A neutralizing monoclonal antibody 10G8 that recognizes CV-A6 and its application

Abstract

The present invention provides a neutralizing monoclonal antibody 10G8 that recognizes CV-A6. The six CDR regions of the monoclonal antibody 10G8 are as follows: (1) the amino sequence of the heavy chain CDR1 is GFTLKNYA; (2) the amino sequence of the heavy chain CDR2 is VSSGGST; (3) the amino sequence of the heavy chain CDR3 is EREEGGYAEAWFAY; (4) the amino sequence of the light chain CDR1 is QSIVQSNGNTY; (5) the amino sequence of the light chain CDR2 is EVS; and (6) the amino sequence of the light chain CDR3 is FQGSHVPFT. The monoclonal antibody 10G8 provided by the present invention can recognize the CV-A6 virus and has neutralizing activity, with a 100% protection rate in mice, and is of great significance for the treatment of diseases caused by the CV-A6 pathogen and vaccine research.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to a neutralizing monoclonal antibody 10G8 capable of identifying Coxsackievirus A6 (CV-A6) and an application thereof. Background Art

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

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

[0004] In light of this, the present invention further obtained the monoclonal antibody 10G8 by immunizing mice with purified inactivated CV-A6 stock solution, then fusing splenic lymphocytes with myeloma cells, and screening to obtain the hybridoma cell 10G8. This monoclonal antibody differs from the existing CV-A6 monoclonal antibody 10G8 in that it binds to a different neutralizing site, capable of recognizing CV-A6 and exhibiting neutralizing activity. It can be used to quantify the CV-A6 virus and reflect the virus content and function, which is crucial for quality control in vaccine development.

[0005] One of the objectives of the present invention is to provide a neutralizing monoclonal antibody 10G8 that recognizes CV-A6. The six CDR regions of the neutralizing monoclonal antibody 10G8 are as follows:

[0006] (1) The amino acid sequence of the heavy chain CDR1 is: GFTLKNYA;

[0007] (2) The amino sequence of the heavy chain CDR2 is: VSSGGST;

[0008] (3) The amino sequence of the heavy chain CDR3 is: EREEGGYAEAWFAY;

[0009] (4) The amino sequence of the light chain CDR1 is: QSIVQSNGNTY;

[0010] (5) The amino sequence of light chain CDR2 is: EVS;

[0011] (6) The amino acid sequence of the light chain CDR3 is: 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.1, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.1;

[0013] The full-length amino acid sequence of the light chain variable region of the monoclonal antibody 10G8 is shown in SEQ ID NO. 2, or an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO. 2.

[0014] In some specific embodiments, preferably, the SEQ ID NO.1 is specifically: EVKLVESGGGLVKPGGSLKLSCAASGFTLKNYAMSWVRQTPEKRLEWVASVSSGGSTYYLDSVKGRFTVSRDNARNILYLQMSSLRSEDTAMYYCEREEGGYAEAWFAYWGQGTLVTVSA.

[0015] In some specific embodiments, preferably, the SEQ ID NO. 2 is specifically: AVLMTQTPLSLTVSLGDQASISCRSSQSIVQSNGNTYLEWYLQKSGQSPKLLLYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK.

[0016] Furthermore, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.3; the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.4.

[0017] In some specific embodiments, preferably, the SEQ ID NO.3 is specifically: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTAAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACCTTAAAAAAACTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCGTTAGTAGTGGTGGTAGCACCTACTA TTTAGACAGTGTGAAGGGCCGATTCACCGTCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGAAAGAGAAGAAGGCGGTTACGCCGAGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0018] In some specific embodiments, preferably, the SEQ ID NO.4 is specifically: GCTGTTTTGATGACCCAAACTCCACTCTCCCTGACTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACAAAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAATCAGGCCAGTCTCCAAAGCTCCTGCTTTATGA AGTTTCCAACCGCTTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAAATAAAA.

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

[0020] A second object of the present invention is to provide a nucleotide sequence encoding the monoclonal antibody 10G8.

[0021] A third object of the present invention is to provide an expression vector comprising a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0022] A fourth object of the present invention is to provide a host cell, wherein the host cell contains the above-mentioned nucleotide sequence or the above-mentioned expression vector.

[0023] A fifth object of the present invention is to provide an antibody conjugate, wherein the antibody conjugate comprises the above-mentioned monoclonal antibody or antigen-binding fragment thereof and a label.

[0024] A sixth object of the present invention is to provide the use of the above-mentioned monoclonal antibody in the preparation of a reagent or kit for detecting CV-A6.

[0025] A seventh object of the present invention is to provide the use of the above-mentioned monoclonal antibody in the preparation of a drug for preventing or treating diseases caused by CV-A6.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The monoclonal antibody 10G8 provided by the present invention can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) 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 preventive vaccine production intermediates and products containing CV-A6 antigen quantitative detection reagents.

[0028] (2) The monoclonal antibody 10G8 provided by the present invention exhibits excellent passive protective therapeutic effects. When mice injected with a lethal dose of the strain were injected with different concentrations of monoclonal antibody 10G8 (162.0 ng / g mice, 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 the 162.0 ng / g experimental group reached 70%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the SDS-PAGE image of the IgM light chain and heavy chain of the monoclonal antibody 10G8.

[0030] Figure 2 This is the result of indirect immunofluorescence detection of antigens in RD cells infected with CV-A6 using monoclonal antibody 10G8.

[0031] Figure 3 The results show the binding activity of monoclonal antibody 10G8 to CV-A6.

[0032] Figure 4 The results show the neutralizing activity of monoclonal antibody 10G8 against CV-A6.

[0033] Figure 5 The results show the protective efficacy of monoclonal antibody 10G8 in mice. DETAILED DESCRIPTION

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

[0035] Sources of key test materials and physical and chemical parameters:

[0036] Freund's complete and incomplete adjuvants were 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) were provided by Wuhan Institute of Biological Products Co., Ltd.

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

[0039] Goat anti-mouse IgG (H+L) Alexa Fluor 488: 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 Bioengineering Co., Ltd.

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

[0043] Example 1

[0044] This example 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. Four intraperitoneal immunizations were performed 14 days apart, and serum antibody titers were measured by blood test. Before fusion, tail vein pulse immunization was performed once, followed by fusion of spleen lymphocytes and myeloma cells.

[0046] (2) Screening: Use the ELISA method to screen the supernatant of fused cells, specifically:

[0047] CV-A6 inactivated antigen was diluted with 0.05 M carbonate buffer and coated on a 96-well microplate at a coating concentration of 2 μg / mL at 4°C overnight. The plate was then blocked with blocking solution (PBST-2% BSA) at 37°C for 1 h.

[0048] Add the sample to be tested at 100 μL / well and incubate at 37°C for 1 hour. Add HRP-labeled goat anti-mouse IgG (1:10,000) at 100 μL / well and incubate at 37°C for 1 hour. After washing, add substrate solution A and solution B at 50 μL / well each and develop at 37°C in the dark for 15 minutes. Add stop solution at 50 μL / well and read the OD at 450 nm on a microplate reader. 450nm value.

[0049] The hybridoma cell 10G8 was obtained through screening through the above steps.

[0050] (3) Subcloning: Subclone the positive mother clone using the limiting dilution method, screen the subclones using the ELISA method, and expand the culture to prepare ascites.

[0051] (4) Antibody production and purification: Female BALB / c mice aged 6-8 weeks were selected and intraperitoneally injected with 0.5 mL of liquid paraffin per mouse. 7-10 days after the injection of liquid paraffin, hybridoma cells were intraperitoneally injected. When the abdomen of the mouse was distended and the mouse was on the verge of death, it was killed by cervical dislocation, and ascites was aseptically extracted in a clean bench.

[0052] Purification of ascites monoclonal antibodies using Protein L affinity chromatography:

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

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

[0055] 3) Flow-through: The loading buffer is used to elute 5 column volumes to remove the impurities in the ascites.

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

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

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

[0059] Example 2

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

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

[0062] Hybridoma cell RNA was extracted using the Vazyme FastPure Cell / Tissue Total RNA Isolation Kit, and then reverse transcribed using the Oligo dT primer in the TaKaRa RimeScript™ II 1st Strand cDNA Synthesis Kit to generate cDNA. The heavy and light chain variable region genes of monoclonal antibody 10G8 were amplified using universal primers for both the light and heavy chains, each with sequences homologous to the cloning vector pUC-Kan. The purified PCR products were cloned into the pUC-Kan vector (purchased from Nanjing GenScript Biotechnology Co., Ltd.). Positive clones were screened and sequenced, and the sequences were analyzed against the Kabat database to determine the correct amino acid sequences for the light and heavy chain variable regions.

[0063] The sequence of the universal forward primer for the heavy chain, VH-F, was ACGGCCAGTGAATTCMARCTGCAGSAGTCWGG; the sequence of the reverse primer, VH-R, was GATTACGCCAAGCTTTGAGGAGACGGTGACCG. The sequence of the universal forward primer for the light chain, VL-F, was ACGGCCAGTGAATTCCGATTGTKCTSACYCARTCTCCA; the sequence of the reverse primer, VL-R, was GATTACGCCAAGCTTCGTTGGATCTCCAGCTTG. Positive clones were screened and sequenced, and the sequenced sequences were analyzed against the Kabat database to obtain the correct amino acid sequences of the light and heavy chain variable regions.

[0064] The sequences analyzed from the sequencing results of monoclonal antibody 10G8 are shown in SEQ ID NOs: 1 to 4.

[0065] Example 3

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

[0067] The monoclonal antibody 10G8 purified in Example 1 was diluted and mixed with protein loading buffer according to the proportion, heated at 95°C for 10 min, and centrifuged briefly to collect the supernatant for sample loading; SDS-PAGE was performed on a 4-20% polyacrylamide gel, and the eStain ® Coomassie brilliant blue staining and destaining were performed using a protein stainer (purchased from Nanjing GenScript Biotechnology Co., Ltd.). SDS-PAGE results are shown in Figure 1 Under reducing conditions, two bands with molecular weights of approximately 75 kDa and 25 kDa were observed, 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, and the results showed that the monoclonal antibody 10G8 was identified as IgM.

[0068] Example 4

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

[0070] (1) Indirect immunofluorescence experiment

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

[0072] (2) Binding activity assay

[0073] The CV-A6 stock solution was diluted to 1 μg / mL with 0.05 M phosphate buffer (pH 7.2) and coated on the ELISA plate at 150 μL / well at 4°C overnight; the plate was washed 5 times with PBST and blocked with blocking solution (PBST-2% BSA) at 37°C for 1 hour; the plate was washed 5 times with PBST, the purified 10G8 monoclonal antibody was diluted to 5.4 μg / mL and 100 μL was added to the first well of the ELISA plate, 3 replicates were set up, and then the antibody was diluted 2-fold and incubated at 37°C for 1 hour; the plate was washed 5 times with PBST, and HRP-labeled goat anti-mouse IgM (1:10,000) was added at 100 μL / well and incubated at 37°C for 1 hour; the plate was washed 5 times with PBST, and 50 μL of substrate A solution and B solution were added to the ELISA plate respectively, and incubated at 37°C for 15 minutes; 2 M sulfuric acid was added to the ELISA plate at 50 μL per well to stop the reaction and placed in the ELISA reader at 450 Read OD at nm wavelength 450nm After analysis, the median effect dose (ECD) of the monoclonal antibody 10G8 50 The results of the binding activity assay of monoclonal antibody 10G8 and CV-A6 are as follows: Figure 3 shown.

[0074] (2) Neutralization activity identification

[0075] After the purified monoclonal antibody 10G8 was diluted to 200 μg / mL, it was added to the first column of a 96-well plate at 100 μL / well. Eight replicate wells were set up, and then the antibody in the first column was serially diluted 2-fold. The CV-A6 stock solution was diluted to 100 CCID 50 / 50μL, pipette 50μL vertically and add it to the 96-well plate with diluted antibody. Place the 96-well plate in a 37℃ incubator for 2 hours. Dilute the diluted CV-A6 stock solution in a 10-fold gradient to 10 CCID 50 / 50μL, 1 CCID 50 / 50μl, 0.1 CCID 50 / 50μL. Add 50μL MEM maintenance solution and 50μL CV-A6 stock solution to each well (a total of 4 dilutions, namely 100, 10, 1, and 0.1 CCID 50 / 50μL), 8 replicate wells for each dilution, as a back titration plate, placed at 4℃. After neutralization, the digested RD cells were plated at 1×10 5 The cells were plated at a density of 1000 / mL in a neutralization plate and a back titration plate.

[0076] Culture at 37°C, 5% CO2 for 4 days. Use the MTT cell proliferation and cytotoxicity assay kit to add 10 μL MTT solution to each well and incubate at 37°C for 4 hours. Add 100 μL Formazan solution to each well, mix appropriately, and continue incubating at 37°C for 4 hours. Place on a microplate reader and read the OD at a wavelength of 570 nm. 570nm Value. Take positive wells (100 CCID 50 / 50μL) OD 570nm The neutralization percentage at different antibody concentrations was calculated by setting the value as 100%. 50 ) was 0.026 μg / mL. The results of the neutralization activity assay of monoclonal antibody 10G8 and CV-A6 are as follows Figure 4 shown.

[0077] Example 5

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

[0079] To evaluate the protective ability 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) at a dose of 300 μL per mouse. Two hours later, the mice were intraperitoneally injected with 100 μL of various concentrations of monoclonal antibody 10G8 (162.0 ng / g mouse and 486.1 ng / g mouse). The mice were observed for 14 days, and the survival rate was recorded daily.

[0080] The results of the protective efficacy of monoclonal antibody 10G8 in mice are as follows Figure 5 As shown: When the monoclonal antibody dose was 486.1 ng / g mouse, 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 of the heavy chain amino acid sequence of monoclonal antibody 10G8, an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence set forth in SEQ ID NO.1, or an amino acid sequence having greater than 95% homology to the amino acid sequence set forth in SEQ ID NO.1, has the same function as the sequence set forth in SEQ ID NO.1; and the amino acid sequence of the light chain amino acid sequence of monoclonal antibody 10G8, an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence set forth in SEQ ID NO.2, or an amino acid sequence having greater than 95% homology to the amino acid sequence set forth in SEQ ID NO.2, has the same function as the sequence set forth in SEQ ID NO.2.

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

[0083] The raw materials not specifically described in the present invention are all existing materials that can be directly purchased from the market.

[0084] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in 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 specifically as follows: (1) The amino sequence of the heavy chain CDR1 is GFTLKNYA; (2) The amino sequence of the heavy chain CDR2 is VSSGGST; (3) The amino sequence of the heavy chain CDR3 is EREEGGYAEAWFAY; (4) The amino sequence of the light chain CDR1 is QSIVQSNGNTY; (5) The amino sequence of the light chain CDR2 is EVS; (6) The amino sequence of the light chain CDR3 is FQGSHVPFT.

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

1.

3. The monoclonal antibody 10G8 according to claim 1, characterized in that The full-length amino acids of the light chain variable region of the monoclonal antibody 10G8 are amino acid sequences that have more than 95% homology with the amino acid sequence shown in SEQ ID NO.

2.

4. 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. 1; The full-length amino acids of the light chain variable region of the monoclonal antibody 10G8 are shown in SEQ ID NO.

2.

5. The monoclonal antibody 10G8 according to claim 4, characterized in that The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.3; The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.

4.

6. The monoclonal antibody 10G8 according to any one of claims 1 to 5, characterized in that The monoclonal antibody 10G8 is an IgM antibody.

7. A nucleic acid molecule encoding the monoclonal antibody 10G8 according to any one of claims 1 to 6.

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

9. A host cell, characterized in that The host cell contains the nucleic acid molecule according to claim 7, or contains the expression vector according to claim 8.

10. An antibody conjugate, characterized in that The antibody conjugate comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and a label.

11. Use of the monoclonal antibody according to any one of claims 1 to 6 in the preparation of a reagent or kit for detecting CV-A6.

12. Use of the monoclonal antibody according to any one of claims 1 to 6 in the preparation of a medicament for preventing or treating diseases caused by CV-A6.

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