A monoclonal antibody for recognizing coxsackievirus A2 and A4 and its application

Abstract

The present invention relates to the fields of molecular biology and immunological analysis technology, and specifically to a monoclonal antibody capable of recognizing coxsackievirus A2 and A4 and its use. The monoclonal antibody comprises a heavy chain complementary determining region (CDR1) as set forth in SEQ ID NO.9, a heavy chain complementary determining region (CDR2) as set forth in SEQ ID NO.10, a heavy chain complementary determining region (CDR3) as set forth in SEQ ID NO.11, and a light chain complementary determining region (CDR1) as set forth in SEQ ID NO.12, a light chain complementary determining region (CDR2) as set forth in SEQ ID NO.13, and a light chain complementary determining region (CDR3) as set forth in SEQ ID NO.14. The monoclonal antibody specifically binds to the first 15 amino acids at the N-terminus of the structural protein VP1 of coxsackievirus A2 and A4, and can be used for clinical patient sample testing, laboratory antibody identification of virus isolates, and quantitative detection of coxsackievirus A2 and A4 antigens in production intermediates and products.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology and immunological analysis, and in particular to a monoclonal antibody 7H11E for identifying Coxsackievirus A2 and A4 (CV-A2 and CV-A4) and an application thereof. Background Art

[0002] Hand, foot, and mouth disease (HFMD) is a Class C infectious disease caused by various human enteroviruses. Symptoms include fever and rash on the hands, feet, and mouth, and are most common in children under five years old. Severe cases can develop complications such as pulmonary edema and aseptic meningitis, and even death. In recent years, HFMD caused by enteroviruses has been characterized by clustered outbreaks and multi-virus cross-infection. Enterovirus A viruses, such as Coxsackieviruses (CV) A2, A4, A5, A6, A10, and A16, and Enterovirus (EV) A71, are the primary pathogens causing HFMD in infants and young children.

[0003] Currently, only a monovalent inactivated EV-A71 whole-virus vaccine is available for the prevention of hand, foot and mouth disease (HFMD). Coxsackievirus A serotypes 4 and 2 (CV-A4 and CV-A2), both belonging to the Picornaviridae family and the Enterovirus genus, are also important pathogens that cause HFMD, herpangina, and acute flaccid paralysis, but relatively little research has been conducted on them. Therefore, developing specific or broad-spectrum monoclonal antibodies against other HFMD-associated enteroviruses, such as CV-A4 and CV-A2, is of great value for rapid virus detection in clinical samples, laboratory virus identification, functional studies, quality control of vaccine development, and antigen quantification in finished vaccine products. Summary of the Invention

[0004] Based on this, one of the objects of the present invention is to provide a monoclonal antibody that recognizes Coxsackievirus A2 and A4, wherein the monoclonal antibody has a heavy chain complementary determining region CDR1 shown in SEQ ID NO.9, a heavy chain complementary determining region CDR2 shown in SEQ ID NO.10, and a heavy chain complementary determining region CDR3 shown in SEQ ID NO.11, and a light chain complementary determining region CDR1 shown in SEQ ID NO.12, a light chain complementary determining region CDR2 shown in SEQ ID NO.13, and a light chain complementary determining region CDR3 shown in SEQ ID NO.14.

[0005] Preferably, the monoclonal antibody has a heavy chain with the amino acid sequence shown in SEQ ID NO.7; and / or, the monoclonal antibody has a light chain with the amino acid sequence shown in SEQ ID NO.8.

[0006] Preferably, the heavy chain amino acid sequence of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.7; and / or, the light chain amino acid sequence of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID No.8.

[0007] Preferably, the monoclonal antibody is of IgG2b subtype.

[0008] This monoclonal antibody can specifically recognize Coxsackievirus A2 and A4, but does not bind to other enteroviruses. It can be used in basic experiments such as indirect immunofluorescence, immunoblotting and enzyme-linked immunosorbent assay. It has the same antigen high titer binding to diseases such as hand, foot and mouth disease, herpangina and acute flaccid paralysis caused by Coxsackievirus A2 and A4.

[0009] The second object of the present invention is to protect the polynucleotide molecules encoding the above-mentioned monoclonal antibodies that recognize Coxsackievirus A2 and A4.

[0010] Preferably, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.5; and / or, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.6.

[0011] A third object of the present invention is to protect a kit for detecting Coxsackievirus A2 and A4, which comprises the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the above-mentioned polynucleotide molecule.

[0012] A fourth object of the present invention is to protect the use of the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the above-mentioned polynucleotide molecule in the preparation of a reagent or kit for detecting one or both of Coxsackievirus A2 and A4.

[0013] A fifth object of the present invention is to protect the use of the above-mentioned monoclonal antibody or the monoclonal antibody encoded by the above-mentioned polynucleotide molecule in the preparation of a single or dual drug for inhibiting, preventing, and treating Coxsackievirus A2 and A4.

[0014] A sixth object of the present invention is to protect the use of the aforementioned monoclonal antibodies or the monoclonal antibodies encoded by the aforementioned polynucleotide molecules in the study of VP1 proteins of Coxsackievirus A2 and A4 and changes in viral structure and conformation. For example, the basic research on the function of intermediates in the morphological and conformational transitions of CV-A2 and CV-A4 virus particles and the role of N-terminal amino acids in penetrating the cell membrane and releasing viral RNA into the cell.

[0015] The monoclonal antibody provided by the present invention is obtained by immunizing Balb / c mice with solid particles (FP) of coxsackievirus A group 4 (CV-A4), preparing and screening hybridoma cells, and then purifying them. The monoclonal antibody can specifically recognize coxsackievirus A group 2 and 4 (CV-A2 and CV-A4), is an IgG2b subtype non-neutralizing antibody, and can specifically bind to the 1st to 15th amino acids at the N-terminus of the structural protein VP1 of CV-A2 and CV-A4. Among them, the VP1 N-terminal amino acids 2-4 (DAI) and 6 (D) are the footprints of the monoclonal antibody that specifically binds to the CV-A4 VP1 N-terminal amino acids.

[0016] This monoclonal antibody can be combined with a conjugate (horseradish peroxidase or fluorescein isothiocyanate, etc.) for direct or indirect detection or rapid diagnosis. It has important research and application value in rapid and economical detection of clinical samples, laboratory identification, virus titration, antigen quantification of vaccine preparation intermediates and products, research and development of structural protein VP1 function and antiviral drug targets, and other applications based on antigen-antibody reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the reducing SDS-PAGE result of IgG light chain and heavy chain of monoclonal antibody 7H11E;

[0018] Figure 2 The results of indirect immunofluorescence assay of RD cells infected with CV-A2 and CV-A4 were detected by monoclonal antibody 7H11E;

[0019] Figure 3 Identification diagram of monoclonal antibody 7H11E recognizing CV-A2 and CV-A4 structural protein VP1;

[0020] Figure 4 The results of ELISA binding titer test of monoclonal antibody 7H11E and different viral antigens;

[0021] Figure 5 The linear epitope peptide library and peptide segment mapping results of monoclonal antibody 7H11E, where A is the result of the linear epitope peptide library that recognizes the full length of VP1 (positions 1 to 305), and B is the result of the linear epitope peptide segment that recognizes amino acids 1 to 50 of VP1. DETAILED DESCRIPTION

[0022] 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.

[0023] The following embodiments are only used to illustrate the present invention, but are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0025] Reagent Source:

[0026] Freund's complete adjuvant and incomplete adjuvant: purchased from Sigma;

[0027] Isotyping Kit for Mouse Monoclonal Antibody: purchased from Beijing Sino Biological Technology Co., Ltd.

[0028] Fast Pure Cell / Tissue Total RNA Isolation Kit: purchased from Novozymes Biotech;

[0029] RD cells and female Balb / c mice were provided by Wuhan Institute of Biological Products Co., Ltd.; goat anti-mouse IgG (H+L) Alexa Fluor 488 fluorescent secondary antibody was purchased from Thermo Fisher Scientific.

[0030] HRP-labeled goat anti-mouse IgG: purchased from Wuhan Boster Bioengineering Co., Ltd.;

[0031] pUC-Kan: purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0032] Example 1 Material Preparation

[0033] CV-A4 (strain CV-A4 / R3179 / XY / 2017, see article for details: Tian Yuxuan, Wang Mengjun and Wang Wenhui et al. Preparation of CV-A4 monoclonal antibody and establishment and validation of candidate vaccine type-specific quantitative analysis method. Modern Immunology, 2023, 43(01):8-15.), CV-A2 (strain CVA2-1388-M14 / XY / CHN / 2017, NCBI database accession number MW846233), CV-A5 (strain CV-A5-3487-M14-XY-CHN-2017, NCBI database accession number MW079817) and Echo 11 virus culture: CV-A4, A2, A5 and Echo 11 viruses were inoculated into serum-free Vero cells respectively. When the cytopathic effect reached 90%, the viruses were harvested and packaged.

[0034] Preparation of CV-A4, A2, A5, and Echo 11 virus full particles (FP) and empty particles (EP): CV-A4, CV-A2, CV-A5, and Echo 11 (MOI = 0.001) were inoculated into a ten-layer Vero cell factory at 100% confluence, cultured at 37°C, and the virus liquid was harvested when the cytopathic effect (CPE) reached 90%. The virus harvest was repeatedly frozen and thawed, and then centrifuged to remove cell debris. The cell supernatant was concentrated 10-fold by ultrafiltration through a 100 kD pore size filter and passed through a 25% (w / v) sucrose pad, centrifuged at 103,745 g for 4 h, and the viral proteins were solubilized with PBS buffer at pH 7.2. The virus was then centrifuged at 103,745 g for 4 h using 15%, 25%, 35%, 45%, and 55% (w / v) sucrose density gradients to obtain full particles (FP) and empty particles (EP). FP and EP were subjected to cesium chloride density gradient centrifugation at 260,000 g for 24 h at 4°C, and opalescent bands were extracted to obtain purified virus particles, including CV-A4, CV-A2, CV-A5, and Echo 11.

[0035] For the preparation of CV-A2 virus-like particles (VLPs), please refer to the literature Yu Yuting, Hu Gang, Luo Zhiyu, et al. Preparation, purification and identification of Coxsackievirus A2 virus-like particles. Chinese Journal of Biological Products, 2021, 34(07): 782-787.

[0036] Cultivation of CV-A6 (EP+FP), CV-A10 (EP+FP), CV-A16 (EP+FP), and EV-A71 (EP+FP) viruses and acquisition of virus particles: CV-A6, CV-A10, CV-A16, and EV-A71 (MOI=0.001) were inoculated into Vero cells, and the virus solution was harvested when the cytopathic effect (CPE) reached 90%. The virus harvest solution was concentrated by ultrafiltration through a 100 kD pore size, and the volume was concentrated. The virus concentrate was subjected to two column chromatography steps to obtain the virus stock solutions of CV-A6, CV-A10, CV-A16, and EV-A71, i.e., mixed virus particles EP+FP.

[0037] CV-A5 DP (Dense Particles): These are viral particles obtained after CV-A5 infection and cesium chloride density gradient centrifugation. These particles, unlike EP and FP, have partially cleaved VP1 protein, i.e., an incomplete VP1 protein with the first 1-40 amino acids of the N-terminus cleaved. Information about these particles has been published in the article Jin WP, Lu J, Zhang XY, et al. Efficacy of Coxsackievirus A5 Vaccine Candidates in an Actively Immunized Mouse Model. J Virol. 2021;95(6):e01743-20.

[0038] Anti-A4 FP mouse serum: immunize SPF-grade 6-8 week old Balb / c mice with CV-A4 FP antigen on days 0 and 14, and collect serum on day 28 to obtain anti-A4 FP mouse serum.

[0039] Normal mouse serum: Obtain SPF level 6-8 week old Balb / c mice and collect serum to obtain normal mouse serum.

[0040] Example 2 Preparation of monoclonal antibodies

[0041] The present invention provides a method for preparing the monoclonal antibody 7H11E, which is specifically as follows:

[0042] S1, 60 μg of CV-A4 FP particles were mixed with Freund's complete adjuvant (V / V = 1:1), and the booster immunization was Freund's incomplete adjuvant mixed with antigen. Female 6-8 week old Balb / c mice were immunized by multiple subcutaneous injections and intraperitoneal injections at a volume of 500 μL / time. Mice were immunized on days 0, 14, 28, and 42. The titer of the serum of mice on day 52 was detected by indirect ELISA. The ELISA serum binding titer exceeded 1×10 6The mice were subjected to shock immunization. The spleen cells of the mice were collected on the third day of immunization. PEG 1500 was used to induce fusion of the spleen cells with SP2 / 0 myeloma cells. The fusion ratio was controlled at (1:5) to (1:10). The hybridoma cells with successful fusion were screened using HAT culture medium.

[0043] S2. The antibody titer of the culture supernatant of the successfully fused hybridoma cells was detected by indirect ELISA method, and the antibodies with ELISA binding titer greater than 1×10 4 Positive hybridoma cells were cloned and purified three times, and then cultured and expanded to prepare ascites. The ascites was purified using a three-step saturated ammonium sulfate method to obtain monoclonal antibodies, and the antibody isotype was identified using the Isotyping Kit for Mouse Monoclonal Antibody.

[0044] Among them, the method of indirect ELISA screening hybridoma cells is:

[0045] CV-A4 FP was prepared into 1 μg / mL coating solution with carbonate buffer at pH 9.6 and added to 96-well microtiter plates, 100 μL / well, and incubated at 4°C overnight. After the incubation, the plates were washed 5 times with PBST at pH 7.4; the plates were washed with PBST containing 1% (W / V) bovine serum albumin at pH 7.4. The cells were blocked with PBST containing albumin (BSA) at 37°C for 1 hour, and the blocking solution was discarded. The supernatant of the hybridoma cells to be tested was diluted tenfold (V / V) and added to the ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 hour and washed 5 times with PBST. 0.1 μg / mL HRP-labeled goat anti-mouse IgG (Boster) antibody was added to the ELISA plate at 100 μL / well. The plates were incubated at 37°C for 1 hour and washed 5 times with PBST. TMB colorimetric solution was added to develop the cells in the dark at 37°C for 30 minutes. The reaction was terminated with 2 M sulfuric acid, and the absorbance at 450 nm was measured with a microplate reader. The titer was calculated as the maximum dilution factor that was greater than 2.1 times the OD value of the negative wells. Negative wells and hybridoma cell culture medium served as negative controls.

[0046] Example 3 Monoclonal Antibody Sequence Analysis

[0047] The hybridoma cells (secreting 7H11E monoclonal antibody) screened in Example 2 were inoculated into RPMI 1640 medium (Gibco) containing 10% fetal bovine serum and cultured at 37°C in 5% CO2. 6Hybridoma cells were harvested and total RNA was extracted using the Fast Pure Cell / Tissue Total RNA Isolation Kit. Single-stranded cDNA was obtained by reverse transcription of total RNA using the Oligo dT primer in the TaKaRa RimeScript II 1st Strand cDNA Synthesis Kit. The heavy and light chain variable region genes of the monoclonal antibody 7H11E were amplified using primers 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 correct variable region amino acid sequences were analyzed by alignment against the Kabat database. Among them, the sequence of the heavy chain universal forward primer VH-F is SEQ ID NO.1: acggccagtgaattcmarctgcagsagtcwgg, and the sequence of the reverse primer VH-R is SEQ ID NO.2: gattacgccaagctttgaggagacggtgaccg; the sequence of the light chain universal forward primer VL-F is SEQ ID NO.3: acggccagtgaattccgattgtkctsacycartctcca, and the sequence of the reverse primer VL-R is SEQ ID NO.4: gattacgccaagcttcgttggatctccagcttg.

[0048] The determined nucleotide sequence of the heavy chain variable region of monoclonal antibody 7H11E is as follows: TCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTACCCAGCTATGATGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAGCACACATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTACTGTGCGGGGCTACGGGCCTTTGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO. 5);

[0049] The nucleotide sequence of the light chain variable region is as follows: CCGATTGTGCTCACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTTCCTGCAGGGCCAGTCAGAGCATTGGCACAAACATACACTGGTATCAGCAAGGAGCAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGA GTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTACTGTCACAGAGTAATAGCTGGCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGATCCAACGAGCT(SEQ ID No.6);

[0050] The amino acid sequence encoded by the nucleotide sequence of the heavy chain variable region is as follows: SGPGLVAPSQSLSITCTVSGFSLPSYDVHWVRQPPGKGLEWLGVIWAGGSTHYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCAGLRAFAMDYWGQGTSVTVSS (SEQ ID No. 7);

[0051] The amino acid sequence encoded by the nucleotide sequence of the light chain variable region is as follows: PIVLTQSPAILSVSPGERVSFSCRASQSIGTNIHWYQQGANGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPWTFGGGTKLEIQRA (SEQ ID No. 8).

[0052] The sequences of the six CDRs (complementarity determining regions) analyzed are as follows:

[0053] The amino acid sequence of the heavy chain complementarity determining region VHCDR1: SYDVH (SEQ ID No. 9);

[0054] The amino acid sequence of the heavy chain complementarity determining region VHCDR2 is: VIWAGGSTHYNSALMS (SEQ ID No. 10);

[0055] The amino acid sequence of the heavy chain complementarity determining region VHCDR3 is LRAFAMDY (SEQ ID No. 11); the amino acid sequence of the light chain complementarity determining region VLCDR1 is RASQSIGTNIH (SEQ ID No. 12); the amino acid sequence of the light chain complementarity determining region VLCDR2 is YASESIS (SEQ ID No. 13);

[0056] The amino acid sequence of the complementarity determining region VLCDR3 of the light chain is: QQSNSWPWT (SEQ ID No. 14).

[0057] Example 4 Identification of subtypes of monoclonal antibody 7H11E

[0058] The monoclonal antibody 7H11E purified from Example 2 was subjected to 4-20% SDS-PAGE. Figure 1 As shown, from Figure 1 It can be seen that the heavy chain and light chain of the 7H11E monoclonal antibody correspond to protein bands of 50kDa and 25kDa respectively, and the subtype is IgG 2b, which is consistent with the expected results.

[0059] Example 5 Functional Analysis of Monoclonal Antibody 7H11E

[0060] The screened monoclonal antibody 7H11E was analyzed using immunofluorescence assay and immunoblotting assay, as follows:

[0061] (1) Indirect immunofluorescence experiment

[0062] CV-A2 and CV-A4 viruses were inoculated into 6-well plates of RD cells with a confluence of 95%. RD cells without virus inoculation served as negative controls and were placed in a 37°C, 5% CO2 incubator. After culturing the 6-well plates for 24 hours, the cell supernatant was discarded and 2 mL / well of 4% paraformaldehyde was added for fixation at room temperature for 1 hour and washed 5 times / 5 min with 0.01 M PBS. 2 mL / well of 2% BSA-PBST (W / V) solution (containing 0.5% Triton-X 100, V / V) was added for permeabilization at room temperature for 30 minutes and washed 5 times / 5 min with 0.01 M PBS. Blocking was performed with 2% BSA-PBST solution at room temperature for 1 hour and the blocking solution was discarded. 1 mL / well of 2 μg / mL monoclonal antibody 7H11E was added and incubated at room temperature for 1 hour and 0.01 M Wash with PBS 5 times / 5 min; add 1 mL / well, 2 μg / mL goat anti-mouse fluorescent antibody IgG (H+L) (Thermo Fisher), incubate at room temperature for 1 h in the dark, wash with 0.01 M PBS 5 times / 5 min, and add 1 mL / well 5 μg / mL DAPI solution (Biyuntian); observe and photograph with a fluorescence microscope. The results are as follows Figure 2shown.

[0063] from Figure 2 As can be seen in the figure, monoclonal antibody 7H11E can be used to identify CV-A2 and CV-A4 in indirect immunofluorescence experiments.

[0064] (2) Immunoblotting

[0065] Identification of the CV-A2 and CV-A4 structural protein regions recognized by the monoclonal antibody 7H11E. Enterovirus structural proteins are VP1, VP2, VP3, and VP4. Western blot analysis can be performed to determine the CV-A2 and CV-A4 structural protein regions recognized by 7H11E. The specific steps are as follows:

[0066] The same amount of antigen, CV-A4 FP, CV-A2 FP, CV-A2 VLP, CV-A5 FP, CV-A5 DP (DP, dense particles) and Vero cell lysate were added to 4× SDS-PAGE loading buffer (Bio-rad), heated at 100°C for 10 minutes for SDS-PAGE detection. After electrophoresis, the antigen was transferred to a 0.45 μm nitrocellulose membrane using a rapid wet transfer instrument (GenScript). After transfer, the membrane was blocked with 2% BSA (W / V) for 30 minutes. After blocking, 10 mL of 0.3 μg / mL 7H11E was added, incubated at 37°C for 1 hour and washed. After washing, 10 mL of HRP-labeled goat anti-mouse IgG was added at a dilution of 1:10,000, incubated at 37°C for 45 minutes and washed. Chemical exposure imaging (Gene) was performed using a colorimetric solution. The antibody recognition results are shown in Figure 2. Figure 3 shown.

[0067] Depend on Figure 3 It can be seen that the 7H11E monoclonal antibody can specifically bind to the structural protein VP1 region of CV-A2 and CV-A4 viruses.

[0068] (3) Binding titer detection of monoclonal antibody 7H11E

[0069] ELISA was used to determine the binding ability of monoclonal antibody 7H11E to enterovirus A swarm viruses CV-A2 (FP), CV-A4 (EP, FP), CV-A5 (FP), CV-A6 (EP+FP), CV-A10 (EP+FP), CV-A16 (EP+FP), and EV-A71 (EP+FP). Purified particles of enterovirus B swarm virus Echo11 (FP) were coated with 1 μg / mL of solution prepared in carbonate buffer (pH = 9.6) and added to the ELISA plate at 100 μL / well. The plates were incubated overnight at 4°C and washed three times after the incubation. The plates were blocked with 1% (W / V) BSA in PBST at 37°C for 1 h, and the blocking solution was discarded.

[0070] Add primary antibody: dilute 1 mg / mL monoclonal antibody 7H11E tenfold (volume ratio), add 100 μL / well of the diluted antibody to the ELISA plate, incubate at 37°C for 1 hour, and wash the plate five times with PBST (pH 7.4);

[0071] Add secondary antibody: add 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody to the ELISA plate, 100 μL / well, incubate at 37°C for 1 h, and wash the plate five times with PBST (pH 7.4);

[0072] TMB colorimetric solution was added and the mixture was developed at 37°C in the dark for 30 min. The reaction was terminated with 2M sulfuric acid and the absorbance at 450 nm was measured.

[0073] Titer calculation: The maximum dilution factor that is greater than 2.1 times the OD value of the negative well. The negative control is the addition of secondary antibody only without 7H11E. The positive control is mouse serum that specifically recognizes CV-A2, CV-A4, CV-A5, CV-A6, CV-A10, CV-A16, and EV-A71 particles.

[0074] Test results such as Figure 4 As shown, according to Figure 4 It can be calculated that the binding titers of the monoclonal antibodies to CV-A4 and CV-A2 are comparable.

[0075] Example 6 Study on the linear epitope of monoclonal antibody 7H11E

[0076] (1) Preliminary mapping of the linear epitope of monoclonal antibody 7H11E

[0077] The amino acid sequence of the linear epitope of the 7H11E monoclonal antibody was preliminarily identified by ELISA. The method is as follows: overlapping peptides from the VP1 region of the CV-A4 structural protein were synthesized, covering the full length of VP1 (305 amino acids). The peptide sequences are shown in Table 1 below:

[0078] Table 1 Overlapping peptide sequences in the VP1 region of the CV-A4 structural protein

[0079]

[0080]

[0081] Initial localization was performed using an indirect ELISA experiment: the peptide was prepared at a concentration of 4 μg / mL using carbonate buffer (pH = 9.6) to prepare a 1 μg / mL coating solution, which was then added to the ELISA plate (100 μL / well). The plate was incubated overnight at 4°C and washed three times with PBST. The plate was blocked with PBST (pH = 7.4) containing 1% (W / V) BSA at 37°C for 1 hour, and the blocking solution was discarded. The monoclonal antibody 7H11E was diluted tenfold (V / V) and 1 μg / mL was used. l monoclonal antibody, 100 μL / well was added to the ELISA plate, incubated at 37℃ for 1 hour, and washed 5 times with PBST; 0.1 μg / mL HRP-labeled goat anti-mouse IgG antibody was added to the ELISA plate, 100 μL / well, incubated at 37℃ for 1 hour, and washed 5 times with PBST; TMB (3,3',5,5'-tetramethylbenzidine) color development solution was added and color was developed at 37℃ in the dark for 30 minutes. The reaction was terminated with 2 M sulfuric acid and the absorbance at 450 nm was detected.

[0082] Titer calculation method: the maximum dilution factor that is greater than 2.1 times the OD value of the negative well; normal mouse serum is used as the negative control, and anti-A4 FP mouse serum is used as the positive control.

[0083] The preliminary results of linear epitope mapping of monoclonal antibodies are shown in Figure 5 A and 5B, from Figure 5 As can be seen from A and 5B, the monoclonal antibody can bind to peptide segment 1 of the CV-A4 structural protein VP1 (i.e., the first 1-15 amino acids of VP1).

[0084] (2) Precise localization of the linear epitope of monoclonal antibody 7H11E

[0085] To further narrow the localization range of the 7H11E monoclonal antibody binding peptide, a single peptide was coated in the wells of a 96-well plate to identify the specific binding peptide. To further determine the binding residue, a set of single amino acid substitutions from X (any amino acid except G) to G were synthesized, with a total of 14 single-point mutation peptides, as shown in Table 2, covering the first 15 amino acids of the N-terminus of VP1. These peptides were used to further locate the residues responsible for the binding of a single monoclonal antibody. The coating concentration range was experimentally determined to be 5 μg-10 μg / ml, with 100 μl / well of synthetic peptide solution. The subsequent ELISA steps were the same as the preliminary localization method described above. The results are shown in Table 2:

[0086] Table 2 Precise positioning of the key binding sites of monoclonal antibody 7H11E

[0087] VP1 amino acid position Mutated amino acid ELISA results 2 DG - 3 AG - 4 IG - 5 AG + 6 DG - 7 AG + 8 IG + 9 QG + 10 NG + 11 TG + 12 VG + 13 TG + 14 TG + 15 TG +

[0088] Among them, + and - indicate positive or negative ELISA results.

[0089] As shown in Table 2, amino acids 2 to 15 at the VP1 N-terminus (VP1-2 to 15) were mutated from their original residues to glycine. This is represented by VP1-2D-G, which indicates that the D at position 2 was mutated to a G, while the first glycine was retained. These synthetic peptides contain single amino acid changes and carry a single point mutation (G) as shown in the table. They were coated onto well plates for detection. The mutated residues involved in 7H11E binding to CV-A4 and CV-A2 are indicated in green, i.e., residues 2-4 (DAI) and 6 (D), respectively.

[0090] The inventors further discovered through experiments that the amino acid sequence of the heavy chain of the monoclonal antibody 7H11E, wherein the amino acid sequence of SEQ ID No. 7 is replaced, deleted, or added with one or more amino acids to form a new sequence, or an amino acid sequence having 95% or more homology to the amino acid sequence of SEQ ID No. 7, has the same function as the sequence of SEQ ID No. 7; and the amino acid sequence of the light chain, wherein the amino acid sequence of SEQ ID No. 8 is replaced, deleted, or added with one or more amino acids to form a new sequence, or an amino acid sequence having 95% or more homology to the amino acid sequence of SEQ ID No. 8, has the same function as the sequence of SEQ ID No. 8.

[0091] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations 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 shall be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that recognizes Coxsackievirus A2 and A4, characterized in that: The monoclonal antibody has a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.9, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.10, a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.11, and The light chain complementary determining region CDR1 is shown in SEQ ID NO.12, the light chain complementary determining region CDR2 is shown in SEQ ID NO.13, and the light chain complementary determining region CDR3 is shown in SEQ ID NO.

14.

2. The monoclonal antibody for recognizing Coxsackievirus A2 and A4 according to claim 1, characterized in that The monoclonal antibody has a heavy chain with an amino acid sequence shown in SEQ ID NO. 7; and / or, the monoclonal antibody has a light chain with an amino acid sequence shown in SEQ ID NO.

8.

3. The monoclonal antibody for recognizing Coxsackievirus A2 and A4 according to claim 2, characterized in that The heavy chain amino acid sequence of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID NO.7; and / or the light chain amino acid sequence of the monoclonal antibody is an amino acid sequence formed by replacing, deleting or adding one or more amino acid sequences to the amino acid sequence shown in SEQ ID NO.8, or an amino acid sequence having more than 95% homology with the amino acid sequence shown in SEQ ID No.

8.

4. The monoclonal antibody that recognizes Coxsackievirus A2 and A4 according to any one of claims 1 to 3, characterized in that The monoclonal antibody is of IgG2b subtype.

5. A polynucleotide molecule encoding the monoclonal antibody recognizing coxsackievirus A2 and A4 according to claim 1, 2 or 3.

6. The polynucleotide molecule according to claim 5, characterized in that The polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.5; and / or, the polynucleotide molecule has the nucleotide sequence shown in SEQ ID NO.

6.

7. A kit for detecting Coxsackievirus A2 and A4, characterized in that: The kit comprises the monoclonal antibody according to claim 1 or 2 or 3 or 4.

8. Use of the monoclonal antibody according to claim 1, 2, 3 or 4 in the preparation of a reagent or kit for detecting one or both of Coxsackievirus A2 and A4.

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