGAAGAAGGCGGTTACGCCGAGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0018] In some specific embodiments, preferably, the SEQ ID NO.10 is specifically: GCTGTTTTGATGACCCAAACTCCACTCTCCCTGACTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACAAAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAATCAGGCCAGTCTCCAAAGCTCCTGCTTTATGA AGTTTCCAACCGCTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAAATAAAA.

[0019] Furthermore, the monoclonal antibody 10G8 is an IgM antibody.

[0020] The second objective of this invention is to provide a nucleotide sequence encoding the aforementioned monoclonal antibody 10G8.

[0021] A third objective of this invention is to provide an expression vector containing a nucleic acid molecule encoding the aforementioned monoclonal antibody.

[0022] The fourth objective of this invention is to provide a host cell containing the above-mentioned nucleotide sequence or the above-mentioned expression vector.

[0023] The fifth objective of this invention is to provide an antibody conjugate comprising the above-mentioned monoclonal antibody or its antigen-binding fragment and a label.

[0024] The sixth objective of this invention is to provide the application of the above-mentioned monoclonal antibody in the preparation of reagents or kits for detecting CV-A6.

[0025] The seventh objective of this invention is to provide the application of the above-mentioned monoclonal antibody in the preparation of medicaments for inhibiting, preventing and treating diseases caused by CV-A6.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The monoclonal antibody 10G8 provided by the present invention can be combined with conjugates (such as horseradish peroxidase or fluorescein isothiocyanate) for direct or indirect detection and rapid diagnosis; for example, it can be used to develop detection reagents or kits for detecting clinical samples caused by CV-A6 infection, and can also be used for laboratory identification of antibodies in clinical virus isolates. It can also be used to prepare quantitative detection reagents for intermediate products and products containing CV-A6 antigen in preventive vaccine production.

[0028] (2) The monoclonal antibody 10G8 provided by the present invention exhibits excellent passive protective therapeutic effects. When mice injected with lethal doses of the strain were injected with different concentrations of monoclonal antibody 10G8 (162.0 ng / g mice and 486.1 ng / g mice) and fed for 14 days, it was found that the survival rate of mice in the 486.1 ng / g experimental group reached 100%, and the survival rate of mice in the 162.0 ng / g experimental group reached 70%. Attached Figure Description

[0029] Figure 1 shows the SDS-PAGE images of the IgM light and heavy chains of monoclonal antibody 10G8.

[0030] Figure 2 shows the results of antigen indirect immunofluorescence detection of CV-A6-infected RD cells by monoclonal antibody 10G8.

[0031] Figure 3 shows the results of the binding activity assay of monoclonal antibody 10G8 with CV-A6.

[0032] Figure 4 shows the results of the neutralization activity assay of monoclonal antibody 10G8 and CV-A6.

[0033] Figure 5 shows the results of the in vivo protective efficacy of the monoclonal antibody 10G8 in mice. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0035] Key experimental material sources and physicochemical parameters:

[0036] Freund's complete and incomplete adjuvants: purchased from Sigma.

[0037] Female Balb / c mice, RD cells, solution A, solution B, CV-A6 stock solution, and CV-A6 challenge strain (CVA6-R69): provided by Wuhan Institute of Biological Products Co., Ltd.

[0038] Isotyping Kit for Mouse Monoclonal Antibody: Purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0039] Goat anti-mouse IgG (H+L) AlexaFluor488: purchased from Thermo Fisher Scientific.

[0040] MTT cell proliferation and cytotoxicity assay kit and DAPI were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0041] HRP-labeled goat anti-mouse IgM: purchased from Wuhan Boster Biological Engineering Co., Ltd.

[0042] CV-A6 is a publicly disclosed strain in the prior art, recorded in the NCBI database, accession number: MW410845.

[0043] Example 1

[0044] This embodiment provides a method for preparing the neutralizing monoclonal antibody 10G8 that recognizes CV-A6. The specific steps are as follows:

[0045] (1) Immunization: Mice were immunized with purified inactivated CV-A6 stock solution using Freund's adjuvant. After four intraperitoneal immunizations at 14-day intervals, serum antibody titers were measured by blood tests. Before fusion, a tail vein pulse immunization was performed, followed by fusion of splenic lymphocytes and myeloma cells.

[0046] (2) Screening: The supernatant of fused cells was screened using the ELISA method, specifically as follows:

[0047] Dilute CV-A6 inactivated antigen with 0.05M carbonate buffer, coat 96-well microplates at a concentration of 2 μg / mL, incubate overnight at 4°C, and then block with blocking buffer (PBST-2% BSA) at 37°C for 1 h.

[0048] Add 100 μL of the test sample to each well and incubate at 37°C for 1 h. Add 100 μL of HRP-labeled goat anti-mouse IgG (1:10000) to each well and incubate at 37°C for 1 h. After washing, add 50 μL of substrate solutions A and B to each well and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well and read the OD at 450 nm using a microplate reader. 450nm value.

[0049] Hybridoma cells 10G8 were obtained through the above steps.

[0050] (3) Subcloning: Positive maternal clones were subcloned using the limiting dilution method, and subcloned clones were screened using the ELISA method. Ascites fluid was prepared by expanding the culture.

[0051] (4) Antibody production and purification: 6-8 week old female BALB / c mice were selected and injected intraperitoneally with liquid paraffin, 0.5 mL per mouse. 7-10 days after the injection of liquid paraffin, hybridoma cells were injected intraperitoneally. When the mice were swollen and near death, they were euthanized by cervical dislocation, and ascites fluid was aseptically extracted in a clean bench.

[0052] Ascites monoclonal antibodies were purified using Protein L affinity chromatography.

[0053] 1) Equilibration: Equilibrate the chromatography column with 5 to 10 column volumes of loading PBS buffer (pH 7.0) at a flow rate of 5 mL / min.

[0054] 2) Sample loading: Take 2 mL of pretreated ascites fluid and load it at a flow rate of 5 mL / min.

[0055] 3) Flow-through: Elute with loading buffer for a total of 5 column volumes to remove contaminating proteins from the ascites fluid.

[0056] 4) Elution: Wash with glycine buffer (pH 3.0) at a flow rate of 5 mL / min, using 10 column volumes. Elute the antibody with glycine buffer. When the baseline begins to rise, indicating the appearance of the elution peak, collect the antibody in a 15 mL centrifuge tube and adjust the pH to 7.0 with 1 M Tris buffer (pH 9.2).

[0057] 5) Washing: Collect the eluent until the elution peak returns to baseline, then continue eluting with 10 column volumes of 0.5M sodium hydroxide at a flow rate of 10 mL / min. Finally, equilibrate the column with 20% ethanol and elute with 10 column volumes at a flow rate of 10 mL / min.

[0058] The monoclonal antibody 10G8 was prepared and purified using the above steps.

[0059] Example 2

[0060] This embodiment focuses on sequence analysis of the monoclonal antibody 10G8 obtained in Example 1. The specific steps are as follows:

[0061] The hybridoma cells (capable of secreting monoclonal antibody 10G8) screened in Example 1 were inoculated into RPMI 1640 medium (Gibco) containing 20% ​​fetal bovine serum and cultured at 37°C.

[0062] RNA was extracted from hybridoma cells using the Vazyme FastPure Cell / Tissue Total RNA Isolation Kit, and then processed using TaKaRa RimeScript. TM cDNA was obtained by reverse transcription using the Oligo dT primers in the 1st Strand cDNA Synthesis Kit. The heavy and light chain variable regions of monoclonal antibody 10G8 were amplified using universal primers with homologous sequences to the cloning vector pUC-Kan. The purified PCR products were then cloned into the pUC-Kan vector (purchased from Nanjing Genscript Biotech Co., Ltd.). Positive clones were obtained through transformation and screening, and sequenced. The sequences were analyzed in the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions.

[0063] The sequences of the universal forward primer VH-F for the heavy chain are: ACGGCCAGTGAATTCMARCTGCAGSAGTCWGG; and the sequence of the reverse primer VH-R is: GATTACGCCAAGCTTTGAGGAGACGGTGACCG. The sequences of the universal forward primer VL-F for the light chain are: ACGGCCAGTGAATTCCGATTGTKCTSACYCARTCTCCA; and the sequence of the reverse primer VL-R is: GATTACGCCAAGCTTCGTTGGATCTCCAGCTTG. Positive clones were obtained through screening and sequenced. The sequenced sequences were analyzed in the Kabat database to obtain the correct amino acid sequences of the variable regions of the light and heavy chains.

[0064] The sequences obtained from the sequencing results of the monoclonal antibody 10G8 are shown in SEQ ID NO:1~10.

[0065] Example 3

[0066] This embodiment focuses on the purity and subtype identification of the monoclonal antibody 10G8 obtained in Example 1. The specific steps are as follows:

[0067] Take the purified monoclonal antibody 10G8 from Example 1, dilute and mix it with protein loading buffer according to the specified ratio, heat at 95°C for 10 min, briefly centrifuge, and then use the supernatant for loading. Perform SDS-PAGE on a 4-20% polyacrylamide gel. After the process, use... A protein staining instrument (purchased from Nanjing Genscript Biotech Co., Ltd.) was used for Coomassie Brilliant Blue staining and destaining. The SDS-PAGE results are shown in Figure 1. Under reducing conditions, two bands appeared, one with a molecular weight of approximately 75 kDa and the other with a molecular weight of approximately 25 kDa, corresponding to the heavy and light chains of the antibody, respectively. A mouse antibody subtype detection kit was used to identify the monoclonal antibody subtype; the results showed that the 10G8 monoclonal antibody subtype was identified as IgM.

[0068] Example 4

[0069] This embodiment focuses on the functional analysis of the monoclonal antibody 10G8 obtained in Example 1. The specific steps are as follows:

[0070] (1) Indirect immunofluorescence assay

[0071] CV-A6 cells were seeded into 6-well plates containing RD cells at 95% confluence. Uninoculated RD cells served as a negative control. After culturing for 24 hours, the cell supernatant was discarded, and the cells were gently washed three times with PBS. The cells were then fixed with 2 mL of 4% paraformaldehyde at room temperature for 1 hour, followed by three washes with PBS for 5 minutes each. 2 mL of PBST solution (containing 0.5% Triton-X) was then added. Cells were permeated at room temperature for 30 min with 100% (v / v) solution, washed 3 times with PBS for 5 min each time; blocked with blocking buffer (PBST-2% BSA) at room temperature for 1 h, then discarded; 1 mL of monoclonal antibody 10G8 (2 μg / mL) was incubated at room temperature for 1 h, washed 3 times with PBS for 5 min each time; 1 mL of goat anti-mouse IgM (H+L) Alexa Fluor 488 (2 μg / mL) was incubated at room temperature in the dark for 1 h, washed 3 times with PBS for 5 min each time, and 1 mL of DAPI (5 μg / mL) was added; washed 3 times with PBS for 5 min each time. The cells were observed and photographed using a fluorescence microscope. The negative control group consisted of cells not infected with CV-A6, with the corresponding antibody added. The results are shown in Figure 2: Monoclonal antibody 10G8 can be used in indirect immunofluorescence experiments to recognize the CV-A6 antigen and can be used for antigen identification experiments.

[0072] (2) Combined with activity assay

[0073] Dilute the CV-A6 stock solution to 1 μg / mL with 0.05 M phosphate buffer (pH 7.2) and coat it onto an ELISA plate at 150 μL / well, incubating overnight at 4°C. Wash the plate 5 times with PBST, then block with blocking buffer (PBST-2% BSA) at 37°C for 1 h. Wash the plate 5 times with PBST, then dilute the purified 10G8 monoclonal antibody to 5.4 μg / mL and add 100 μL to the first well of the ELISA plate, setting up 3 replicates. Dilute the antibody 2-fold and incubate at 37°C for 1 hour; wash 5 times with PBST, add 100 μL / well of HRP-labeled goat anti-mouse IgM (1:10000), and incubate at 37°C for 1 hour; wash 5 times with PBST, add 50 μL of substrate A and B solutions to each well of the microplate, and incubate at 37°C for 15 minutes; add 50 μL of 2M sulfuric acid to each well to stop the reaction, and read the OD at 450 nm using a microplate reader. 450nm Value. Analysis showed that the median effect dose (EC50) of monoclonal antibody 10G8 was... 50 The concentration was 18.57 ng / mL. The results of the binding activity assay of monoclonal antibody 10G8 to CV-A6 are shown in Figure 3.

[0074] (2) Neutralization activity identification

[0075] Dilute the purified monoclonal antibody 10G8 to 200 μg / mL and add 100 μL / well to column 1 of a 96-well plate, setting up 8 replicates. Then, serially dilute the antibody in column 1 twofold. Dilute the CV-A6 stock solution to 100 CCID. 50 / 50μL, pipette 50μL and vertically suspend it in the air to add to a 96-well plate containing the diluted antibody. Incubate the 96-well plate at 37°C for 2 hours. Dilute the diluted CV-A6 stock solution 10-fold serially to 10 CCID. 50 / 50μL, 1CCID 50 / 50μl, 0.1CCID 50 / 50μL. Add 50μL of MEM maintenance solution and 50μL of CV-A6 stock solution to each well (4 dilutions in total: 100, 10, 1, 0.1 CCID). 50 / 50μL), 8 replicates per dilution, used as a back-drop plate, incubated at 4°C. After neutralization, the digested RD cells were then... 5 Spread at a density of 1 / mL onto the neutralization plate and the re-dropping plate.

[0076] Cells were cultured at 37°C and 5% CO2 for 4 days. Using an MTT assay kit, 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C for 4 hours. Then, 100 μL of Formazan lysate was added to each well, mixed well, and incubated for another 4 hours at 37°C. OD values ​​were read at 570 nm using a microplate reader. 570nm Value. With positive well (100 CCID) 50 / 50μL)OD 570nm The value was set to 100%, and the neutralization percentage at different antibody concentrations was calculated. Analysis revealed the half-maximal inhibitory concentration (IC50) of monoclonal antibody 10G8. 50 The concentration was 0.026 μg / mL. The results of the neutralizing activity assay of monoclonal antibody 10G8 and CV-A6 are shown in Figure 4.

[0077] Example 5

[0078] This embodiment evaluates the protective efficacy of the monoclonal antibody 10G8 obtained in Example 1 in mice. The specific steps are as follows:

[0079] To evaluate the protective efficacy of monoclonal antibody 10G8 in mice, 14-day-old Balb / c mice were intraperitoneally injected with a lethal dose of the CV-A6 challenge strain (CVA6-R69), 300 μL per mouse. Two hours later, these mice were intraperitoneally injected with different concentrations of monoclonal antibody 10G8 (162.0 ng / g mice and 486.1 ng / g mice), 100 μL per mouse. The mice were observed for 14 consecutive days, and their survival rate was recorded daily.

[0080] The results of the in vivo protective efficacy of the monoclonal antibody 10G8 in mice are shown in Figure 5: when the monoclonal antibody dose was 486.1 ng / g mice, all mice survived during the 14-day observation period, showing a 100% protection rate.

[0081] The inventors also discovered through experiments that the amino acid sequence formed by substituting, deleting, or adding one or more amino acid sequences to the heavy chain amino acid sequence of monoclonal antibody 10G8, or an amino acid sequence with more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, has the same function as the sequence shown in SEQ ID NO.7; the amino acid sequence formed by substituting, deleting, or adding one or more amino acid sequences to the light chain amino acid sequence of monoclonal antibody 10G8, or an amino acid sequence with more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, has the same function as the sequence shown in SEQ ID NO.8.

[0082] Through the above series of investigations and verifications, it has been proven that the monoclonal antibody 10G8 provided in this application can recognize CV-A6 and has neutralizing activity. It can be used for CV-A6 virus quantification and reflects the content and function of the virus, which is crucial for quality control in vaccine development.

[0083] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0084] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A neutralizing monoclonal antibody 10G8 that recognizes CV-A6, characterized in that, The six CDR regions of the monoclonal antibody 10G8 are as follows: (1) The amino sequence of the heavy chain CDR1 is shown in SEQ ID NO.1; (2) The amino sequence of the heavy chain CDR2 is shown in SEQ ID NO.2; (3) The amino sequence of the heavy chain CDR3 is shown in SEQ ID NO.3; (4) The amino sequence of the light chain CDR1 is shown in SEQ ID NO.4; (5) The amino sequence of the light chain CDR2 is shown in SEQ ID NO.5; (6) The amino sequence of the light chain CDR3 is shown in SEQ ID NO.

6.

2. The monoclonal antibody 10G8 according to claim 1, characterized in that, The full-length amino acid sequence of the heavy chain variable region of the monoclonal antibody 10G8 is shown in SEQ ID NO.7, or an amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.

7. The full-length amino acid sequence of the light chain variable region of the monoclonal antibody 10G8 is as shown in SEQ ID NO.8, or an amino acid sequence that has more than 95% homology with the amino acid sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody 10G8 according to claim 2, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.7 is shown in SEQ ID NO.9; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 8 is shown in SEQ ID NO.

10.

4. The monoclonal antibody 10G8 according to any one of claims 1-3, characterized in that, The monoclonal antibody 10G8 is an IgM antibody.

5. The nucleotide sequence encoding the monoclonal antibody 10G8 according to any one of claims 1 to 4.

6. An expression carrier, characterized in that, The vector contains a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4.

7. A host cell, characterized in that, The host cell contains the nucleotide sequence of claim 5, or the expression vector of claim 6.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the monoclonal antibody or its antigen-binding fragment and a marker as described in any one of claims 1 to 4.

9. The use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of reagents or kits for detecting CV-A6.

10. The use of the monoclonal antibody according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting, preventing and treating diseases caused by CV-A6.

Images