Monoclonal antibody against coxsackie virus A6 and preparation method and application thereof

Abstract

The present application relates to the technical field of antibodies, in particular to a monoclonal antibody against coxsackie virus A6 and a preparation method and application thereof.The heavy chain complementarity determining region CDR1, CDR2 and CDR3 of the antibody or antigen binding fragment thereof have the amino acid sequences shown as SEQ ID NO.1-3, and the light chain complementarity determining region CDR1, CDR2 and CDR3 have the amino acid sequences shown as SEQ ID NO.4-6.The antibody or antigen binding fragment thereof has CV-A6 specificity and intratypic broad spectrum, can cross bind and neutralize different subtype strains of CV-A6, has a good protective effect on animals infected with CV-A6, and has important significance for the detection of CV-A6, the research and development, evaluation and clinical diagnosis of vaccines, and the treatment of CV-A6 infection and diseases related thereto.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to a monoclonal antibody against Coxsackievirus A6, its preparation method, and its application. Background Technology

[0002] In recent years, hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A6 (CV-A6) has been on the rise globally. In some countries, CV-A6 has even replaced EV71 and CV-A16 as the most prevalent pathogen causing HFMD. Children infected with CV-A6 mainly present with fever, rash, and nail loss. Studies have found that the proportion of patients infected with CV-A6 who develop fever is significantly higher than those infected with EV71 and CV-A16, with an average body temperature of approximately 38.8°C. Adults can also be infected with CV-A6, exhibiting the above symptoms plus rashes on the palms, soles, and around the mouth. These are common clinical manifestations of HFMD, but not all patients present with these symptoms; some patients may be asymptomatic. Currently, research on CV-A6 is incomplete, and no corresponding vaccine is available. The currently prevalent CV-A6 strain globally is a recombinant variant. CV-A6 is also prone to co-infection with other enteroviruses. Recombinant CV-A6 infection may cause more extensive skin lesions than non-recombinant CV-A6 infection, with more frequent skin lesions appearing on the upper limbs, lower limbs, and forequarters.

[0003] With the advancement of gene sequencing technology, studies have found that the VP1 segment in the viral genome encodes genes associated with the main neutralizing epitopes of the virus. Therefore, the homology of the VP1 segment and the results of neutralization experiments show a high correlation. Thus, homology comparison based on the VP1 segment of the viral genome has become one of the gold standards for virus identification and typing. Currently, there is no internationally recognized CV-A6 genotyping standard. Based on the standard of a difference of >15% in the whole genome sequence (915bp) of the VP1 coding region and a difference of >8% in nucleotides between subtypes, Song et al. (Song Y, Zhang Y, Ji T, et al. Persistent circulation of Coxsackievirus A6 of genotype D3 in mainland of China between 2008 and 2015[J]. Scientific reports,2017,7(1): 1-11.) divided CV-A6 into four genotypes: A, B, C, and D, as well as genotypes B1 and B2, C1 and C2, D1, D2, and D3. Genotype A contains a single strain, namely the CVA6 prototype strain Gdula, which was isolated in the United States in 1949. In recent years, the prevalent CV-A6 strains are mainly subtypes D2 and D3, with subtype D3 and its D3a evolutionary branch being more common.

[0004] Studies have shown that immunoglobulins can significantly reduce the levels of cytokines such as interferon-gamma, interleukin-6, and interleukin-8. However, immunoglobulins also have limitations, such as limited broad-spectrum activity, inability to neutralize all serotypes, and potential for enhanced infection. Current research on therapeutic drugs focuses on monoclonal antibodies against specific viruses and small molecule drugs that inhibit viral entry or replication.

[0005] Monoclonal antibodies, characterized by high targeting and low toxicity, are crucial for the clinical diagnosis and treatment of viral infections and are essential tools in modern life sciences, widely used in clinical diagnosis, treatment, and vaccine evaluation. Therefore, developing monoclonal antibodies against CV-A6 is of great significance for the diagnosis, treatment, and vaccine production of this viral infection. Summary of the Invention

[0006] The purpose of this invention is to provide an antibody against Coxsackievirus A6 and its antigen-binding fragment, as well as a method for preparing the antibody and its antigen-binding fragment and their applications.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or the antigen-binding fragment thereof have amino acid sequences as shown in SEQ ID NO. 1-3, and / or, the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or the antigen-binding fragment thereof have amino acid sequences as shown in SEQ ID NO. 4-6.

[0009] In this invention, an antigen-binding fragment refers to a polypeptide containing a fragment of a full-length antibody, which maintains the ability to specifically bind to the same antigen bound by the full-length antibody, or can compete with the full-length antibody for specific binding to the antigen.

[0010] In some embodiments of the present invention, the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment have amino acid sequences as shown in SEQ ID NO. 1-3, and the light chain complementarity-determining regions CDR1, CDR2, and CDR3 have amino acid sequences as shown in SEQ ID NO. 4-6.

[0011] In some embodiments of the present invention, the amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 1-3, and the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO. 4-6.

[0012] The antibodies or antigen-binding fragments provided by this invention possess both CV-A6 specificity and intratype broad-spectrum activity, enabling them to cross-bind with and neutralize strains of different CV-A6 subtypes.

[0013] Preferably, the heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.7 or has an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.7; and / or, the light chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.8 or has an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.8.

[0014] In some embodiments of the present invention, the heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.7, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO.8.

[0015] In some embodiments of the present invention, when the heavy chain and light chain complementarity-determining regions CDR1, CDR2, and CDR3 described above are present, the heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with the amino acid sequence shown in SEQ ID NO. 8.

[0016] In some embodiments of the present invention, the antibody or its antigen-binding fragment further includes a signal peptide at the N-terminus of the heavy chain variable region.

[0017] In some embodiments of the present invention, the signal peptide at the N-terminus of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO.9.

[0018] In some embodiments of the present invention, the antibody or its antigen-binding fragment further includes a signal peptide at the N-terminus of the light chain variable region.

[0019] In some embodiments of the present invention, the signal peptide at the N-terminus of the light chain variable region has an amino acid sequence as shown in SEQ ID NO.10.

[0020] In this invention, the antibody may be a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0021] The monoclonal antibody may be a murine antibody, a chimeric antibody, or a humanized antibody.

[0022] In this invention, the antigen-binding fragment may be Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity-determining region fragment, or a single-chain antibody.

[0023] In some embodiments of the present invention, the antibody further comprises a heavy chain constant region.

[0024] In some embodiments of the present invention, the heavy chain of the antibody is IgG2b.

[0025] In some embodiments of the present invention, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.11.

[0026] In some embodiments of the present invention, the antibody further comprises a light chain constant region.

[0027] In some embodiments of the present invention, the light chain of the antibody is Kappa.

[0028] In some embodiments of the present invention, the amino acid sequence of the light chain constant region is shown in SEQ ID NO.12.

[0029] Secondly, the present invention provides a labeled antibody, which is obtained by labeling the antibody or its antigen-binding fragment described above with an enzyme, biotin, fluorescent dye, chemiluminescent dye and / or radioisotope.

[0030] Thirdly, the present invention provides a nucleic acid molecule that encodes the antibody or its antigen-binding fragment.

[0031] Based on the amino acid sequence of the antibody or its antigen-binding fragment described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence encoding the nucleic acid molecule of the antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the antibody or its antigen-binding fragment are within the scope of protection of this invention.

[0032] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain of the antibody is shown in SEQ ID NO.13, and the nucleotide sequence of the nucleic acid molecule encoding the light chain of the antibody is shown in SEQ ID NO.14.

[0033] Fourthly, the present invention provides a biomaterial containing the nucleic acid molecules described above; the biomaterial is an expression cassette, a vector, or a host cell.

[0034] The expression cassette described above can be a recombinant DNA obtained by operatively linking the nucleic acid molecule to a promoter.

[0035] The aforementioned vectors include, but are not limited to, plasmid vectors, bacteriophage vectors, viral vectors, and artificial chromosome vectors.

[0036] The host cells mentioned above include microbial cells, animal cells, or cell lines. Among them, microbial cells include, but are not limited to, Escherichia coli and yeast, animal cells include, but are not limited to, insect cells, and cell lines include, but are not limited to, CHO cells and 293T cells.

[0037] Fifthly, the present invention provides a method for preparing the antibody or its antigen-binding fragment, the method comprising: culturing host cells capable of expressing the antibody or its antigen-binding fragment, and separating the antibody or its antigen-binding fragment.

[0038] In a sixth aspect, the present invention provides any of the following applications of the antibody or its antigen-binding fragment, the labeled antibody, the nucleic acid molecule, or the biological material:

[0039] (1) Application in the preparation of products for detecting the presence or level of Coxsackievirus A6 in a sample;

[0040] (2) Use in the preparation of products for the diagnosis of Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection;

[0041] (3) Application in the preparation of products for neutralizing the virulence of Coxsackievirus A6 in samples;

[0042] (4) Use in the preparation of medicines for the prevention or treatment of Coxsackievirus A6 infection or diseases caused by Coxsackievirus A6 infection;

[0043] (5) Application in detecting the antigenicity and immunogenicity of Coxsackievirus A6 vaccine;

[0044] (6) Application in quality control of Coxsackievirus A6 vaccine production;

[0045] (7) Application in detecting the specificity of Coxsackievirus A6 antigen.

[0046] In the application described in (1) above, detecting the presence of Coxsackievirus A6 in a sample means detecting whether the sample contains Coxsackievirus A6, and detecting the level of Coxsackievirus A6 means detecting the content of Coxsackievirus A6 in the sample.

[0047] In the applications described in (1) and (3) above, the sample can be a sample from a living person or animal (including blood, excrement, oral and nasal secretions, etc.) or a cell sample cultured in vitro.

[0048] In the applications described in (1) and (2) above, the product may be a detection reagent or a kit.

[0049] In the applications described in (2) and (4) above, diseases caused by Coxsackievirus A6 infection include hand-foot-mouth disease, etc.

[0050] In the application described in (5) above, immunogenicity testing specifically refers to testing the performance of the Coxsackievirus A6 vaccine in inducing an immune response in animals, including the evaluation of the humoral immune function of immunized animals (e.g., neutralizing antibodies and their levels, antibody affinity).

[0051] In the application described in (6) above, the quality control of the Coxsackievirus A6 vaccine specifically involves testing whether the quality, content, and stability of the antigen in the Coxsackievirus A6 vaccine are up to standard.

[0052] In the above applications, detection methods can include enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, and immunochromatography. The antibody or its antigen-binding fragment provided by this invention can be used as a binding antibody in the above detection methods for the detection of Coxsackievirus A6 vaccine antigen.

[0053] In a seventh aspect, the present invention provides a kit comprising the antibody or its antigen-binding fragment or the labeled antibody.

[0054] The above-mentioned reagent kit has any of the following uses:

[0055] (1) Detect the presence or level of Coxsackievirus A6 in the sample;

[0056] (2) Diagnosis of Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection;

[0057] (3) Detect the antigenicity and immunogenicity of the Coxsackievirus A6 vaccine;

[0058] (4) Quality control in the production of Coxsackievirus A6 vaccine;

[0059] (5) Specificity of detection of Coxsackievirus A6 antigen.

[0060] In addition to the antibody or its antigen-binding fragment or the labeled antibody, the above-mentioned kit may also contain a second antibody carrying a detectable label to detect the antibody or its antigen-binding fragment of the present invention. The detectable label includes, but is not limited to, enzymes, radioisotopes, fluorescent dyes, etc.

[0061] In an eighth aspect, the present invention provides a medicament comprising the antibody or an antigen-binding fragment thereof.

[0062] The above-mentioned drugs have any of the following uses:

[0063] (1) Prevention of Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection;

[0064] (2) Treatment of Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection.

[0065] In addition to the antibody or its antigen-binding fragment, the aforementioned drugs may also contain pharmaceutically acceptable carriers, excipients or other active ingredients.

[0066] Ninthly, the present invention provides a method for detecting the presence or level of Coxsackievirus A6 in a sample, the method comprising detecting the presence or level of Coxsackievirus A6 in a sample using an antibody or antigen-binding fragment thereof of the present invention. The method may be used for diagnostic purposes (samples derived from living humans or animals) or for non-diagnostic purposes (samples are in vitro cultured cell samples, rather than samples derived from living humans or animals).

[0067] In a tenth aspect, the present invention provides a method for diagnosing Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection, the method comprising: using the antibody of the present invention or its antigen-binding fragment thereof to diagnose Coxsackievirus A6 infection or disease caused by Coxsackievirus A6 infection.

[0068] Eleventhly, the present invention provides a method for neutralizing the virulence of Coxsackievirus A6 in a sample, the method comprising contacting a sample containing Coxsackievirus A6 with an antibody or antigen-binding fragment of the present invention. The method can be used for therapeutic or non-therapeutic purposes (the sample is a cultured cell sample, not a sample from a living human or animal).

[0069] In a twelfth aspect, the present invention provides a method for preventing or treating diseases (including hand-foot-mouth disease, etc.) caused by Coxsackievirus A6 infection, the method comprising: administering to a subject a preventive or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention or a medicament comprising said antibody or antigen-binding fragment thereof.

[0070] In a thirteenth aspect, the present invention provides a quality control method for the production of a Coxsackievirus A6 vaccine, the method comprising the step of detecting Coxsackievirus A6 using the antibody or antigen-binding fragment of the present invention.

[0071] The beneficial effects of the present invention include at least the following aspects:

[0072] (1) The antibody or its antigen-binding fragment provided by the present invention has both CV-A6 specificity and intratype broad spectrum, and can specifically bind to CV-A6 without cross-reactivity with other type antigens and Vero host cell proteins; and can also cross-bind to different subtypes of CV-A6 strains.

[0073] (2) The antibody or its antigen-binding fragment provided by the present invention has broad-spectrum neutralizing activity and can cross-neutralize different subtypes of CV-A6.

[0074] (3) The antibody or its antigen-binding fragment provided by the present invention can competitively inhibit human naturally infected serum.

[0075] (4) The antibody or its antigen-binding fragment provided by the present invention has a good protective effect on CV-A6 infected animals, and the protective effect is related to the antibody dose.

[0076] The antibodies or antigens provided by this invention can be used to detect the presence or level of CV-A6, evaluate the antigenicity and immunogenicity of CV-A6 vaccines, and are of great significance for the detection of CV-A6, the research and development, evaluation and clinical diagnosis of CV-A6 vaccines; they can also be used in the development of drugs for the prevention or treatment of CV-A6, and are of great significance for the treatment of CV-A6 infection and related diseases. Attached Figure Description

[0077] Figure 1 The images show the electron microscopy identification of solid and hollow virus particles after sucrose density gradient centrifugation in Example 1 of this invention. The left image shows hollow particles, and the right image shows solid particles. The scale bar in the images is 100 nm.

[0078] Figure 2 The results are SDS-PAGE assays of the monoclonal antibody in Example 3 of this invention.

[0079] Figure 3 This is a competitive inhibition test between monoclonal antibody and human serum in Example 3 of the present invention.

[0080] Figure 4 This is the reaction curve of the enzyme-labeled antibody in Example 5 of the present invention.

[0081] Figure 5 These are the results of three evaluations of the linear range of the antigen detection method in Embodiment 7 of the present invention.

[0082] Figure 6 This is the specificity evaluation result of the antigen detection method in Example 7 of the present invention.

[0083] Figure 7 These are the survival curves for each group in Embodiment 8 of the present invention. Detailed Implementation

[0084] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0085] Example 1: Preparation of CV-A6 antigen

[0086] Virus culture: Take one CV-A6 strain and inoculate it into four 10-layer cell factories with confluent Vero cells at an MOI of 0.001, using 2 L of each cell. Incubate at 35°C for 3–5 days. Harvest the virus solution when the cytopathic effect reaches 100%. Aliquot the virus harvested solution into centrifuge cups, centrifuge at 6000 rpm for 40 min, and collect the supernatant to obtain the CV-A6 supernatant.

[0087] Ultrafiltration concentration: The supernatant from centrifugation was concentrated by ultrafiltration using a 300 kDa membrane. The membrane was washed twice with 10 mmol / L PBST solution, and the washing solution was combined with the unfiltered solution to form the ultrafiltration product.

[0088] Sucrose density gradient centrifugation: The ultrafiltration product was diluted 3.5 times with 10 mmol / L PBS solution (0.15 mol / L NaCl) and subjected to gradient centrifugation. The sucrose concentration was 40%–55%. Centrifugation was performed at 30,000 rpm for 15–18 h, collecting 35 ml samples per tube, for a total of 24 tubes. SDS-PAGE analysis was performed on the samples. Hollow and solid particles were combined based on the results, and the hollow and solid particles were identified using electron microscopy.

[0089] Comparative electron micrographs of solid and hollow virus particles identified by electron microscopy after sucrose density gradient centrifugation are shown below. Figure 1 .

[0090] Example 2: Preparation of Monoclonal Antibodies

[0091] Hybridoma cell line preparation: The hollow and solid particles obtained in Example 1 were collected, desaccharified, and combined in a 1:1 volume ratio to obtain the purified CV-A6 virus solution. NIH mice were immunized with the above purified CV-A6 virus solution; five mice were immunized subcutaneously at multiple sites on the back at weeks 0, 2, 4, 6, and 8. Immunization dosage: 30 μg / mouse for the first dose with Freund's complete adjuvant; 15 μg / mouse for the second and third doses with Freund's incomplete adjuvant; 15 μg / mouse for the fourth dose without adjuvant, administered via tail vein injection. Blood testing: Blood samples were collected one week after the second and third immunizations to detect the indirect enzyme-linked immunosorbent assay (ELISA) titer. Antibody titers reaching 10 were considered positive. 4 The mice were given a fourth intraperitoneal injection for booster immunization.

[0092] Cell fusion: Cell fusion was performed using an electrofusion method. The average fusion efficiency was approximately 2500 spleen cells to produce one hybridoma cell.

[0093] Screening and Validation: The supernatant of fusion cells was initially screened using an indirect ELISA method, selecting those positive for the immunogen. All positive clones obtained in the initial screening stage were confirmed using an indirect ELISA method. The neutralizing activity of the confirmed positive clones was verified using a neutralizing antibody assay. Positive clones with neutralizing activity and high binding titers were then selected.

[0094] Subcloning, expansion culture, and cryopreservation: Positive maternal clones were transferred to 24-well plates for expansion culture, and the cell supernatant was collected for further verification. Finally, the positive maternal clones were subcloned twice using a limiting dilution method, and screened using indirect ELISA and neutralizing antibody assays. The positive clones confirmed after the second subcloning were expanded cultured, and ascites fluid was prepared by immunizing mice.

[0095] Example 3: Purification and Identification of Monoclonal Antibodies

[0096] Antibody purification: Ascites fluid from mice immunized in Example 2 was centrifuged at 2–8°C and 4000–8000 rpm for 5–15 minutes. The supernatant was filtered through a 0.45 μm filter and the antibody was purified using Protein A / G affinity chromatography. The purified antibody was then dialyzed and stored in phosphate-buffered saline (PBS). The protein concentration was determined to be 4.15 mg / ml using the Lowry method. The purity and titer of the purified monoclonal antibody were then measured.

[0097] SDS-PAGE purity determination: Analysis showed that the heavy chain and light chain of monoclonal antibody 3A9 (hereinafter referred to as 3A9) were approximately 50KD and 25KD, respectively. Figure 2 As shown.

[0098] For antibody titer determination: EV-A71, CV-A16, CV-A6, and CV-A10 viral purification solutions and Vero cell host protein were diluted to 1.0 μg / ml with carbonate buffer and coated onto 96-well ELISA plates overnight at 4°C. The plates were washed 3–5 times with 0.01 M phosphate-buffered saline (PBST) to a final concentration of 0.05% Tween 20. Each well was then blocked with 150 μl of PBST containing 3–6% BSA at 37°C for 1–2 hours. The blocking solution was discarded and the plates were patted dry. The test antibody and negative control (sample dilution) were serially diluted 10-fold to 10-fold. 8 Add 10 times, then add 10 times in sequence. -2 Up to 10 -8 Diluted samples were added to each well of the above-mentioned 96-well plate at a concentration of 100 μl, incubated at 37°C for 1 hour, followed by 300 μl of PBST per well. The plate was washed 2–5 times, then 100 μl of goat anti-mouse IgG-HRP diluted 1:5000 was added to each well. The plate was incubated at 37°C for 45 minutes, washed 2–5 times with PBST, blotted dry, and then substrate chromogenic buffer was added. The plate was incubated at room temperature in the dark for 10–15 minutes. The reaction was terminated with 1 M H₂SO₄, and the reading was taken at 450 nm using a microplate reader. The cutoff value for positive and negative results was ≥ 2.1 times the mean value of the negative control. The results showed that monoclonal antibody 3A9 specifically binds to the CV-A6 antigen, with a binding antibody titer of 1.6 × 10⁻⁶. 5It does not bind to other types and Vero cell host proteins, indicating that it has good specificity.

[0099] Neutralizing antibody titer assay: The test sample (1:8 dilution) was added to a 96-well cell culture plate and serially diluted 2-fold, 0.05 ml / well, and then titrated with 100 CCIDI. 50 (Half-cell infectious dose) of CV-A6 virus suspension was neutralized at 37°C for 2 hours; 1–2 × 10⁻⁶ cells / mL were added. 5 RD cell suspension was prepared at 0.1 ml / well and incubated for 7 days at 35 ± 0.5℃ in a 5% CO2 incubator. The highest dilution that inhibited cytopathic effects by 50% was defined as the neutralizing antibody titer, expressed as the reciprocal of the dilution factor. A viral backtiing assay was performed for each experiment, with backtiing results ranging from 32 to 320 CCID. 50 The test was considered valid at a time. A neutralizing titer ≥8 was considered positive for neutralizing antibodies, and <8 was considered negative. The geometric mean titer (GMT) of negative antibodies was calculated as 4. The neutralizing antibody titer of monoclonal antibody 3A9 was 1:4096, indicating that this monoclonal antibody has high in vitro neutralizing activity.

[0100] Subtype identification: The purified antibody was diluted to 200 ng / ml with PBST, and 50 μl / well was added to a pre-coated ELISA plate (manufacturer: Proteintech). Then, 50 μl / well of 1x goat anti-mouse IgM + IgG-HRP was added to each well. The plate was gently mixed and incubated at room temperature for 1 hour. The liquid in the wells was discarded, and the plate was washed three times with PBST and patted dry. 100 μl / well of chromogenic reagent was added. The plate was incubated at room temperature in the dark for 10–20 min. 100 μl of stop solution was added to each well to terminate the reaction. The OD at 450 nm was read using an ELISA reader; the well with the darkest OD value corresponds to the subtype. The subtype detection results for monoclonal antibody 3A9 are shown in Table 1. The heavy chain was IgG2b, and the light chain was Kappa.

[0101] Table 1 Subtype Identification Results

[0102] Subtype classification IgGl IgG2a IgG2b IgG2c IgG3 IgM Kappa Lambda Absorbance 450 nm 0.72 0.696 3.208 0.062 0.117 0.088 1.755 0.214

[0103] In vitro neutralizing activity: The in vitro neutralizing activity of monoclonal antibody 3A9 was detected using the microcytopathic effect assay, as follows: 1 mg / ml of monoclonal antibody 3A9 was serially diluted 2-fold from 1:8 to 1:16384 in a 96-well plate, and then mixed with 100 CCID... 50The CV-A6 strain Gdula (genotype A, genbank No. AY421764) and 11 D genotype strains (isolated from clinical patients in China between 2014 and 2019, of which R01170631 was deposited on July 13, 2021, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), were classified and named Coxsackievirus A6, with accession number CGMCC No. 19532. This strain has been disclosed in patent application CN113564131A; cross-neutralization testing was entrusted to the China National Institutes for Food and Drug Control, and the remaining strains used were all from the China National Institutes for Food and Drug Control and were used for testing) were neutralized at 37℃ for 2 hours; the concentration added was (1-2)×10 5 RD cell suspension was cultured at 100 μL / well at 35°C in a CO2 incubator for 7 days. Cytopathic effect (CPE) was observed, and the titer of the neutralizing antibody was determined by the reciprocal of the highest dilution that inhibited 50% CPE. The experiment included both cell controls and viral backtiing. Only the cell control showed no CPE, and the viral backtiing ranged from 30 to 300 CCIDs. 50 The experiment can only be considered valid if the number of wells is equal to the number of wells. The neutralizing titers of 12 viral strains, including the prototype strain, ranged from 48 to 3072, with a MAX / MIN ratio of 64, demonstrating relatively uniform broad-spectrum neutralizing ability. The results are shown in Table 2, indicating that monoclonal antibody 3A9 has broad-spectrum cross-neutralizing ability against CV-A6, and 3A9 was selected as the detection monoclonal antibody.

[0104] Table 2 Neutralization Capacity Study

[0105]

[0106] Competitive inhibition study with human serum (healthy human serum, human naturally mixed infection serum, and human naturally infected serum 1): CV-A6 antigen was diluted to 0.01 μg / ml and coated onto 96-well ELISA plates. Human serum was serially diluted 1:4 with sample diluent, 100 μL / well, and incubated at 37°C for 1 h. HRP-labeled monoclonal antibody 3A9 was diluted 1:200 with antibody diluent, 100 μL / well, and incubated at 37°C for 1 h. The mean absorbance of the control wells was used as B0, and the mean absorbance of the test sample group was used as B1. Inhibition rate % = (B0-B1) / B0*100. The inhibition rate was used as the ordinate, and the dilution of the test serum (logarithm to base log2) was used as the abscissa. The four-parameter method was used to fit and determine the reaction curve. Figure 3 R 2 The values ​​are 0.9842, 0.9801, and 0.9801, respectively.

[0107] The above results indicate that serum from healthy individuals does not exhibit significant competitive inhibition against monoclonal antibody 3A9. However, serum from naturally occurring mixed infections and naturally infected individuals do exhibit significant competitive inhibition against monoclonal antibody 3A9. This demonstrates that the epitope corresponding to monoclonal antibody 3A9 plays an important role in the immune response induced by human infection.

[0108] Gene sequence isolation and identification of monoclonal antibody 3A9: Total RNA was extracted from well-grown hybridoma cell lines using Trizol reagent, and cDNA was synthesized by reverse transcription. The light and heavy chain variable regions were then amplified by PCR, and their amino acid sequences were determined. Sequencing results showed that the amino acid sequences of the heavy chain complementarity-determining regions (CDR1, CDR2, and CDR3) of monoclonal antibody 3A9 are shown in SEQ ID NO. 1-3, and the amino acid sequences of the light chain complementarity-determining regions (CDR1, CDR2, and CDR3) are shown in SEQ ID NO. 4-6; the amino acid sequences of the heavy chain variable region are shown in SEQ ID NO. 7, the light chain variable region in SEQ ID NO. 8, the heavy chain in SEQ ID NO. 15, the coding gene sequence in SEQ ID NO. 13, the light chain in SEQ ID NO. 16, and the coding gene sequence in SEQ ID NO. 14.

[0109] Example 4: Monoclonal antibody 3A9 can specifically bind to CV-A6 viral particle antigen.

[0110] Sandwich ELISA assay: Dilute anti-CV-A6 rabbit polyclonal antibody (1:1000, 1:5000, 1:10000, 1:20000) with coating buffer carbonate buffer (CBS pH 9.6), add 100 μl to each well of a Corning ELISA plate (removable), and coat overnight at 2–8°C; wash three times with PBST and blot dry; block with 150 μl of 3% BSA PBST per well and incubate at 37°C for 2 h; wash three times with PBST and blot dry; then block with sample dilution buffer (0.5% BSA PBS solution). Dilute the antigen standard to 100 ng / ml as a positive control, and the sample diluent as a negative control, 100 μl per well, and incubate at 37°C for 2 h; wash 3 times with PBST and blot dry; dilute the monoclonal antibody to an appropriate concentration (1:1000, 1:5000, 1:10000, 1:20000) with antibody diluent, 100 μl per well, and incubate at 37°C for 1 h; wash 3 times with PBST and blot dry; dilute goat anti-mouse-HRP to 1:8000 with antibody diluent, 100 μl per well, and incubate at 37°C for 30–60 min; wash 4 times with PBST and blot dry; add 100 μl of TMB substrate chromogenic solution to each well and incubate at room temperature in the dark for 15 min; add 50 μl of 1 mol / L H2SO4 to each well. Read values ​​at 450 nm / 630 nm. The results showed that when the polyclonal antibody was diluted 1:5000, the monoclonal antibody 3A9, as the detection antibody, had the highest P / N value at a dilution of 1:1000, indicating that this monoclonal antibody can be used as a detection antibody.

[0111] Example 5 Horseradish peroxidase-labeled monoclonal antibody 3A9

[0112] Dissolve 5 mg of HRP in 0.5 ml of 0.2 mol / L, pH 5.6 acetate buffer; add 0.25 ml of freshly prepared 0.1 mol / L NaIO4, at which point the solution changes from brown to dark green. Incubate at 4°C for 30 min, at which point the solution changes from brown to dark green again. Add 0.5 ml of 2.5% ethylene glycol and gently stir at room temperature for 1 h to terminate the reaction. Add 10 mg of the antibody to be labeled, and adjust the pH to 9.0 with 1.0 mol / L, pH 9.5 CBS; mix well and incubate at 4°C overnight. Add 0.1 ml of sodium borohydride solution, mix well, and incubate at 4°C for 3 h. Dialyze overnight at 4°C with 0.15 mol / L, pH 7.4 PBS solution, changing the medium 3 times. Centrifuge at 3000 rpm for 30 min to remove the precipitate. Collect the supernatant, which is the enzyme-labeled antibody. Add an equal volume of 60% glycerol, aliquot, and store at -20°C or below.

[0113] Assay for enzyme-labeled antibody titer: First, dilute the CV-A6 antigen to approximately 10 μg / ml with 0.05 mol / L pH 9.6 coating buffer. Add 100 μl to each well of a polystyrene plate and incubate overnight at 4°C. Wash three times with PBST. Block the plate with 150 μl of 3% BSA-PBST per well and incubate at 37°C for 2 hours. Wash three times with PBST. Dilute the enzyme-labeled antibody 1:100 starting at 0.5% BSA-PBST, then serially dilute 2-fold to 12800. Add 100 μl to each well (two wells per dilution), incubate at 37°C for 1 hour, and wash. Then add 100 μl of chromogenic reagent per well and incubate at room temperature in the dark for 10 minutes. Terminate the reaction with 50 μl of 1 mol / L H₂SO₄ per well.

[0114] The OD values ​​at 450nm and 630nm for each well were read using an ELISA reader, and a titration curve was plotted with OD (450nm-630nm) as the ordinate and the enzyme-labeled antibody dilution as the abscissa. Figure 4 The antibody dilution with an OD value of approximately 1.0 and the steepest curve, obtained from the curve, represents the antibody titer of the labeled antibody. Table 3 shows the antibody titer to be 600.

[0115] Table 3 Absorbance values ​​of enzyme-labeled antibody reactions

[0116] Dilution Absorbance 1 Absorbance 2 Mean 100 2.422 2.084 2.253 200 1.758 1.538 1.648 400 1.259 1.232 1.2455 800 0.841 0.691 0.766 1600 0.563 0.461 0.512 3200 0.309 0.269 0.289 6400 0.169 0.137 0.153 12800 0.097 0.081 0.089

[0117] Example 6: Establishment of Antigen Detection Method

[0118] A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established using rabbit polyclonal antibody as the coating antibody and monoclonal antibody 3A9 as the detection antibody to detect CV-A6 antigen. The checkerboard method was used for the experiment, and the specific procedure was as follows: Rabbit polyclonal antibody was diluted with carbonate buffer (pH 9.6) to 1.6 μg / ml, 0.32 μg / ml, and 0.16 μg / ml, and coated onto 96-well ELISA plates at 100 μl / well, incubating at 37°C for 2 hours. The plates were washed three times with PBST, and non-specific binding sites were blocked with blocking buffer (0.01M PBS containing 3% BSA, 0.5‰ Tween 20) at 150 μl / well, incubating at 37°C for 2 hours. CV-A6 vaccine antigen was diluted with PBS (containing 0.5% BSA) to 100 ng / ml, and added to the ELISA plate at 100 μl / well. Sample dilution buffer was used as a negative control, and two blank controls were also set up. The plates were incubated at 37°C for 2 hours. Wash the plate four times with PBST, add HRP-labeled 3A9 monoclonal antibody (1:1000, 1:5000), and incubate at 37°C for 30–45 min. Wash the plate five times with PBST, add 100 μl of TMB chromogenic solution to each well, and incubate at room temperature in the dark for 15 min. Then add 50 μl of stop solution (1M sulfuric acid solution) to each well to stop the reaction. Read the values ​​at 450 / 630 nm using a microplate reader. The detection results are shown in Table 4. The optimal combination for achieving the highest P / N value was 0.32 μg / ml coating antibody and 1:1000 dilution of enzyme-labeled antibody. This demonstrates that the double-antibody sandwich method using monoclonal antibody 3A9 as the detection antibody can be used for the detection of CV-A6 antigen.

[0119] Table 4 Chessboard Method

[0120]

[0121] Note: P represents the mean of the test samples, and N represents the mean of the negative control.

[0122] Example 7 Evaluation of Antigen Detection Method

[0123] The antigen detection method established in Example 6 was evaluated as follows:

[0124] Linear Range: Following the established antigen detection method, the antigen reference standard was serially diluted 2-fold at 8 dilutions starting from 150 ng / ml (300 ng / ml, 150 ng / ml, 75 ng / ml, 37.5 ng / ml, 18.75 ng / ml, 9.375 ng / ml, 4.6875 ng / ml, 2.34375 ng / ml), with each dilution performed in replicates. Six independent measurements were performed. A standard curve was plotted with the logarithm of the standard concentration on the x-axis and the logarithm of the absorbance on the y-axis to examine the R-value of the six measurements. 2 Consistency. 2.1 times the mean negative result was used as the criterion for determining positive or negative.

[0125] R of the results of 6 trials 2 >0.98, see Figure 5 This indicates that the method has good linearity and parallelism, and the reference sample shows good linearity in the range of 150–9.375 ng / ml.

[0126] Specificity evaluation: Referring to the method in Example 6, EV-A71, CV-A16, and CV-A10 antigens (all belonging to the same family as CV-A6 in the Picornaviridae family) and Vero cell host protein used in production were selected and diluted to 1000 ng / mL as test samples (calculated based on protein content). Other possible components present in the poliovirus (PV) antigen and CV-A6 antigen samples, such as M199, DMEM, PEG6000, 58% sucrose, and 10 mmol / L PBST (1M NaCl, 0.1% Tween 80), were also selected as test samples (abbreviated as S). CV-A6 antigen was used as a positive control, and the sample dilution was used as a negative control. The cut-off value (abbreviated as CO) was 2.1 times that of the negative control. This verified the specificity of the antigen detection system for enterovirus detection.

[0127] See results Figure 6 This antigen evaluation system has excellent specificity for the detection of CV-A6 virus, but does not react to other types of enteroviruses.

[0128] Evaluation of the binding capacity of the antigen detection kit to hollow and solid particles: According to the established antigen detection method, hollow and solid virus particles are serially diluted at certain concentrations. Under the condition of achieving the same OD value, the reciprocal of the ratio of the concentrations of hollow and solid virus proteins added is the ratio of the antigen detection kit's reactivity to hollow and solid virus particles.

[0129] The results are shown in Table 5. The reactivity ratio of hollow to solid particles is 1:2, indicating that this antigen detection kit can effectively detect both hollow and solid particles.

[0130] Table 5 Comparison of reaction capabilities between hollow and solid particles

[0131]

[0132] Example 8: Experiment on the treatment of CV-A6 infection with monoclonal antibody 3A9 in mice.

[0133] Use lethal dose R01170631 / CV-A6 (38 CCID) 50One-day-old BALB / c suckling mice were infected, and eight groups were set up, including a virus control group. After 0.5 hours, the experimental groups were injected with 16.0 μg / mouse, 4.0 μg / mouse, 1.0 μg / mouse, 0.25 μg / mouse, 0.0625 μg / mouse, and 0.015625 μg / mouse, respectively, of monoclonal antibody 3A9. The blank control group was injected with the same dose of MEM. The survival status of the suckling mice was observed and recorded for 21 consecutive days, and survival curves were plotted.

[0134] The results are as follows Figure 7 As shown, all mice in the MEM control group survived, while 100% of the mice in the virus control group died 8 days after challenge. The results showed that all suckling mice in the 0.25 μg group survived without exhibiting clinical symptoms such as emaciation or limb paralysis; all suckling mice in the 0.0625 μg and 0.015625 μg groups died.

[0135] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Beijing Minhai Biotechnology Co., Ltd. <120> A monoclonal antibody against Coxsackievirus A6, its preparation method and application <130> KHP221112871.5 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Ser Tyr Asp Met Ser 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <400> 2 Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Thr Thr Val Val Val Pro Phe Asp Tyr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <400> 4 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <400> 5 Ser Thr Ser Asn Leu Ala Ser 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <400> 6 Gln Gln Arg Ser Thr Tyr Pro Pro Tyr Ala 1 5 10 <210> 7 <211> 118 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Arg Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Thr Thr Val Val Val Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Leu Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <400> 8 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Pro Tyr 85 90 95 Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 19 <212> PRT <213> Artificial Sequence <400> 9 Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Val Leu Ile Leu Lys Gly 1 5 10 15 Val Gln Cys <210> 10 <211> 22 <212> PRT <213> Artificial Sequence <400> 10 Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly 20 <210> 11 <211> 336 <212> PRT <213> Artificial Sequence <400> 11 Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly 1 5 10 15 Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 20 25 30 Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 35 40 45 Ser Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met 50 55 60 Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val 65 70 75 80 Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys 85 90 95 Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys 100 105 110 Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser 115 120 125 Val Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu 130 135 140 Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro 145 150 155 160 Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala 165 170 175 Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Ile Arg Val Val 180 185 190 Ser Thr Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe 195 200 205 Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro Ile Glu Arg Thr 210 215 220 Ile Ser Lys Ile Lys Gly Leu Val Arg Ala Pro Gln Val Tyr Ile Leu 225 230 235 240 Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys Asp Val Ser Leu Thr Cys 245 250 255 Leu Val Val Gly Phe Asn Pro Gly Asp Ile Ser Val Glu Trp Thr Ser 260 265 270 Asn Gly His Thr Glu Glu Asn Tyr Lys Asp Thr Ala Pro Val Leu Asp 275 280 285 Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys Leu Asn Met Lys Thr Ser 290 295 300 Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys Asn Val Arg His Glu Gly 305 310 315 320 Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile Ser Arg Ser Pro Gly Lys 325 330 335 <210> 12 <211> 107 <212> PRT <213> Artificial Sequence <400> 12 Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 13 <211> 1422 <212> DNA <213> Artificial Sequence <400> 13 atgaactttg ggctgagctt gattttcctt gtcctaattt taaaaggtgt ccagtgtgaa gtgaagctgg tggagtctgg gggaggctta gtgaagcctg gagggtccct gaaactctcc tgtgcagcct ctggattcgt tttcagtagc tatgacatgt cttgggttcg ccagactccg gagacgaggc tggagtgggt cgcaaccatt agtagtggtg gtacttacac ctactatcca gacagtgtga agggccgatt caccatctcc agagacactg ccaggagac cctgtacctg caaatgagca gtctgaggtc tgaggacacg gccttgtatt actgtgcaac tactacggta gtagtcccct ttgactactg gggccaaggc acctctctca cagtctcctc agccaaaaca 420 acacccccat cagtctatcc actggcccct gggtgtggag atacaactgg ttcctccgtg 480 actctgggat gcctggtcaa gggctacttc cctgagtcag tgactgtgac ttggaactct 540 ggatccctgt ccagcagtgt gcacaccttc ccagctctcc tgcagtctgg actctacact 600 atgagcagct cagtgactgt cccctccagc acctggccaa gtcagaccgt cacctgcagc 660 gttgctcacc cagccagcag caccacggtg gacaaaaaac ttgagcccag cgggcccatt 720 tcaacaatca acccctgtcc tccatgcaag gagtgtcaca aatgcccagc tcctaacctc 780 gagggtggac catccgtctt catcttccct ccaaatatca aggatgtact catgatctcc 840 ctgacaccca aggtcacgtg tgtggtggtg gatgtgagcg aggatgaccc agacgtccag 900 atcagctggt ttgtgaacaa cgtggaagta cacacagctc agacacaaac ccatagagag 960 gattacaaca gtactatccg ggtggtcagc accctcccca tccagcacca ggactggatg 1020 agtggcaagg agttcaaatg caaggtcaac aacaaagacc tcccatcacc catcgagaga 1080 accatctcaa aaattaaagg gctagtcaga gctccacaag tatacatctt gccgccacca 1140 gcagagcagt tgtccaggaa agatgtcagt ctcacttgcc tggtcgtggg cttcaaccct 1200 ggagacatca gtgtggagtg gaccagcaat gggcatacag aggagaacta caaggacacc 1260 gcaccagtcc tggactctga cggttcttac ttcatatata gcaagctcaa tatgaaaaca 1320 agcaagtggg agaaaacaga ttccttctca tgcaacgtga gacacgaggg tctgaaaaat 1380 tactacctga agaagaccat ctcccggtct ccgggtaaat ga 1422 <210> 14 <211> 711 <212> DNA <213> Artificial Sequence <400> 14 atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60 agaggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc aggggagaag 120 gtcaccataa cctgcagtgc cagctcaagt gtaagttaca tgcactggtt ccagcagaag 180 ccaggcactt ctcccaaact ctggatttat agcacatcca acctggcttc tggggtccct 240 gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag ccgaatggag 300 gctgaagatg ctgccactta ttactgccag caaaggagta cttacccacc ctacgcgttc 360 ggagggggga ccaagctgga aataaaacgg gctgatgctg caccaactgt atccatcttc 420 ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 480 ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc 540 gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc 600 ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac 660 aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgtta g 711 <210> 15 <211> 454 <212> PRT <213> Artificial Sequence <400> 15 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Ser Ser Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Thr Pro Glu Thr Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Arg Lys Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr Thr Thr Val Val Val Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro 115 120 125 Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly 130 135 140 Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn 145 150 155 160 Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Ala Leu Leu Gln 165 170 175 Ser Gly Leu Tyr Thr Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr 180 185 190 Trp Pro Ser Gln Thr Val Thr Cys Ser Val Ala His Pro Ala Ser Ser 195 200 205 Thr Thr Val Asp Lys Lys Leu Glu Pro Ser Gly Pro Ile Ser Thr Ile 210 215 220 Asn Pro Cys Pro Pro Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn 225 230 235 240 Leu Glu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Asn Ile Lys Asp 245 250 255 Val Leu Met Ile Ser Leu Thr Pro Lys Val Thr Cys Val Val Val Asp 260 265 270 Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn 275 280 285 Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn 290 295 300 Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Gln His Gln Asp Trp 305 310 315 320 Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro 325 330 335 Ser Pro Ile Glu Arg Thr Ile Ser Lys Ile Lys Gly Leu Val Arg Ala 340 345 350 Pro Gln Val Tyr Ile Leu Pro Pro Pro Ala Glu Gln Leu Ser Arg Lys 355 360 365 Asp Val Ser Leu Thr Cys Leu Val Val Gly Phe Asn Pro Gly Asp Ile 370 375 380 Ser Val Glu Trp Thr Ser Asn Gly His Thr Glu Glu Asn Tyr Lys Asp 385 390 395 400 Thr Ala Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Ile Tyr Ser Lys 405 410 415 Leu Asn Met Lys Thr Ser Lys Trp Glu Lys Thr Asp Ser Phe Ser Cys 420 425 430 Asn Val Arg His Glu Gly Leu Lys Asn Tyr Tyr Leu Lys Lys Thr Ile 435 440 445 Ser Arg Ser Pro Gly Lys 450 <210> 16 <211> 214 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 16 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Pro Tyr 85 90 95 Ala Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210

Claims

1. An antibody against Coxsackievirus A6 or an antigen-binding fragment thereof, characterized in that, The amino acid sequences of the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 1-3, and the amino acid sequences of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO. 4-6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.7; And / or, the light chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence that is at least 80% homologous to the amino acid sequence shown in SEQ ID NO.

8.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.7; And / or, the light chain variable region of the antibody or its antigen-binding fragment has an amino acid sequence as shown in SEQ ID NO.

8.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or a single-chain antibody.

5. A labeled antibody, characterized in that, It is obtained by labeling the antibody or its antigen-binding fragment as described in any one of claims 1 to 4 with an enzyme, biotin, fluorescent dye, chemiluminescent dye and / or radioisotope.

6. A nucleic acid molecule, characterized in that, It encodes the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

7. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule of claim 6, wherein the biomaterial is an expression cassette, a vector, or a host cell.

8. A method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-4, characterized in that, The method includes: culturing host cells capable of expressing the antibody or its antigen-binding fragment, and then isolating the antibody or its antigen-binding fragment.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of a product for detecting the presence or level of Coxsackievirus A6 in a sample.

10. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of a product for diagnosing Coxsackievirus A6 infection.

11. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of products for diagnosing diseases caused by Coxsackievirus A6 infection.

12. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of a product for neutralizing the virulence of Coxsackievirus A6 in a sample.

13. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of a medicament for the prevention or treatment of Coxsackievirus A6 infection.

14. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-4, the labeled antibody according to claim 5, the nucleic acid molecule according to claim 6, or the biological material according to claim 7 in the preparation of a medicament for the prevention or treatment of diseases caused by Coxsackievirus A6 infection.

15. A reagent kit, characterized in that, The kit contains the antibody or antigen-binding fragment thereof as described in any one of claims 1-4 or the labeled antibody as described in claim 5.

16. A drug, characterized in that, The drug comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

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