Enterovirus 71 type capsid protein vp1 monoclonal antibody and application thereof

Abstract

The application discloses a high-specificity and high-affinity monoclonal antibody and antigen-binding fragment thereof for recognizing enterovirus 71 capsid protein VP1. The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 5. The monoclonal antibody or antigen-binding fragment thereof can be used for qualitative and quantitative detection of enterovirus 71 VP1 protein.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a monoclonal antibody with high specificity and high affinity for recognizing enterovirus 71 capsid protein VP1 and its applications. Background Technology

[0002] Hand, foot, and mouth disease (HFMD) is a common childhood illness, primarily caused by enterovirus 71 (EV71) and coxsackievirus (CV), both belonging to the enterovirus family of the piconerivirus family. Clinically, it manifests as blisters on the hands, feet, and mouth, pharyngitis, and other symptoms. HFMD caused by EV71 can lead to damage to the central nervous system, with a significantly higher proportion of severe cases and mortality rate compared to other enteroviruses. Children may develop symptoms such as meningoencephalitis, limb paralysis, and pulmonary edema. Therefore, accurate differentiation is crucial in clinical diagnosis and treatment.

[0003] Currently, enterovirus detection methods include virus isolation, immunofluorescence assays, nucleic acid detection, and specific IgM antibody detection. While virus isolation is the gold standard for diagnosing enterovirus infection, it is time-consuming and costly, and rarely used clinically. Immunofluorescence assays require expensive fluorescence microscopes and are complex and time-consuming, similarly unsuitable for clinical needs. Nucleic acid detection, which has emerged in the last decade or so, still requires advanced equipment and personnel, making it difficult for ordinary primary care hospitals to implement. While specific IgM antibody detection has lower technical and equipment requirements and is simple and rapid, suitable for most primary healthcare institutions, its detection target is antibodies, resulting in a longer window period compared to antigen or nucleic acid detection methods, potentially leading to false negatives. To date, no enterovirus 71 antigen detection reagent has been approved for clinical use by the China National Medical Products Administration, primarily due to the lack of highly specific and high-affinity antibodies against enterovirus 71.

[0004] The lack of highly specific and high-affinity antibodies against enterovirus 71 (EV71) is due, in part, to the fact that while EV71 capsid protein VP1 (EV71-VP1) possesses good antigenicity and immunogenicity, effectively inducing the body to produce EV71-specific antibodies and serving as an important target antigen for EV71 infection detection, EV71-VP1 and CVA16-VP1 of Coxsackievirus A16 (CVA16) share approximately 70% amino acid homology. The presence of identical or similar antigenic epitopes between the two leads to cross-reactivity between EV71-specific detection and CVA16. Therefore, interference from CVA16-VP1 homology sequences must be eliminated during the preparation of EV71-VP1 antibodies to obtain highly specific monoclonal antibodies against EV71-VP1. On the other hand, in recent years, infections with Coxsackievirus A6 (CVA6) and Coxsackievirus A10 (CVA10) have shown a significant upward trend and are becoming the dominant circulating strains. Therefore, in preparing EV71-VP1 antibodies, it is also necessary to eliminate interference from the homologous sequences of the newly circulating dominant strains CVA6-VP1 and CVA10-VP1 in order to obtain truly EV71-VP1 specific monoclonal antibodies.

[0005] Therefore, the present invention aims to prepare and screen monoclonal antibodies with high specificity and high affinity for EV71-VP1 by eliminating interference from the homology sequences of CVA16-VP1, CVA6-VP1 and CVA10-VP1, using EV71-VP1 specific epitope peptides as immunogens, and to establish an EV71-VP1 antigen detection method to promote the early diagnosis of enterovirus EV71 infection. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a monoclonal antibody with high affinity for EV71-VP1 prepared using a fused hybridoma cell line. The monoclonal antibody obtained through experiments does not cross-react with CVA16-VP1, CVA6-VP1 and CVA10-VP1 and has high specificity. Therefore, it can be used for the qualitative and quantitative detection of enterovirus 71 capsid protein VP1.

[0007] Therefore, one aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof with high specificity and high affinity for enterovirus 71 capsid protein VP1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein,

[0008] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2;

[0009] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3;

[0010] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4;

[0011] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6;

[0012] The amino acid sequence of the light chain CDR2 is LVS;

[0013] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7.

[0014] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1 and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0015] The present invention also relates to the above-mentioned monoclonal antibodies or their antigen-binding fragments, wherein the antibodies or antigen-binding fragments are Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain antibodies or humanized antibodies. These antibodies or antigen-binding fragments, because they retain the variable regions of the light chain and heavy chain, or only retain the variable region of the heavy chain, can recognize EV71-VP1 with high specificity and high affinity, and do not have cross-reactivity with CVA16-VP1, CVA6-VP1 and CVA10-VP1.

[0016] Furthermore, this invention relates to a nucleic acid molecule comprising a nucleic acid encoding the aforementioned antibody or its antigen-binding fragment, and an expression vector comprising the aforementioned nucleic acid molecule, the expression vector being capable of expressing the aforementioned antibody or its antigen-binding fragment. This invention also relates to recombinants comprising the aforementioned nucleic acid molecule or the aforementioned expression vector, which can produce the aforementioned antibody or its antigen-binding fragment. On another note, this invention relates to a monoclonal antibody hybridoma cell line with high specificity and high affinity for EV71-VP1, namely mouse hybridoma cell line 3F93, with accession number CGMCC No. 46585.

[0017] Furthermore, this invention relates to the application of the aforementioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for detecting enterovirus 71 capsid protein VP1. More specifically, this invention relates to a kit for detecting enterovirus 71 capsid protein VP1, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for high-specificity and high-affinity binding to EV71-VP1; preferably, the kit is a double-antibody sandwich assay kit, wherein the monoclonal antibody or its antigen-binding fragment serves as a capture antibody.

[0018] Instructions for the Preservation of Biological Materials

[0019] The monoclonal antibody hybridoma cell line of this invention has been deposited with the China General Microbiological Culture Collection Center (CGMCC) under the registration number CGMCC 46585, on September 8, 2025, and classified as a monoclonal antibody mouse hybridoma cell line. The address of the China General Microbiological Culture Collection Center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0020] Figure 1 This is an amino acid sequence alignment analysis of EV71-VP1 with CVA16-VP1, CVA6-VP1, and CVA10-VP1.

[0021] Figure 2 This is an SDS-PAGE electrophoresis image showing prokaryotic expression of EV71-VP1 with CVA16-VP1, CVA6-VP1 and CVA10-VP1, where M is the marker, 1 is EV71-VP1, 2 is CVA16-VP1, 3 is CVA6-VP1 and 4 is CVA10-VP1.

[0022] Figure 3 This demonstrates the binding specificity of three monoclonal antibodies against the VP1 capsid protein of enterovirus 71.

[0023] Figure 4 This is a graph showing the limit of detection results for the EV71-VP1 double antibody sandwich ELISA method.

[0024] Figure 5 This is a result interpretation diagram for the EV71-VP1 double antibody sandwich colloidal gold immunochromatographic assay. The C line is the control line, and the T line is the test line.

[0025] Figure 6 This is a graph showing the limit of detection results for the EV71-VP1 double antibody sandwich colloidal gold immunochromatographic assay.

[0026] Figure 7 This is a diagram showing the identification results of the 3F93 subtype of the anti-EV71-VP1 monoclonal antibody. Detailed Implementation

[0027] The purpose of this invention is to provide a highly specific and high-affinity monoclonal antibody that recognizes the capsid protein VP1 of enterovirus 71, prepared using a fused hybridoma cell line. The specific preparation process involves first performing homology analysis on the amino acid sequences of EV71-VP1 with CVA16-VP1, CVA6-VP1, and CVA10-VP1 to identify EV71-VP1-specific amino acid sequence regions; then, using B-cell epitope analysis of EV71-VP1, specific dominant antigenic epitopes of EV71-VP1 are screened; finally, these epitopes are used as immunogens to immunize mice to prepare monoclonal antibodies. The prokaryotically expressed EV71-VP1 is then screened with CVA16-VP1, CVA6-VP1, and CVA10-VP16 to obtain a highly specific and high-affinity monoclonal antibody that recognizes EV71-VP1. This monoclonal antibody binds only to EV71-VP1 with high specificity and high affinity, and shows no cross-reactivity with CVA16-VP1, CVA6-VP1, and CVA10-VP16. The mouse hybridoma cell line secreting this monoclonal antibody, named 3F93, exhibits very high specificity and affinity for EV71-VP1. This cell line was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 8, 2025, with accession number CGMCC No. 46585.

[0028] Subsequently, the inventors sequenced and analyzed the immunoglobulin domain sequence of the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No. 46585, and found that its heavy chain variable region amino acid sequence was: EVQLQQSGPELMKPGTSVEISCKASGYSFTTYYIHWVKQSHGESLEYIGYIDPLNGGIRYNQKFKGKATLTVDKSSSTAYMLLSSLTSDDSAVYYCARGMDYWGQGTTVTVSS (SEQ ID NO.1), of which the heavy chain CDR1 amino acid sequence was GYSFTTYY (SEQ ID NO.2); the heavy chain CDR2 amino acid sequence was IDPLNGGI (SEQ ID NO.3); and the heavy chain CDR3 amino acid sequence was ARGMDY (SEQ ID NO.4). The amino acid sequence of the light chain variable region is: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGHQAGNQ (SEQ ID NO.5), of which the amino acid sequence of the light chain CDR is KSVSTSGYSY (SEQ ID NO.6); the amino acid sequence of the light chain CDR2 is LVS; and the amino acid sequence of the light chain CDR3 is QHIRELTR (SEQ ID NO.7).

[0029] The inventors evaluated the binding specificity of the aforementioned monoclonal antibody to EV71-VP1 using enzyme-linked immunosorbent assay (ELISA). They found that this monoclonal antibody could only specifically recognize EV71-VP1, and showed no cross-reactivity with CVA16-VP1, CVA6-VP1, or CVA10-VP16. The inventors also tested the affinity of the anti-EV71-VP1 monoclonal antibody for the EV71-VP1 antigen using Biocore kinetic experiments, finding that this monoclonal antibody could bind to the EV71-VP1 antigen with high affinity. The EV71-VP1 antigen in pharyngeal swabs, vesicular fluid, and fecal samples was specifically detected using colloidal gold immunochromatography, exhibiting very high specificity and sensitivity.

[0030] As is well known in the art, the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens. Furthermore, a conserved substitution of a single amino acid in the amino acid sequence of these CDR regions may not alter the protein structure; therefore, a single amino acid substitution within these regions may still possess the property of binding to the corresponding antigen. Thus, monoclonal antibodies or their antigen-binding fragments obtained by making a conserved substitution of a single amino acid in heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 may still recognize enterovirus 71 capsid protein VP1. In this patent application, the term "conservative substitution of amino acids" refers to the substitution of one amino acid in a protein by another chemically similar amino acid. Examples include substitutions between aromatic amino acids Phe, Trp, and Tyr; substitutions between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; substitutions between polar amino acids Gln and Asn; substitutions between basic amino acids Lys, Arg, and His; substitutions between acidic amino acids Asp and Glu; and substitutions between hydroxy amino acids Ser and Thr.

[0031] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing enterovirus 71 capsid protein VP1, from the monoclonal antibodies of this invention. These fragments include, but are not limited to, Fab, Fab', and F(ab')2. The Fab fragment is the antigen-binding region of the antibody structure, consisting of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, linked by disulfide bonds. The antigen-binding fragments can be prepared, for example, by enzymatic digestion with papain, where antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is further degraded into an F(ab')2 fragment and an Fc fragment, with the F(ab')2 fragment further reduced to form two Fab' fragments. Because these antigen-binding fragments can still bind the corresponding antigens, they can be used to prepare kits for detecting enterovirus 71 capsid protein VP1.

[0032] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. The single-chain antibody can be expressed, for example, in *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the property of recognizing enterovirus 71 capsid protein VP1 and can be applied to the detection of enterovirus 71 capsid protein VP1.

[0033] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of monoclonal antibodies that recognize enterovirus 71 capsid protein VP1 with high specificity and high affinity, based on the aforementioned amino acid sequence. They can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors that can express monoclonal antibodies or antigen-binding fragments that recognize enterovirus 71 capsid protein VP1 with high specificity and high affinity. Those skilled in the art can also introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, and yeast to obtain recombinant bodies, and express the antibodies or antigen-binding fragments of the present invention through these recombinant bodies. The expressed antibodies or antigen-binding fragments can recognize enterovirus 71 capsid protein VP1; therefore, the aforementioned nucleic acid molecules, expression vectors, and recombinant bodies are within the scope of protection of the claims of this invention. Furthermore, the above-mentioned techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0034] As described above, the antibody or its antigen-binding fragment of the present invention can recognize enterovirus 71 capsid protein VP1 with high specificity and high affinity. Therefore, it can be used to prepare a kit for detecting enterovirus 71 capsid protein VP1. The kit can be any kit that utilizes the binding reaction between the antibody or its antigen-binding fragment of the present invention and enterovirus 71 capsid protein VP1, such as, but not limited to, kits using enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold immunochromatography, fluorescence immunochromatography, Western blotting, immunohistochemistry, and immunofluorescence methods. Furthermore, ELISA kits, chemiluminescence kits, colloidal gold immunochromatography kits, and fluorescence immunochromatography kits can employ methods such as a double-antibody sandwich assay.

[0035] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0036] Example 1: Screening and determination of the dominant epitope specific to enterovirus 71 capsid protein VP1

[0037] Because Coxsackievirus and Enterovirus 71 (EV71) share a high amino acid homology (approximately 70%) in their capsid protein VP1, which is also a pathogen causing hand-foot-mouth disease, and have numerous identical or similar antigenic epitopes, making cross-reactivity likely, the amino acid sequences of their capsid proteins VP1 were first searched in the GenBank database (https: / / www.ncbi.nlm.nih.gov / ) of the National Center for Biotechnology Information (NCBI). The results were: GenBank: JN256068.1 (EV71-VP1), GenBank: ON840107.1 (CVA16-VP1), GenBank: KY424356.1 (CVA6-VP1), and GenBank: AMQ36990.1 (CVA10-VP1). The GenBank accession numbers and sequence summary information for these sequences are shown in Table 1 below.

[0038] Table 1. Amino acid sequences of the capsid protein VP1 of enterovirus 71 and coxsackievirus.

[0039] Serial number Pathogen GenBank accession number Amino acid sequence of capsid protein SEQ ID NO 1 EV71 JN256068.1 GDRVADVIESSIGDSVSRALTHALPAPTGQNTQVSSHRLDTGKVPALQAAEIGASSNASDESMIETRCVLNSHSTAETTLDSFFSRAGLVGEIDLPLEGTTNPNGYANWDIDITGYAQMRRKVELFTYMRFDAEFTFVACTPTGEVVPQLLQYMFVPPGAPKPDSRESLAWQTATNPSVFVKLSDPPAQVSVPFMSPASAYQWFYDGYPTFGEHKQEKDLEYGACPNNMMGTFSVRTVGTSKSKYPLVVRIYMRMKHVRAWIPRPMRNQNYLFKANPNYAGNSIKPTGASRTAITTL 8 2 CVA16 ON8 ​ 9 3 ​ ​ ​ 10 4 ​ AMQ36990.1 GDPVEDIIHDALGNTARRAISGATNVESAANTTPSSHRLETGRVPALQAAETGATSNATDENMIETRCVVNRNGVLETTINHFFSRSGLVGVVNLTDGGTDTTGYATWDIDIMGFVQLRRKCEMFTYMRFNAEFTFVTTTENGEARPYM LQYMYVPPGAPKPTGRDAFQWQTATNPSVFVKLTDPPAQVSVPFMSPASAYQWFYDGYPTFGQHPETSNTTYGLCPNNMMGTFAVRVVSREASQLKLQTRVYMKLKHVRAWVPRPIRSQPYLLKNFPNYDSSKIANSARDRSSIKQANM 11

[0040] The amino acid sequences of EV71-VP1, CVA16-VP1, CVA6-VP1, and CVA10-VP1 were compared and analyzed using the bioinformatics analysis software DNAMAN 6.0. The results are as follows: Figure 1 As shown, the amino acid sequence homology of the four capsid proteins VP1 is 70.50%, and the regions with significant differences in amino acid sequence are: 1-29, 71-81, 90-103, 137-149, 163-169, 212-220, 231-248 and 264-297.

[0041] The distribution of B-cell epitopes in EV71-VP1 (GenBank: JN256068.1) was analyzed using the bioinformatics analysis software BIOSUN. First, its full-length amino acid sequence was input, and then B-cell epitopes were analyzed. The predicted B-cell epitopes and their scores are shown in Table 2. All epitopes with scores ≥3 were identified as dominant epitopes, specifically epitopes 4, 10, and 14. Combined with the differentially expressed sequences identified by the amino acid homology analysis above, epitopes 10 and 14 were determined to be specific dominant epitopes of enterovirus 71 capsid protein VP1.

[0042] Table 2. B-cell epitope analysis of enterovirus 71 capsid protein VP1.

[0043] Serial Number Epitope sequence number Epitope sequence Score SEQ ID NO 1 11-17 SIGDSVS 2.172 12 2 28-34 TGQNTQV 2.914 13 3 34-40 VSSHRLD 2.834 14 4 57-63 NASDESM 3.122 15 5 74-80 STAETTL 2.756 16 6 99-105 GTTNPNG 2.889 17 7 117-123 AQMRRKV 2.566 18 8 129-135 MRFDAEF 1.631 19 9 141-147 TPTGEVV 1.472 20 10 161-167 PKPDSRE 3.647 21 11 170-176 AWQTATN 2.182 22 12 183-189 LSDPPAQ 2.174 23 13 198-204 ASAYQWF 1.716 24 14 214-220 HKQEKDL 3.805 25 15 229-235 MMGTFSV 1.453 26 16 239-245 GTSKSKY 2.817 27 17 253-259 MRMKHVR 1.675 28 18 265-271 PMRNQNY 2.665 29 19 276-282 NPNYAGN 2.601 30 20 284-290 IKPTGAS 2.660 31

[0044] To prepare a highly specific and high-affinity monoclonal antibody against the dominant epitope sequence of enterovirus 71 capsid protein VP1, the epitope sequences of epitopes 10 and 14 were first tandemly linked. To ensure epitope integrity, one extra amino acid was added before and after each epitope, and a cysteine ​​residue (C) was added at the amino terminus for easier conjugation. The final sequence of the peptide synthesized from the dominant epitope of enterovirus 71 capsid protein VP1 is CA. PKPDSRE SE HKQEKDL E (SEQ ID NO. 32), where the sequence indicated by the straight underline is the 10th epitope sequence and the sequence indicated by the wavy underline is the 14th epitope sequence. Shanghai Dechi Biotechnology Co., Ltd. was commissioned to prepare a synthetic peptide specific to the dominant epitope of enterovirus 71 capsid protein VP1 and conjugated it with KLH and BSA, respectively.

[0045] Example 2: Preparation of monoclonal antibody against enterovirus 71 capsid protein VP1 specific dominant epitope

[0046] Using the KLH conjugate, a peptide synthesized from the VP1-specific dominant epitope of enterovirus 71 capsid protein prepared in Example 1, as an immunogen, 6-8 week old female BALB / c mice were immunized with 100 µg of antigen per mouse plus an equal volume of Freund's complete adjuvant. After thorough emulsification using a stirrer, the mice were immunized subcutaneously in the back and intraperitoneally. Three mice were immunized. A second immunization was performed 4 weeks later, and a third immunization was performed 8 weeks later. For each third immunization, 50 µg of antigen per mouse plus incomplete Freund's adjuvant was emulsified thoroughly using a stirrer and then injected subcutaneously in the back and intraperitoneally. One week after the third immunization, blood was collected from the tail vein of the mice to detect the titer of the immune serum. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection (50 µg per mouse). Three days later, spleen cells were harvested for fusion. SP20 myeloma cells were resuscitated and cultured until they reached the logarithmic growth phase. Spleen cells were collected from the immunized BALB / c mice, and a spleen cell suspension was prepared. The above-mentioned spleen cells and myeloma cells were mixed in serum-free DMEM medium at a ratio of 9:1, centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were gently shaken to disperse them. The cells were then fused in a 37°C water bath. 1 mL of preheated 50% PEG fusion cells was added within 1 minute, while gently shaking to mix. After the addition was complete, the cells were allowed to stand for 90 seconds, and serum-free DMEM medium was added to terminate the fusion. The cells were then allowed to stand at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, and the pellet was resuspended in HAT medium. The pellet was then aliquoted into 96-well cell plates containing feeder cells and cultured in a cell culture incubator at 37°C and 5% CO2 for 5 days. The medium was changed once with HAT medium. The medium was changed again on day 10. When the fusion cells covered about 60% of the bottom of the wells, the cell culture supernatant was collected, and high-affinity positive clones were screened using a rapid reaction screening method. The specific method is as follows: Dilute the BSA conjugate of the enterovirus 71 capsid protein VP1-specific dominant epitope with carbonate coating buffer to a concentration of 2.0 μg / ml, and coat each well with 150 μl, incubating overnight at 4°C; wash the plate twice with washing buffer; add 200 μl / well blocking buffer and block at room temperature for 6 hours; wash the plate 5 times with washing buffer. Add 100 μl of sample diluent to each well, then add 10 μl of cell culture supernatant, and incubate with shaking at room temperature for 5-15 min, then discard the supernatant. Wash the plate 5 times, invert the washed ELISA plate onto absorbent paper to dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate with shaking at room temperature for 15 min. Wash the plate 5 times. Add 50 μL each of TMB chromogenic solutions A and B to each well, and incubate at room temperature in the dark. Continuously observe the cells, selecting the three positive clones that first show significant color changes as high-affinity monoclonal antibody hybridoma cell lines, named 2D61, 3F93, and 7A34. Culture these three hybridoma cell lines in 1640 medium containing 10% fetal bovine serum. Intraperitoneally inject 0.5 mL of liquid paraffin into each BALB / c male mouse. Collect cells after 10 days, resuspend them in 10 mL of physiological saline, and maintain a cell density of 1 × 10⁻⁶ cells / mL. 70.5 mL of antibody was administered intraperitoneally to each mouse at a concentration of 1 / mL. Ascites fluid was collected after 2 weeks. Antibody purification was performed using the Thermo Melon Gel Monoclonal IgG Purification Kit, and the purified antibody was aliquoted and stored at -20°C.

[0047] Example 3: Prokaryotic expression of EV71-VP1, CVA16-VP1, CVA6-VP1 and CVA10-VP1

[0048] To identify the specificity of high-affinity monoclonal antibodies against the dominant epitope of enterovirus 71 capsid protein VP1, EV71-VP1 (GenBank: JN256068.1), CVA16-VP1 (GenBank: ON840107.1), CVA6-VP1 (GenBank: KY424356.1), and CVA10-VP1 (GenBank: AMQ36990.1) were expressed in prokaryotes. First, based on the amino acid sequence of the mature protein and the genetic code preference of *E. coli*, optimized nucleotide sequences for the four capsid protein VP1 expression systems suitable for expression in *E. coli* were derived and summarized in Table 3 below.

[0049] Table 3 Optimized nucleotide sequences of four capsid proteins VP1

[0050] name Optimize nucleotide sequences SEQ ID NO EV71-VP1 GGTGATCGTGTTGCAGACGTTATTGAAAGCTCTATCGGTGATTCCGTGTCGCGCGCCCTGACCCATGCGTTGCCGGCACCAACGGGCCAGAACACTCAAGTCAGCAGTCACCGTCTGGATACCGGTAAAGTTCCGGCTTTACAGGCGGCCGAGATTGGCGCATCTTCGAATGCTAGCGACGAAAGCATGATCGAAACTCGCTGCGTACTGAACTCTCATTCCACGGCGGAGACCACTCTTGATTCGTTTTTCAGCCGTGCCGGTCTGGTGGGCGAAATTGACCTCCCACTGGAAGGTACCACCAATCCGAACGGCTATGCGAATTGGGATATCGATATTACGGGCTACGCACAGATGCGCCGTAAGGTTGAGCTGTTTACTTATATGCGCTTCGATGCTGAATTTACCTTCGTCGCGTGTACTCCAACGGGTGAAGTGGTTCCGCAATTGCTGCAGTACATGTTTGTGCCACCGGGCGCCCCGAAACCAGACAGTCGTGAGTCTTTAGCATGGCAGACCGCTACTAACCCGTCGGTCTTCGTTAAACTGAGCGATCCACCGGCGCAAGTAAGCGTGCCATTTATGTCTCCGGCCTCCGCGTATCAGTGGTTTTACGATGGTTATCCAACCTTCGGCGAACACAAGCAGGAAAAAGACCTTGAGTACGGTGCATGCCCGAATAACATGATGGGCACCTTTTCGGTTCGTACGGTCGGTACTAGCAAAAGTAAGTATCCGCTGGTGGTTCGCATCTATATGCGTATGAAACATGTGCGCGCTTGGATTCCACGTCCGATGCGCAACCAAAATTACCTCTTCAAAGCGAACCCAAATTATGCCGGCAACTCTATTAAGCCGACCGGCGCATCGCGTACTGCTATCACGACCCTG 33 CVA16-VP1 GGTGATCCGATTGCAGACATGATCGATCAGACCGTGAACAGCCAAGTTAATCGTTCTCTGACGGCCTTGCAGGTGCTGCCAACTGCGGCAAACACCGAAGCTTCCTCGCATCGCTTAGGTACTGGCGTCGTTCCGGCGCTGCAGGCCGCAGAGACGGGTGCTAGCAGTAATGCGTCTGATAAAAACCTTATTGAAACCCGTTGCGTACTGAATCACCATTCGACTCAAGAAACCGCCATCGGCAACTTTTTCAGCCGCGCGGGTCTCGTGAGCATTATCGCAATGCCAACCACGGGCACTCAGAATACCGACGGTTATGTTAACTGGGATATTGACCTGATGGGCTACGCTCAGCTGCGTCGCAAGTGTGAGTTGTTTACTTATATGCGTTTCGATGCGGAATTTACGTTCGTCGTGGCCAAACCGAACGGCGTTCTGGTGCCACAATTACTGCAGTACATGTATGTCCCGCCAGGTGCACCGAAACCGACCTCTTCCGATTCGTTTGCTTGGCAGACTGCGACCAATCCAAGCGTTTTCGTAAAGATGACCGATCCGCCAGCCCAAGTGAGTGTTCCGTTTATGTCTCCAGCGTCGGCATACCAGTGGTTTTATGACGGCTACCCGACGTTCGGTGAACACCTTCAGGCTAACGATCTGGATTATGGCCAATGCCCAAATAACATGATGGGTACTTTTAGCATCCGCACCGTCGGCACTGAGAAAAGCCCGCATTCTATTACGCTCCGTGTGTATATGCGTATTAAACACGTTCGCGCGTGGATTCCGCGTCCACTGCGCAATCAGCCGTACCTGTTCAAGACCAACCCAAATTATAAAGGTAACGACATTAAATGCACTTCCACCTCGCGTGATAAGATCACCACGTTG 34 CVA6-VP1 AACGATCCGATTACCAATGCGGTGGAAAGCGCCGTTTCTGCGCTGACGGACACTACCATCTCCCGTGTGACTGCAGCTAACACGGCGGCCTCGACCCATAGCTTGGGCACTGGTCGCGTCCCAGCACTGCAGGCTGCGGAGACCGGCGCCAGTTCTAATGCGTCGGATGAAAACTTAATTGAAACCCGTTGCGTTATGAATCGCAACGGTGTAAATGAGGCAAGCGTGGAACACTTTTATAGCCGTGCTGGCCTGGTTGGTGTCGTGGAAGTTAAAGATTCTGGCACGTCCCTTGACGGTTACACTGTGTGGCCGATCGATGTCATGGGCTTCGTTCAACAGCGCCGTAAGCTGGAGCTCTCGACCTATATGCGCTTTGACGCGGAGTTCACTTTTGTAAGCAACCTGAACGATAGTACGACCCCAGGCATGCTGTTGCAGTACATGTATGTGCCGCCAGGTGCCCCGAAACCAGATTCTCGTAAATCGTACCAATGGCAGACTGCAACCAATCCGAGCGTTTTCGCTAAGCTGAGCGATCCGCCACCGCAGGTCTCTGTGCCATTTATGTCCCCGGCGACCGCCTATCAATGGTTCTACGACGGCTATCCAACGTTTGGTGAACATAAACAGGCGACTAACTTACAGTATGGCCAATGTCCGAATAACATGATGGGTCACTTTGCAATTCGTACCGTTTCGGAGAGCACTACGGGCAAAAATGTGCATGTCCGCGTTTACATGCGTATCAAGCACGTACGCGCTTGGGTGCCACGTCCGCTGCGCAGTCAGGCGTATATGGTTAAAAACTACCCGACCTATTCTCAGACTATTACCAATACCGCCACGGATCGTGCATCGATCACTACCACTGATTACGAAGGTGGCGTCCCAGCTAACCCACAACGCACGAGC 35 CVA10-VP1 GGTGATCCAGTGGAAGACATTATCCATGATGCGCTGGGTAACACCGCCCGTCGCGCGATTAGCGGCGCAACGAATGTTGAGTCTGCTGCGAACACTACCCCATCCTCGCACCGTTTGGAAACTGGTCGCGTGCCGGCCCTGCAGGCAGCTGAAACGGGCGCGACCAGCAATGCCACTGATGAGAACATGATCGAAACCCGTTGCGTCGTTAATCGCAACGGTGTATTAGAAACCACGATTAATCATTTTTTCAGTCGTTCTGGCCTGGTGGGTGTTGTCAACCTTACTGACGGCGGCACCGATACTACGGGTTATGCGACCTGGGACATCGATATTATGGGCTTTGTGCAACTGCGCCGTAAATGTGAGATGTTCACTTACATGCGTTTTAACGCAGAGTTCACCTTTGTTACCACGACTGAAAATGGTGAGGCTCGCCCATATATGCTCCAGTACATGTATGTGCCGCCAGGCGCGCCGAAGCCAACCGGTCGTGATGCCTTCCAGTGGCAAACTGCAACGAACCCGTCGGTCTTTGTTAAACTGACCGATCCGCCAGCTCAGGTAAGCGTGCCGTTTATGAGCCCAGCGTCTGCCTACCAGTGGTTCTATGACGGCTACCCGACTTTTGGTCAACACCCAGAAACCTCCAATACCACGTATGGCCTGTGCCCGAACAATATGATGGGCACTTTCGCGGTTCGCGTCGTGTCGCGTGAAGCAAGCCAGTTGAAACTGCAGACCCGCGTTTATATGAAGTTAAAACATGTGCGTGCTTGGGTCCCACGCCCGATCCGTAGTCAACCGTACCTGCTTAAAAACTTTCCAAATTATGATTCTTCGAAGATTGCGAACAGCGCCCGTGATCGCAGCTCTATTAAGCAGGCAAACATG 36

[0051] Then, using BamH I and EcoR I restriction sites, they were ligated into pCold I to construct recombinant expression plasmids pC-EV71, pC-CVA16, pC-CVA6, and pC-CVA10, respectively. The correctly sequenced recombinant expression plasmids were transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and cultured overnight at 37°C with shaking in 5 mL of LB broth containing ampicillin sodium. The next day, the cells were inoculated into 250 mL of fresh LB broth and cultured until the logarithmic growth phase. The temperature was then adjusted to 16°C, and after 30 minutes, 150 μl of 1 mol / L IPTG induction solution was added. Induction was continued at 16°C for 12-14 h. Induced bacterial cells were collected by centrifugation, resuspended in 25 mmol / L Tris-HCl (pH 8.5), and sonicated. Inclusion bodies were collected by centrifugation at 12,000 rpm for 20 min at 4 °C. The inclusion bodies were washed with 25 mmol / L Tris-HCl containing 2 mol / L urea, and then dissolved in 25 mmol / L Tris-HCl containing 6 mol / L urea. The supernatant was collected by centrifugation at 12,000 rpm for 20 min and purified using a Ni column. The target protein was collected by elution with washing buffer containing 250 mmol / L imidazole and subjected to SDS-PAGE gel electrophoresis. The results are shown below. Figure 2 As shown, M is the marker, 1 is EV71-VP1, 2 is CVA16-VP1, 3 is CVA6-VP1, and 4 is CVA10-VP1. EV71-VP1, CVA16-VP1, CVA6-VP1, and CVA10-VP1 are all inclusion bodies expressed, with a molecular weight of approximately 43 kDa, consistent with the expected molecular weight.

[0052] Example 4: Enzyme-linked immunosorbent assay (ELISA) for detecting the binding specificity of monoclonal antibodies against enterovirus 71 capsid protein VP1.

[0053] Using prokaryotically expressed EV71-VP1, CVA16-VP1, CVA6-VP1, and CVA10-VP1 as coating antigens, the binding specificity of the three high-affinity antibodies prepared for EV71-VP1 was detected by indirect ELISA. Specifically, the EV71-VP1, CVA16-VP1, CVA6-VP1, and CVA10-VP1 antigens were diluted with carbonate coating buffer to a concentration of 2.0 μg / mL, with 150 μL coated per well, and incubated overnight at 4°C; the plates were washed twice with washing buffer; 200 μL / well blocking buffer was added, and the plates were blocked at room temperature for 6 hours; the plates were then washed five times with washing buffer. The three monoclonal antibodies 2D61, 3F93, and 7A34 prepared in Example 2 were diluted to 1.0 μg / mL, with 100 μL of each antigen added to each coated well in parallel, and incubated with shaking at room temperature for 45 min, after which the solution was discarded. Wash the plate 5 times, invert it onto absorbent paper to dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate at room temperature with shaking for 45 min. Wash the plate 5 times. Add 50 μl each of TMB chromogenic solution A and B to each well, and incubate at room temperature in the dark for 10 min. Add 50 μl of 2 M H2SO4 to each well to stop the reaction. Measure the absorbance of each well using a microplate reader at a wavelength of 450 nm, and read the values ​​within 10 minutes after termination. Results are as follows. Figure 3 As shown, monoclonal antibodies 2D61 and 3F93 exhibit high specificity against EV71-VP1 and show no cross-reactivity with CVA16-VP1, CVA6-VP1, and CVA10-VP1; monoclonal antibody 7A34 shows weak cross-reactivity with CVA6-VP1 and CVA10-VP1. Therefore, monoclonal antibodies 2D61 and 3F93 are identified as highly specific monoclonal antibodies against enterovirus 71 capsid protein VP1.

[0054] Example 5: Biacore kinetic assay to detect the binding affinity of monoclonal antibody against enterovirus 71 capsid protein VP1.

[0055] The binding affinity of the highly specific monoclonal antibodies 2D61 and 3F93 against enterovirus 71 capsid protein VP1 was analyzed using a BIACORE 3000 biomolecular interaction analyzer via Biacore kinetic experiments. Antibodies were diluted with phosphate buffer and then introduced at a rate of 5 μL / min for 2 min to bind. Subsequently, 1 mg / mL of prokaryotically expressed EV71-VP1 antigen was introduced at a rate of 30 μL / min for 7 min. The binding and dissociation times were the same. Biacore binding kinetic data are shown in Table 4. ka represents the binding rate constant, indicating the rate of binding between macromolecules; kd represents the dissociation rate constant, indicating the rate of dissociation of the molecular complex; and KD represents the dissociation equilibrium constant (KD = kd / ka). Generally, a smaller KD value indicates a higher affinity between the two molecules. Table 4 shows that the dissociation equilibrium constant of monoclonal antibody 3F93 is smaller than that of monoclonal antibody 2D61, indicating that monoclonal antibody 3F93 has a stronger binding affinity for the EV71-VP1 antigen than monoclonal antibody 2D61. Therefore, monoclonal antibody 3F93 is identified as a highly specific and high-affinity monoclonal antibody against enterovirus 71 capsid protein VP1.

[0056] Table 4. Affinity constants of monoclonal antibodies to EV71-VP1 antigen

[0057] antibody <![CDATA[ka(10 4 1 / Ms)]]> <![CDATA[kd (10 -4 1 / s)]]> <![CDATA[KD (10 -9 M)]]> 2D61 27.36±3.04 9.23±1.11 3.37±0.59 3F93 30.17±3.93 7.45±1.81 2.46±0.87

[0058] Example 6: Preparation of polyclonal antibody against enterovirus 71 capsid protein VP1

[0059] Polyclonal antibodies against enterovirus 71 capsid protein VP1 were prepared using EV71-VP1 as an immunogen. Healthy New Zealand white rabbits were selected. 1.0 mg of EV71-VP1 was mixed with 1.0 mL of Freund's complete adjuvant, thoroughly emulsified using a stirrer, and then subcutaneously injected at two points along the spine of the rabbits, 0.2 mL at each point. Four weeks later, 1.0 mg of EV71-VP1 was mixed with 1.0 mL of Freund's incomplete adjuvant, thoroughly emulsified using a stirrer, and then administered as a second immunization at different points on the same site. A third booster immunization was performed four weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After the blood clots and contracted, the blood was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20°C for later use. The indirect ELISA method for determining the titer of polyclonal antibodies is as follows: ELISA plates are coated with EV71-VP1 at a concentration of 2.0 μg / ml, with 150 μL coated per well, and incubated overnight at 4°C; the plates are washed twice with washing buffer; 200 μL / well blocking buffer is added and the plates are blocked at room temperature for 6 hours; the plates are then washed 5 times with washing buffer. Rabbit serum was diluted with PBS at dilution ratios of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000, and 1:4096000, with 100 μL added to each well. The plate was incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. HRP-labeled goat anti-rabbit secondary antibody was added and incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. Freshly prepared substrate solution was added, 100 μL per well, and the plate was incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 2 M H2SO4 to each well. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm, and the readings were taken within 10 minutes after termination. Using pre-immunization rabbit serum diluted serially as a negative control, the titer of the prepared anti-EV71-VP1 polyclonal antibody reached 1:512000.

[0060] Example 7: Establishment of an enzyme-linked immunosorbent assay (ELISA) for enterovirus 71 capsid protein VP1

[0061] Using the highly specific and high-affinity monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1 prepared in this invention as the capture antibody, and the rabbit polyclonal antibody against enterovirus 71 capsid protein VP1 prepared in Example 6 as the detection antibody, an enzyme-linked immunosorbent assay (ELISA) method for the specific detection of enterovirus 71 capsid protein VP1 based on a double-antibody sandwich method was established. Simultaneously, a commercially available monoclonal antibody against enterovirus 71 capsid protein VP1 (abnova, catalog number MAB1255-M05) was used as the capture antibody for a control study. The specific steps are as follows: Coat the ELISA plate with a monoclonal antibody against enterovirus 71 capsid protein VP1 at a concentration of 2.0 μg / mL, using 100 μL per well. Incubate overnight at 4°C, and wash twice with washing buffer. Add 120 μL / well blocking buffer and block at room temperature for 6 hours, then wash five times with washing buffer. Serially dilute the prokaryotically expressed EV71-VP1 antigen from Example 3 with double-distilled water to concentrations of 1000, 100, 10, 5.0, 1.0, 0.5, 0.1, and 0.05 ng / mL. Add 100 μL of each to the wells and incubate at 37°C for 45 min, then discard the diluted solution. Wash the plate five times with washing buffer. Add 100 μL of horseradish peroxidase-labeled rabbit anti-enterovirus 71 capsid protein VP1 polyclonal antibody to each well using the conventional method, and incubate at 37°C for 45 min. Wash the plate 5 times, blot dry, add 50 μL each of TMB chromogenic solution A and B to each well, and incubate at room temperature in the dark for 15 min. Add 50 μL of 2 M H2SO4 stop solution per well to terminate the reaction. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm, and read the value within 10 minutes after termination.

[0062] The results are as follows Figure 4 As shown, using three times the negative control detection value as the cutoff (cutoff = 0.146), the double-antibody sandwich detection method established using the anti-enterovirus 71 capsid protein VP1 monoclonal antibody 3F93 prepared in this invention as the capture antibody achieved a limit of detection (LOD) of 0.1 ng / mL for EV71-VP1 antigen. In contrast, the LOD of the commercially available control antibody for EV71-VP1 antigen was 1.0 ng / mL. The LOD of the anti-enterovirus 71 capsid protein VP1 monoclonal antibody 3F93 prepared in this invention for EV71-VP1 antigen is significantly higher than that of the commercially available antibody. It is also significantly higher than the limit of quantitation of 0.23 μg / mL of the double-antibody sandwich ELISA method for detecting EV71 antigen content reported in the literature (Xu Jing, Cui Wenyu, Li Shuxiang, Liu Jing, Wang Xinyi, Chen Lei, Shen Xinliang. Establishment and preliminary validation of a method for detecting enterovirus 71 antigen content. Chinese Journal of Experimental and Clinical Virology, 2009, 23(5): 388-390).

[0063] Example 8: Establishment of a colloidal gold immunochromatographic method for detecting enterovirus 71 capsid protein VP1.

[0064] A colloidal gold immunochromatographic assay was established for the detection of Campylobacter using the monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1 prepared according to this invention and the polyclonal antibody against enterovirus 71 capsid protein VP1 prepared in Example 6. The rabbit polyclonal antibody against enterovirus 71 capsid protein VP1 prepared according to this invention was coated at the T-line on a nitrocellulose membrane. The colloidal gold-labeled monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1 prepared according to this invention was immobilized on a gold-labeled pad. Goat anti-mouse IgG (purchased from Phytobio Biotechnology Co., Ltd.) was coated at the C-line.

[0065] Similarly, the EV71-VP1 antigen expressed in prokaryotic cells in Example 3 was serially diluted with double-distilled water to concentrations of 1000, 100, 10, 5.0, 1.0, 0.5, 0.1, and 0.05 ng / mL. 100 μL of each solution was added to the sample wells. Specifically, the test card was placed flat on a dry surface, and 100 μL of the above sample was added vertically and slowly to each well. Results were read after 5–15 minutes; readings after 15 minutes were invalid. When the sample is added to the sample well of the test card, it will move forward along the test card under capillary action. Upon migration to the gold-labeled pad, if the sample contains EV71-VP1, it will be specifically captured by the colloidal gold-labeled mouse anti-enterovirus 71 capsid protein VP1 monoclonal antibody 3F93, forming an immune complex. The resulting immune complex continues to migrate forward and is captured by the rabbit anti-enterovirus 71 capsid protein VP1 polyclonal antibody (T line) immobilized on the nitrocellulose membrane, forming an immune complex of "mouse anti-enterovirus 71 capsid protein VP1 monoclonal antibody 3F93 - EV71-VP1 antigen - rabbit anti-enterovirus 71 capsid protein VP1 polyclonal antibody," producing a red T line. Regardless of whether the sample contains EV71-VP1 antigen, the goat anti-mouse IgG coated in the C line region will bind to an excess of mouse anti-enterovirus 71 capsid protein VP1 monoclonal antibody 3F93, forming a red band. Result interpretation is as follows: Figure 5 As shown, where

[0066] Positive: A red band appears at line C, and red bands of varying intensity also appear at line T.

[0067] Negative: A red band appears only at line C, and no band appears at line T.

[0068] Invalid: No band at line C, regardless of whether a red band appears at line T.

[0069] Experimental results are as follows Figure 6As shown, the detection limit of the colloidal gold immunochromatographic method for EV71 capsid protein VP1 of enterovirus 71 was also determined to be 0.5 ng / mL, which is significantly higher than the quantitative limit of 0.23 μg / mL of the double-antibody sandwich ELISA method for detecting EV71 antigen content reported in the literature (Xu Jing, Cui Wenyu, Li Shuxiang, Liu Jing, Wang Xinyi, Chen Lei, Shen Xinliang. Establishment and preliminary verification of the detection method for enterovirus 71 antigen content. Chinese Journal of Experimental and Clinical Virology, 2009, 23(5): 388-390).

[0070] Example 9: Detection of clinical samples

[0071] Pharyngeal swab samples, vesicle fluid samples, and stool samples were collected in parallel from children with hand-foot-mouth disease (HFMD) diagnosed with enterovirus EV71 infection via nucleic acid testing. These samples were diluted with PBS sample processing solution before testing. The specific procedure was as follows: the test card was placed flat on a dry surface, and 100 µL of the sample was slowly and vertically added to each well. Results were interpreted after 5–15 minutes; results after 15 minutes were invalid. The results are shown in Table 5. For the pharyngeal swab and vesicle fluid samples from HFMD patients, 10 samples were positive for bacteria, with a detection rate of 100%. For the corresponding 10 stool samples, 9 were positive, with a detection rate of 90%.

[0072] Table 5. Detection results of clinical samples from children with hand-foot-mouth disease

[0073] patient number throat swab herpes fluid feces 1 ++ ++ + 2 ++ ++ + 3 +++ ++++ +++ 4 +++ +++ + 5 ++ ++ + 6 + + — 7 ++ ++ + 8 ++ +++ ++ 9 ++ ++ + 10 +++ +++ +

[0074] Example 10: Subtype analysis of monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1

[0075] The heavy and light chain subtypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a-10 / 20) from Antaiji (Beijing) Biotechnology Co., Ltd. First, the detection card was placed flat on the table, then 80 μl of 3F93 monoclonal antibody hybridoma cell line culture supernatant was added to each well. After standing for 5-10 min, the results were observed and recorded. The results are as follows: Figure 7 As shown, the monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0076] Example 11: Sequencing of the variable region of monoclonal antibody 3F93 against enterovirus 71 capsid protein VP1

[0077] Mouse hybridoma cell line 3F93 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab fragment of mouse monoclonal antibodies synthesized by Beijing Qingke Biotechnology Co., Ltd. The cells were preheated at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The resulting cells were ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data was compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website's BLAST module.

[0078] Sequence analysis revealed that the heavy chain variable region contains 113 amino acids, with the following sequence: EVQLQQSGPELMKPGTSVEISCKAS GYSFTTYY IHWVKQSHGESLEYIGY IDPLNGGI RYNQKFKGKATLTVDKSSSTAYMLLSSLTSDDSAVYYC ARGMDY WGQGTTVTVSS (SEQ ID NO.1), where CDRs are underlined, heavy chain CDR1 is located at 26-33 amino acids, with the amino acid sequence GYSFTTYY (SEQ ID NO.2); heavy chain CDR2 is located at 51-58 amino acids, with the amino acid sequence IDPLNGGI (SEQ ID NO.3); heavy chain CDR3 is located at 97-102 amino acids, with the amino acid sequence ARGMDY (SEQ ID NO.4). The light chain variable region has 109 amino acids, and its sequence is as follows: DIVMTQSPASLAVSLGQRATISYRAS KSVSTSG YSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGHQAGNQ (SEQ ID NO.5), wherein CDRs are underlined, light chain CDR1 is located at 27-36 aa, with an amino acid sequence of KSVSTSGYSY (SEQ ID NO.6); light chain CDR2 is located at 54-56 aa, with an amino acid sequence of LVS; and light chain CDR3 is located at 93-100 aa, with an amino acid sequence of QHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof with high specificity and high affinity to recognize enterovirus 71 capsid protein VP1, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the light chain variable region comprising a light chain CDR1, a light chain CDR2 and a light chain CDR3, characterized in that, the amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO. 2; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO. 3; the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO. 4; the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO. 6; the amino acid sequence of the light chain CDR2 is LVS; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO. 7; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

5. It is secreted by the monoclonal antibody mouse hybridoma cell strain 3F93 with the accession number of CGMCC No. 46585. The monoclonal antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab') 2 fragment or a humanized antibody. It comprises a nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. It comprises the nucleic acid molecule according to claim 5. It comprises the nucleic acid molecule according to claim 5 or the expression vector according to claim 6. It is the monoclonal antibody mouse hybridoma cell strain 3F93 with the accession number of CGMCC No. 46585.

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, 9.Use of the monoclonal antibody or antigen-binding fragment thereof with high specificity and high affinity to recognize enterovirus 71 capsid protein VP1 according to any one of claims 1 to 4 in the preparation of a kit for detecting enterovirus 71 capsid protein VP1.

3. The monoclonal antibody according to claim 2, characterized in that, It comprises the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

4. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that, It is a double antibody sandwich method detection kit, and the monoclonal antibody or antigen-binding fragment thereof is used as a capture antibody.

5. A nucleic acid molecule, characterized in that, ​ 6. An expression vector, characterized by, ​ 7. A cell, bacterial or yeast recombinant, characterized in that, ​ 8. A hybridoma cell line secreting a monoclonal antibody of high specificity and high affinity for recognizing the capsid protein VP1 of Enterovirus 71, characterized in that, ​ ​ 10. A kit for detecting Enterovirus 71 capsid protein VP1, characterized in that, ​ 11. The kit of claim 10, wherein ​

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