Broad-spectrum monoclonal antibody recognizing the linear epitope at the c-terminal of the vp2 protein of foot-and-mouth disease virus and its use

Abstract

The application belongs to the technical field of biology and relates to a broad-spectrum monoclonal antibody for recognizing a linear antigen epitope at the C-terminal of foot-and-mouth disease virus VP2 protein and application thereof. The monoclonal antibody pOTB-1 is successfully constructed, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 6, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 7. The biological activity of the antibody is verified by indirect immunofluorescence test, enzyme-linked immunosorbent assay and Western-blot. The antibody exhibits broad-spectrum reactivity to O type and A type FMDV and has strong binding affinity. The antibody recognizes a continuous linear B cell epitope at the C-terminal of the structural protein VP2. The epitope sequence is highly conserved among FMDV O type, A type and Asia1 type, which indicates that there is an inter-type common antigen site in the capsid protein VP2. A competitive ELISA detection method is established based on biotinylated pOTB-1, which provides a tool for the prevention and control of FMDV and serological detection, and lays a foundation for the design of FMDV marker vaccine and the evaluation of immunization effect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a broad-spectrum monoclonal antibody that recognizes the C-terminal linear antigenic epitope of the foot-and-mouth disease virus VP2 protein and its application. Background Technology

[0002] Foot-and-mouth disease virus (FMDV) is a highly virulent pathogen that infects cloven-hoofed animals such as pigs, cattle, and sheep. It is characterized by its high mutation rate, wide range of infecting hosts, and tendency to cause persistent and latent infections, resulting in significant economic losses to my country's livestock industry. FMDV belongs to the Picornaviridae family (…). Picornaviridae Foot-and-mouth disease virus genus ( Aphthovirus The FMDV family comprises seven serotypes: O, A, Asia1, C, and SAT1-3. Currently, serotypes O and A are the main serotypes threatening my country's livestock industry. According to official WOAH epidemic data, since 2005, there have been over 27,000 outbreaks, with serotypes O, A, Asia1, and C accounting for 49.8%, 28.9%, and 7.7% respectively. Asia and Africa are high-incidence areas for FMDV, accounting for over 97% of global outbreaks. Serotype O FMDV can be divided into 11 topologies, and my country has four lineages of three topologies circulating: O / Mya / 98 (SEA topology), O / Ind / 2001 and O / PanAsia lineages (ME-SA topology), and a new swine virus lineage (O / Cathay topology). Significant differences in the antigenic structure of different lineages lead to decreased vaccine efficacy or immunization failure.

[0003] Determining the structure and variation of viral antigens is crucial in virology and immunology research, aiding in the design of targeted vaccines and antibody drugs. The structural proteins VP1, VP2, VP3, and VP4 of foot-and-mouth disease virus (FMDV) each consist of 60 molecules, forming the icosahedral capsid structure. VP4 is located within the capsid. VP1-VP3 are structurally similar, each containing eight β-sheet structures and loop structures connecting these sheets. These contain numerous linear or conformational antigenic epitopes. A region composed of several epitopes or antigenic determinants is called an antigenic site. Different antigenic sites collectively constitute the viral antigenic structure. FMDV surface antigenic epitopes are primarily determined using mouse monoclonal antibodies, including neutralizing and non-neutralizing epitopes. Through cross-neutralization experiments, neutralizing epitopes covering five antigenic sites on FMDV serum type O VP1-VP3 have been identified. Site 1 consists of a linear epitope at the VP1 GH loop (133–157) and the VP1 C-terminus (200–213). Site 2 is located near the triple axis of the viral capsid structure and contains more than one epitope. Site 3 is located around the pentagonal axis on the viral capsid surface. Site 4 is located on the β-B junction of VP3 adjacent to the pentagonal axis. Site 5 is formed by the interaction of the VP1 GH loop with other adjacent amino acids on the capsid surface. Except for site 1, which contains a linear neutralizing epitope, all other sites consist of conformational neutralizing epitopes. Compared to neutralizing epitopes, the non-neutralizing epitopes of FMDV are mainly concentrated on VP2 and can be recognized by several mouse monoclonal antibodies targeting the N-terminal linear epitope of VP2 for different FMDV serotypes. The novel discovery of epitope-targeting antibodies can be identified and applied as biomarkers to study novel labeled vaccines, enhance the monitoring of vaccine immunization effects, differentiate between vaccine-immunized and naturally infected animals, and promote the clearance and eradication of FMDV.

[0004] Monoclonal antibodies are crucial tools for elucidating antigen structures. Their development has progressed through three technological stages: hybridoma cell technology, phage display antibody library technology, and single-B cell antibody technology. Single-B cell antibody technology is currently the most important method for high-throughput screening of functionally diverse antibodies. Its core principle involves isolating antigen-specific B cells from immunized animal tissues or peripheral blood, then combining this with single-cell immune repertoire sequencing to obtain naturally paired antibody hypervariable region sequences in antibody-secreting B cells. These sequences are then expressed in mammalian cells to obtain biologically active monoclonal antibodies. By ensuring the natural pairing of light and heavy chain variable regions, antibody genes targeting different antigenic sites can be screened, exhibiting structures similar to antibodies produced naturally in animals. Single-B cell antibody technology plays a vital role in the development of therapeutic antibodies for tumors and major human infectious diseases.

[0005] This invention utilizes the type O FMDV146S antigen to screen antigen-specific antibody-secreting B cells, and obtained a broad-spectrum monoclonal engineered antibody that can recognize the conserved C-terminal sequence of the FMDV VP2 protein. This provides materials for the research of a universal FMDV antigen detection method and can provide a reference for clinical diagnosis and the preparation of reagents or drugs to inhibit foot-and-mouth disease. Summary of the Invention

[0006] The purpose of this invention is to provide a broad-spectrum monoclonal antibody that recognizes the C-terminal linear antigenic epitope of the foot-and-mouth disease virus VP2 protein and its application.

[0007] This invention provides a broad-spectrum monoclonal antibody pOTB-1 that recognizes the C-terminal linear antigenic epitope of the foot-and-mouth disease virus VP2 protein, wherein the antibody pOTB-1 includes a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region includes three heavy chain complementarity determination regions: HCDR1, HCDR2, and HCDR3.

[0009] HCDR1 amino acid sequence: GFTFSTTY (SEQ ID No. 1)

[0010] HCDR2 amino acid sequence: ISTKGGTT (SEQ ID No. 2)

[0011] HCDR3 amino acid sequence: GRTEWSADWTGCGSGYCLGKRSG (SEQ ID No. 3);

[0012] The light chain variable region includes three light chain complementarity determination regions LCDR1, LCDR2, and LCDR3;

[0013] LCDR1 amino acid sequence: SGSVTSSNV (SEQ ID No. 4)

[0014] LCDR2 amino acid sequence: STN

[0015] LCDR3 amino acid sequence: ALYKIGGNP (SEQ ID No. 5).

[0016] The amino acid sequence of the variable region of the heavy chain of the antibody pOTB-1 is: EEKLVESGGGLVQPGGSLRLSCVGSGFTFSTTYITWLRQAPGKGLEWLASISTKGGTTDYADSVRGRFTISRDNSQNTASLQMNSLRTEDTARYYCGRTEWSADWTGCGSGYCLGKRSGWGPGVEVVV (SEQ ID No. 6)

[0017] The amino acid sequence of the variable region of the light chain of the antibody pOTB-1 is: QTVIQEPAMSVSPGGTVTLTCAFSSGSVTSSNVPSWYQQTPGQPPRQLIYSTNSRPTGVPSRFSGVISGNKAALTITGAQAEDEADYFCALYKIGGNPFGGGTHLTVLGQPKAAPTVNLFPPSSEELGTNK (SEQ ID No. 7).

[0018] This invention also provides the use of monoclonal antibody pOTB-1 in the preparation of drugs for the prevention or treatment of foot-and-mouth disease, and the use of monoclonal antibody pOTB-1 in the preparation of competitive ELISA detection reagents or competitive ELISA detection kits for foot-and-mouth disease virus.

[0019] This invention successfully constructed a broad-spectrum monoclonal antibody, pOTB-1, that recognizes a linear antigenic epitope at the C-terminus of the VP2 protein of foot-and-mouth disease virus (FMDV). The antibody's biological activity was verified by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blot. This antibody exhibited broad-spectrum reactivity against both type O and type A FMDV, with strong binding affinity. Through truncation, mutation, and complementation techniques, the antibody's recognition of a continuous linear B-cell epitope located at the C-terminus of the structural protein VP2 was determined, with the smallest recognition unit being VP2 217-218aa. This epitope sequence is highly conserved among FMDV types O, A, and Asia1, indicating the existence of a type-universal antigenic site in the capsid protein VP2. A competitive ELISA detection method was developed based on biotinylated pOTB-1 and another cross-reactive monoclonal antibody, E32, providing a tool for FMDV prevention and serological detection, and laying the foundation for the design and efficacy evaluation of FMDV-labeled vaccines. Attached Figure Description

[0020] Figure 1 Flow cytometry sorting of antigen-specific B cells;

[0021] Figure 2 O / PanAsia / Xizang99 specific antibody library sequence subtype classification;

[0022] Figure 3 Antibody structure and amino acid sequence of the CDR region of pOTB-1 V region;

[0023] Figure 4 SDS-PAGE detection of antibody expression products;

[0024] Figure 5 IFA assay to detect antibody reactivity to FMDV;

[0025] Figure 6 Indirect ELISA detection of the reactivity of pOTB-1 with type O antigen;

[0026] Figure 7 FMDV type O inactivated antigen identifies the type of antibody-recognized epitope;

[0027] Figure 8 Identification of GST fusion structural proteins by pOTB-1 recognition;

[0028] Figure 9 GST integrates VP2 truncated body verification scheme;

[0029] Figure 10 Identification of the pOTB-1-bound GST fusion VP2 structural protein truncated form;

[0030] Figure 11 Comparison of amino acid sequences of VP2, a structural protein of FMDV type O and type A representative strains;

[0031] Figure 12 Workflow of a competitive ELISA method based on pOTB-1;

[0032] Figure 13 Sensitivity testing of competing ELISA methods;

[0033] Figure 14 Specificity testing of competing ELISA methods;

[0034] Figure 15 Competitive ELISA assays for detecting FMDV in immunized pig serum; animal serum assays immunized with antigen A. O / Mya98 / HNNY / 2022; animal serum assays immunized with antigen BO / PanAsia / Xizang / 99. Detailed Implementation

[0035] The present invention will be further described below through specific embodiments.

[0036] BHK-21 cells, FMDV type O (O / PanAsia / Xizang99, O / Mya98 / HNNY-2, O / Mya98 / BY / 2010, O / Cathay / 18074, O / ZK) strains, and FMDV type A (A / WH / 09, A / HNXX, A / F72) strains were all preserved by the National Foot-and-Mouth Disease Reference Laboratory.

[0037] FMDV type O inactivated antigen (O / PanAsia / Xizang99) was kindly provided by Zhongnong Weite Biotechnology Co., Ltd. The biotin-labeled kit EZ-Link™ Sulfo-NHS-LC-Biotin was purchased from Thermo Fisher Scientific; Histopaque (R)-1.077 sterile-filtered lymphocyte separation medium (density: 1.077 g / mL) was purchased from Sigma-Aldrich; APC fluorescently labeled anti-biotin antibody (Anti-biotin-APC) was purchased from Miltenyi Biotec; the endotoxin-free plasmid extraction kit was purchased from TIGEN; FITC-labeled goat anti-pig IgG antibody was purchased from Sigma-Aldrich; and horseradish peroxidase (HRP)-labeled goat anti-pig antibody was purchased from Bioss.

[0038] Example 1: Preparation of porcine monoclonal antibody pOTB-1

[0039] 1. Animal immunization and isolation of PBMCs

[0040] Healthy 2-month-old three-way crossbred pigs were intramuscularly injected with an inactivated FMDV type O Xizang99 strain vaccine, immunized three times at 28-day intervals. The vaccine was prepared using Montanide ISA201 adjuvant and O / PanAsia / Xizang99 antigen 146S. Four days after the last immunization, EDTA-anticoagulated blood was collected from the anterior vena cava of the pigs, and peripheral blood mononuclear cells (PBMCs) were isolated using HISTOPAQUE 1.077 according to the manufacturer's instructions. The isolation procedure was as follows: Lymphocyte separation medium and PBS were equilibrated to room temperature before use. 6 mL of lymphocyte separation medium was added to a 15 mL centrifuge tube, and 6 mL of anticoagulated blood diluted 1:1 with PBS was slowly added to the top layer of the lymphocyte separation medium. The mixture was centrifuged at 1200×g for 30 min at 4°C. After centrifugation, the cells showed clear stratification based on density. The white, cloudy layer of PBMCs was aspirated and added to a 15 mL centrifuge tube containing 1 / 2 volume of cell sorting medium. The cells were washed by centrifugation at 600×g for 5 min. Discard the supernatant, add 1 mL of erythrocyte lysis buffer, lyse at room temperature for 2 min, then add cell sorting buffer and centrifuge at 300×g for 10 min. Discard the supernatant and wash the cells twice with cell sorting buffer. Resuspend the cells in cell sorting buffer to obtain PBMCs, which are then counted using the GE Cytell Cell Imaging System.

[0041] Flow cytometry sorting of antigen-specific B cells

[0042] To obtain FMDV antigen-specific antibody-secreting B cells, biotin-labeled antigen 146S was used as a decoy for flow cytometry sorting.

[0043] Biotin is a small, bioactive molecule with a molecular weight of 224 Da. It can bind to proteins without altering their biological activity; therefore, biotin was chosen as the labeled protein for preparing the bait antigen. Following the manufacturer's instructions, high-purity FMDV 146S antigen was biotinylated using EZ-Link NHSLC-Biotin reagent. The resulting Biotin-FMDV 146S was used as the bait antigen to screen for antigen-specific B cells. Approximately 10... 7 PBMCs were resuspended in 200 μL of cell sorting medium, and Biotin-FMDV 146S was added. The mixture was incubated at 4°C for 30 min, followed by centrifugation at 500 x g for 5 min and washing once. Then, 1 μL of mouse avidin (APC) was incubated at 4°C for 30 min, followed by another washing of the cells. The cells were resuspended in 500 μL of cell sorting medium and stored on ice in the dark, ready for analysis. Additionally, a set of sample tubes containing the same cells but without bait antigen was prepared as a fluorescence-minus-one (FMO) control. Appropriate phyla were delineated in the FMO control sample for FACS sorting of type O antigen-specific B cells.

[0044] FACS statistics showed that 730,000 lymphocytes were identified from 1 million PBMCs, of which 700,000 lymphocytes were gated as monocytes. Figure 1 FMDV antigen-specific B cells are few in number in porcine peripheral blood, accounting for approximately 0.028% of porcine PBMCs. Therefore, 10⁷ PBMCs were collected to obtain 2 × 10⁴ FMDV-specific antibody-secreting B cells.

[0045] Establishment of a specific BCR library

[0046] The sorted FMDV O / PanAsia / Xizang99 antigen-specific B cells were sent to the company for single-cell transcriptome and immunomic sequencing using the 10X Genomics Chromium system. First, single cells were rapidly encapsulated in gel bead emulsion (GEM) droplets, seeding specific DNA fragments, including barcodes, UMIs, and PolyTs, onto the gel beads. Barcodes distinguish different gel beads, UMIs distinguish raw cDNA molecules, and PolyTs capture mRNA. Each cell was individually mixed with a gel bead to form a water-in-oil droplet. After the cell membrane ruptured, the mRNA was released and came into contact with reverse transcriptase, nucleic acid primers on the gel beads, and dNTP substrates, undergoing a reverse transcription reaction to generate cDNA. The tagged cDNA molecules were then extracted, adapters were added, and PCR amplification was performed to prepare a sequencing library suitable for the Illumina sequencing platform for high-throughput sequencing. The raw sequencing data were processed using Cell Ranger software provided by 10X Genomics to obtain gene expression profiles and differential analysis at the single-cell level.

[0047] FMDV-specific antibody-secreting B cells collected via FACS were sent to 10X Genomice for single-cell 5' BCR immunohistometry sequencing. The obtained full-length cDNA was fragmented by enzyme digestion, end repaired, A adapters were added, and reads sequencing primers were ligated before antibody BCR sequence classification and pairing. The results are shown in Table 1, with 8870 antibody sequences obtained from the O / PanAsia / Xizang99 group. Figure 2 A. The heavy chain is mainly composed of the IgG subtype (3046 / 4326), while the IgE subtype BCR sequence, mainly derived from plasma cells secreted by the submucosal cells of the respiratory and digestive tracts, accounts for only 0.05% of all heavy chain BCR sequences. A total of 4544 light chain sequences were detected. Figure 2 B), of which the Kappa subtype accounts for 41% (1849 / 4544), and the Lambda subtype accounts for the majority of the light chain types (2695 / 4544).

[0048]

[0049] The classified BCR sequences were uploaded to the IMGT website (https: / / www.imgt.org / HighV-QUEST / home.action) for analysis of immune receptor gene rearrangements, including identification and alignment of immune receptor genes, VDJ rearrangement analysis, mutation hotspot analysis, and functional annotation. Further pairing of the heavy and light chain sequences of the BCRs (Table 2) yielded a specific antibody library of the FMDV type O Xizang99 strain with a capacity of 4611 sequences. Figure 2 As shown in C, IgG, which plays a central role in the body's immune response and immune defense, accounts for 71% of the paired BCRs. IgD and IgA account for 727 and 621 pairs, respectively, representing 16% and 13% of the entire library.

[0050]

[0051] The variable region amino acid sequence of the porcine monoclonal antibody pOTB-1

[0052] The variable region (V region) of an antibody is the segment within the antibody molecule responsible for recognizing and binding antigens. Antibodies consist of two heavy chains and two light chains, each with a V region at its N-terminus, crucial for recognizing foreign substances. Each V region contains three highly variable regions called complementarity-determining regions (CDRs), with CDR3 exhibiting the highest degree of variability and playing a decisive role in antibody specificity. In addition to these three hypervariable regions, the V region also contains relatively conserved framework regions (FRs), which show less variation among different antibodies, providing structural support for the hypervariable regions. Different combinations of V region CDRs and FRs produce a rich diversity of antibodies; this diversity forms the basis for the immune system's recognition and defense against a wide range of pathogens. By uploading the FMDV-specific IgG antibody sequences obtained through sequencing to the International Immunogenomics Database (IMGT) website (https: / / www.imgt.org / HighV-QUEST / ), the sequencing data was annotated in detail, including information on gene rearrangements and variations.

[0053] Among the five types of Ig, IgG is considered the most important due to its high concentration, wide distribution, long half-life, diverse functions, and crucial role in immune memory. Therefore, the obtained IgG antibody sequence was selected for expression and bioactivity validation. The V region is the part of the antibody molecule responsible for recognizing and binding antigens. It consists of a heavy chain variable region (VH) and a light chain variable region (VL), linked by disulfide bonds to form a unique three-dimensional structure, creating an antigen-binding site and providing broad-spectrum immune protection. The porcine monoclonal antibody screened for expression validation was named pOTB-1, and Table 3 lists the amino acid sequence of the pOTB-1 antibody variable region (V region).

[0054]

[0055] The three complementarity-determining regions (CDR1, CDR2, and CDR3) in region V are highly variable, especially CDR3, which directly participates in the interaction with the antigen, ensuring highly specific binding between the antibody and the antigen. The framework region (FR) of region V is relatively conserved and participates in stabilizing antibody-antigen binding. The variable regions of region V are not only the sites where antibodies recognize antigens, but also key to antibody diversity and specificity. Figure 3 In the pOTB-1 antibody, the hypervariable region HCDR1 of the heavy chain (VH) has the sequence (GFTFSTTY), HCDR2 (ISTKGGTT), and HCDR3, which plays the most important role in antigen binding, is 23 amino acids long and has the sequence (GRTEWSADWTGCGSGYCLGKRSG). The hypervariable region LCDR1 of the light chain (VL) has the amino acid sequence (SGSVTSSNV), LCDR2 (STN), and LCDR3 (ALYKIGGNP).

[0056] Construction, expression, and purification of antibody gene expression vectors

[0057] The codons of the antibody heavy chain variable region (VH) gene were optimized and inserted into the CH-pcDNA3.4 vector containing the porcine IgG heavy chain framework region. Similarly, the antibody light chain variable region (VL) was also codon optimized and inserted into the CL-pcDNA3.4 vector containing the porcine IgG light chain constant region. A 6×His tag was added to the antibody constant region. Sequence synthesis and vector construction were commissioned to Genewiz Biotechnology Co., Ltd. The recombinant expression plasmid was transformed into DH5α competent cells to amplify the plasmid, and the plasmid was extracted using an endotoxin-free plasmid extraction kit according to the kit instructions.

[0058] The antibody heavy chain and light chain plasmids were co-transfected into 293f cells in suspension culture for expression. The heavy chain and light chain plasmids were diluted in D-PBS at a ratio of 2:3 and mixed thoroughly. PEI 40000MW was used as the transfection reagent, and the PEI reagent was mixed with the total plasmid DNA at a ratio of 3:1 to prepare the PEI-DNA transfection mixture. After the transfection complex was incubated at room temperature for 10 min, the volume was slowly increased to 2.0 × 10⁻⁶ cells / mL. 6 Transfected cells were cultured in 293f cells at 37°C in a constant temperature suspension incubator. After 48 hours, 293f SUP feed was added, and expression samples were collected after 7-8 days of culture. The cells were centrifuged at 10,000 rpm for 30 minutes at 4°C, and the culture supernatant was filtered through a 0.22 μm filter for antibody purification. A pre-packed column packed with Ni-NTA 6FF packing material was used for purification. After eluting the target protein with high-concentration imidazole, the cells were incubated overnight at 4°C and then dialyzed against PBS buffer, changing the dialysate 4-6 times to remove imidazole from the protein. The dialyzed antibody was embedded in PEG 8000 for concentration and then identified by SDS-PAGE electrophoresis.

[0059] The obtained IgG variable region sequence was cloned into the pcDNA3.4 vector containing the fused antibody framework region. VH and VL plasmids were extracted and purified, and co-transfected into 293f suspension cells for eukaryotic expression. The expression supernatant was purified using a Ni-NTA pre-packed column and identified by SDS-PAGE electrophoresis. The results are as follows: Figure 4 As shown, the whole-porcine IgG antibody molecule was successfully expressed. The antibody VH chain size was approximately 55 kDa, and the VL chain size was approximately 25 kDa. The bands were clear and consistent with the expected size, with no impurities.

[0060] Example 2: Validation of Antibody Bioactivity

[0061] 1. Indirect immunofluorescence (IFA) detection of antibody reactivity

[0062] BHK-21 cells were inoculated into 24-well plates. When the cells reached 80%-90% confluence, representative strains of FMDV type O and A were inoculated into the cells, with normal cell controls provided. After infection in a 37°C cell culture incubator for 4 h, the supernatant was collected for inactivation. Cells were washed three times with PBS, fixed with 4% paraformaldehyde pre-chilled at -20°C, and incubated at room temperature for 20 min. Cells were then penetrated through the cell membrane with 0.1% Triton for 10 min, blocked with 1% BSA, and then analyzed. Cells were washed three times with PBS, and a 5 μg / mL diluted antibody was added, followed by incubation at 37°C for 1 h. After washing three times with PBS, FITC-labeled anti-pig IgG antibody at the working concentration as indicated in the manufacturer's instructions was added, and the cells were incubated at 37°C for 1 h. After washing five times with PBS, the fluorescence signal was observed under a fluorescence microscope and photographed.

[0063] Cells were infected with antigens from various lineages of FMDV type O and type A, incubated with pOTB-1 (5 μg / mL) at 4℃, and then labeled with anti-pig FITC fluorescent secondary antibody for observation. Figure 5 The results showed that after BHK-21 cells were infected with seven experimental strains of FMDV (type O and type A), pOTB-1 could specifically bind to the viral antigens in the cells, exhibiting specific green fluorescence and demonstrating broad-spectrum reactivity to FMDV OA.

[0064] Indirect ELISA detection of antibody binding affinity

[0065] The affinity of antibody binding was detected using inactivated FMDVO / PanAsia purified antigen as the coating antigen. The specific operation steps are as follows: The concentrated purified antigen was diluted to 1 μg / mL with carbonate buffer, and 100 μL was added to each well of the ELISA plate. Coating was performed overnight at 4°C for 16 h. The plate was washed 5 times with PBST, and 100 μL of blocking buffer was added to each well. After incubation at 37°C for 2 h, the plate was washed 5 times with PBST, and the liquid in the wells was patted dry. The antibody to be tested was serially diluted 2-fold starting from 20 μg / mL, and 100 μL was added to each well of the ELISA plate. Incubation was performed at 37°C for 1 h; a PBS negative control group was set up. The plate was washed 5 times with PBST, and 100 μL of HRP-labeled anti-pig antibody was added to each well. Incubation was performed at 37°C for 30 min. The plate was washed 5 times with PBST, and 100 μL of TMB chromogenic solution was added to each well. Incubation was performed at 37°C for 15 min. The color development was stopped by adding stop solution, and the absorbance at 450 nm was measured using an ELISA reader. The S / CO method is used to calculate the statistical results. S is the sample OD value, and CO is the Cutoff value (positive cutoff value). CO = 2.1 × N (N is the negative control OD value).

[0066] ELISA plates were coated with FMDV type O and type A purified antigens to detect antibody binding affinity. The antibody concentration corresponding to the cut-off value was used as the cut-off value, divided into five intervals: 0-0.05 μg / mL, 0.05-0.1 μg / mL, 0.1-1 μg / mL, 1-5 μg / mL, and >5 μg / mL. The reaction intensity within these intervals was labeled ++++, +++, ++, +, and -, respectively. Based on these criteria, the critical concentration for pOTB-1 reaction with type O antigen was 0.0195 μg / mL, with a reaction intensity of ++++, while the critical concentration for reaction with type A antigen was approximately 0.0976 μg / mL, with a reaction intensity of +++. Figure 6 ).

[0067] In summary, by sorting antigen-specific B cells from FMDV-immunized pigs, a total of 4611 specific BCR sequences were obtained through sequencing, including 3261 IgG sequences. Antibody characterization (SHM, Frequency, Clonotype) analysis revealed the successful screening and expression of the monoclonal antibody pOTB-1. This antibody was validated to bind to both FMDV type O and type A antigens with strong affinity. Monoclonal antibodies, capable of recognizing and binding to specific antigens, are of significant importance for disease diagnosis and treatment, drug development, and immunotherapy research.

[0068] Example 3: Identification of pOTB-1-recognized antigenic epitopes

[0069] 1. Identification of the type of antigenic epitope recognized by pOTB-1

[0070] An antigenic epitope is a specific region on an antigen molecule that an antibody recognizes. They are classified into linear epitopes and conformational epitopes. A linear epitope consists of a continuous amino acid chain, while a conformational epitope is formed by the three-dimensional folding of a protein on the antigen. Identification of linear epitopes is particularly important in linear protein sequence analysis and synthetic vaccine design. To determine the epitope type recognized by pOTB-1, purified antigen 146S from inactivated FMDV was used. 4× Loading Buffer was added, and the sample was boiled at 100°C for 10 min to prepare a linearized antigen protein sample for Western blot analysis. After separating the bands by SDS-PAGE electrophoresis, the protein bands were transferred to a PVDF membrane. The membrane was blocked with TBST buffer containing 5% skim milk for 2 h. After washing, the antibody was diluted to the working concentration (2 μg / mL) with TBST buffer containing 5% skim milk, incubated at room temperature for 4 h, and then washed five times with TBST for 5 min each time. HRP-labeled anti-pig antibody was added, diluted 1:5000, and incubated at room temperature for 1 h. After washing the membrane with TBST, ECL chemiluminescent substrate was added and then exposed for imaging.

[0071] To further determine which structural protein the epitope recognized by pOTB-1 is located in, a Western blot experiment was conducted using GST fusion proteins of truncated FMDV type O structural proteins VP1, VP2, and VP3 expressed in E. coli competent cells.

[0072] To identify whether the epitope recognized by the antibody is a linear epitope or a conformational epitope, Western blot analysis was performed using FMDV type O inactivated antigen. The results showed that pOTB-1 could react with the inactivated antigen, and a specific band appeared at approximately 23 kDa. Figure 7This indicates that pOTB-1 recognizes a linear epitope. According to literature references, during electrophoresis of the FMDV denatured protein, the structural proteins VP1 are approximately 32 kDa, VP2 is approximately 28 kDa, and VP3 is approximately 27 kDa. Figure 7 The approximate location of the stripe is shown, suggesting that pOTB-1 may have identified a location at VP2 or VP3.

[0073] To pinpoint the exact location of the pOTB-1 anchoring epitope, Western blotting was performed on the VP1, VP2, and VP3 proteins of the O / 18074 strain fused with the GST tag, as well as the GH loop of VP1 and the C-terminal protein of VP1. Figure 8 As shown, the epitope recognized by pOTB-1 is located in the VP2 protein.

[0074] Key amino acids for linear epitope recognition

[0075] To determine the specific location of the pOTB-1-recognized epitope in the VP2 protein, a series of truncation protocols were designed to progressively shorten the amino acid length. The truncated protein was fused with a GST tag, expressed in prokaryotes, and then subjected to Western blotting. The specific experimental procedures were the same as those for identifying the type of epitope recognized by pOTB-1.

[0076] To determine the specific epitope recognized by pOTB-1, site-directed mutagenesis was used to mutate the identified epitope amino acid to alanine (A), which has a minimal impact on protein structure. A stepwise validation process involving truncation of the VP2 protein was designed, as follows: Figure 9 .

[0077] A series of GST-VP2 truncated proteins were analyzed to determine the epitopes recognized by the pOTB-1 antibody. Western blot results showed that after truncating the FMDV VP2 protein into four segments, pOTB-1 could react with 180-218 amino acids of the VP2 protein. Figure 10 A), therefore, this segment was further truncated at that location. Subsequently, after truncating both the N and C ends of VP2180-218aa, a Western blot (WB) was performed, revealing that the epitope identified by pOTB-1 was located at the C end ( Figure 10 B). Based on this, gradually reduce the C-terminal amino acids of VP2. When it is shortened to VP2 213-218 aa, as follows... Figure 10 As shown in Figure C, pOTB-1 can still recognize only 6 amino acids, presenting a specific band. The final results show that the pOTB-1 antibody reacts with the C-terminus of FMDV VP2, with the smallest recognition unit being the C-terminal 217-218 amino acids (…). Figure 10 D).

[0078] Comparison of amino acid sequences of VP2 from representative strains of different serotypes

[0079] The VP2 sequences of nine representative strains of FMDV serotypes O, A, and Asia1 were selected. The VP2 sequences of each serotype representative strain were aligned using the bioinformatics software MEGA to identify variant sites and conserved regions. The sequence alignment results were imported into the ESPript 3.0 website, and the results were output and saved.

[0080] After comparing the amino acid sequences of the VP2 structural protein of representative FMDV type O and type A strains, it was found that ( Figure 11 The C-terminal amino acid sequence 215-218 of the VP2 protein is conserved across different serotypes (215 PSKE 218). These conserved amino acid residues may play a crucial role in antigen-antibody interactions. To anchor the key amino acid site for pOTB-1 binding, amino acids 217 and 218 of VP2 were mutated. Additionally, an alanine residue was added at the end of position 218 to verify whether amino acid 218 must be in a free state for successful binding. The specific mutation scheme is as follows: Figure 9 Western blot analysis of the truncated mutant revealed that the antibody failed to bind when the E at position 218 of VP2 was mutated to A or when an A was added to the end; pOTB-1 could still bind when the K at position 217 of VP2 was mutated to a simple amino acid A or G, but the antibody immediately lost its reactivity when the K at position 217 was mutated to an acidic amino acid D. Figure 10 E).

[0081] In summary, pOTB-1 recognizes a linear conserved epitope at the C-terminus of FMDV VP2, with the epitope sequence 215PSKE218. The key amino acid sites of the smallest recognition unit are located at position 217K and position 218E at the C-terminus of VP2. Epitope identification is of great significance for vaccine design, disease diagnosis, and basic immunological research. Based on the key immunogenic regions on FMDV identified by pOTB-1, it can be used to design FMDV-labeled vaccines and develop matching ELISA diagnostic reagents for FMDV purification.

[0082] Example 4: Establishment and application of a competitive ELISA antibody detection method for foot-and-mouth disease virus

[0083] 1. Establishment of a competitive ELISA method

[0084] High-purity pOTB-1 was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo Fisher Scientific, USA), and the resulting biotinylated mAb pOTB-1 was named Bio-pOTB-1. Purified broad-spectrum non-neutralizing monoclonal antibody E32 was used to coat ELISA plates, with concentrations of 0.25, 0.5, and 1 μg / mL. After capturing purified FMDV O, A, and Asia1 antigens, the optimal Bio-pOTB-1 concentration for each antigen was determined using an indirect ELISA assay. OD was selected. 450 For Bio-pOTB-1 concentrations with readings around 2.0, subsequent experimental conditions were optimized to determine the optimal reaction conditions. Optical density (OD) readings were measured at 450 nm using an automated microplate reader (BioTek), and the inhibition rate of the test samples was calculated as (control value - measured value / control value - blank value × 100%).

[0085] The ELISA plate was coated with 0.5 μg / mL E32 antibody (broad-spectrum non-neutralizing mAb) at 4°C and incubated for 14 h. The plate was washed three times with PBST (PBS containing 0.1% Tween), and then 1 μg / mL 146S antigen diluted in 100 μL PBS was captured at room temperature for 2 h, followed by three washes with PBST. Subsequently, the ELISA plate was blocked with blocking buffer (PBS solution of 5% sucrose and 1% BSA) at 37°C for 1 h, followed by three washes with PBST. Serum samples were serially diluted in 50 μL PBS (from 1:4 to 1:512) and incubated at 37°C for 1 h with an equal volume of pre-titrated dose (0.5 μg / mL) of Bio-pOTB-1 PBS solution. After five washes with PBST, 100 μL of HRP-conjugated streptavidin (33 ng / mL) was added, and the plate was incubated at 37°C for 15 min. After washing five times with PBST, 100 μL of tetramethylbenzidine (TMB) was added to each well for color development for 15 min. The color development reaction was terminated with 100 μL of 2MH2SO4. Figure 12 The workflow of this method is outlined in the document.

[0086] Sensitivity testing of competing ELISA methods

[0087] FMDV-positive sera were serially diluted 2-fold starting at a ratio of 1:4, resulting in eight dilutions: 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512. Two replicate wells were set up for each positive serum sample, with a negative control provided by PBS. After repeated assays, the highest dilution that yielded a detectable positive result (>50% inhibition rate) was defined as the sensitivity of this method.

[0088] To improve the sensitivity of the detection method, animal serum (n=6) collected 3 days after booster immunization with FMDV type O antigen was selected. Serum with cross-neutralization titers >1:45 was considered FMDV antibody-positive. The established competitive ELISA method was used for detection, and the results were as follows (…). Figure 13 The results showed that the maximum serum dilution was 1:128 when the inhibition rate was higher than 50%.

[0089] Specificity testing of competing ELISA methods

[0090] Using an established competitive ELISA method, positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine parvovirus (PCV) were detected. Two replicate wells were set up for each serum sample. Positive sera for FMDV were also detected as controls to determine the specificity of the method.

[0091] The cross-reactivity of pOTB-1 with other viral antigen-immunized sera was assessed using sera from pigs immunized with PRRSV (n=10), CSFV (n=10), PRV (n=10), and PCV (n=10). Figure 14 As shown, none of the competitive ELISA tests on the tested sera showed an inhibition rate higher than 50% against this FMDV epitope, indicating no cross-reactivity with other viral immune sera.

[0092] Detection of field serum using competing ELISA methods

[0093] To verify the practical application effect and practicality of this method, serum from pigs immunized with inactivated vaccines containing different topological types (ME-SA and SEA) of FMDV O strains was selected. Serum samples were collected at 10 time points from before immunization to after booster immunization for testing. Two replicate wells were set for each sample to evaluate the stability and reproducibility of the competitive ELISA method based on pOTB-1 in practical applications.

[0094] After labeling pOTB-1 antibody with biotin, the abundance of antibodies against the VP2C-terminal epitope in FMDV-immunized pig serum was detected by competitive ELISA. Figure 15Two groups of animals, one immunized with the O / PanAsia lineage and the other with the O / Mya98 lineage, were selected for the experiment, with serum samples taken from each group at 10 time points. The results showed that from 21-28 days after the initial immunization, serum antibodies in each group exhibited an inhibition rate higher than 50%. The inhibition rate gradually increased after booster immunization, reaching 90%-100% by day 5 after the booster immunization. This demonstrates that pigs immunized with the O-type FMDV vaccine can recognize the VP2 C-terminal antigenic epitope and produce specific antibodies. It also verifies that this method has certain effectiveness and applicability, meeting the needs of practical applications.

[0095] In summary, a competitive ELISA method for monitoring FMDV antibodies was successfully established based on biotinylated pOTB-1. The method showed no reaction with other pathogens such as CSFV, PRRSV, PCV, and PRV after specificity testing. The sensitivity was 1:128, capable of detecting low concentrations of antibodies. Field trials, starting 21-28 days after initial immunization, showed serum antibody inhibition rates exceeding 50%. This method demonstrates practical application value, particularly in early disease diagnosis, disease monitoring, and biomarker research, while ensuring accuracy and reliability. The conserved antigenic epitope at the C-terminus of VP2 was identified, which can be eliminated through genetic engineering techniques, potentially for use in the design of labeled vaccines and the establishment of corresponding ELISA detection methods. This could differentiate between vaccine-immunized and naturally infected animals, promoting the clearance and eradication of FMDV.

Claims

1. A broad-spectrum monoclonal antibody, pOTB-1, that recognizes the C-terminal linear antigenic epitope of the foot-and-mouth disease virus VP2 protein, characterized in that: The antibody pOTB-1 includes a heavy chain variable region and a light chain variable region; The heavy chain variable region includes three heavy chain complementarity determination regions: HCDR1, HCDR2, and HCDR3. HCDR1 amino acid sequence: GFTFSTTY HCDR2 amino acid sequence: ISTKGGTT HCDR3 amino acid sequence: GRTEWSADWTGCGSGYCLGKRSG; The light chain variable region includes three light chain complementarity determination regions LCDR1, LCDR2, and LCDR3; LCDR1 amino acid sequence: SGSVTSSNV LCDR2 amino acid sequence: STN LCDR3 amino acid sequence: ALYKIGGNP.

2. The broad-spectrum monoclonal antibody pOTB-1 that recognizes the C-terminal linear antigenic epitope of the foot-and-mouth disease virus VP2 protein according to claim 1, characterized in that: The amino acid sequence of the variable region of the pOTB-1 heavy chain antibody: EEKLVESGGGLVQPGGSLRLSCVGSGFTFSTTYITWLRQAPGKGLEWLASISTKGGTTDYADSVRGRFTISRDNSQNTASLQMNSLRTEDTARYYCGRTEWSADWTGCGSGYCLGKRSGWGPGVEVVV; The amino acid sequence of the variable region of the antibody pOTB-1 light chain: QTVIQEPAMSVSPGGTVTLTCAFSSGSVTSSNVPSWYQQTPGQPPRQLIYSTNSRPTGVPSRFSGVISGNKAALTITGAQAEDEADYFCALYKIGGNPFGGGTHLTVLGQPKAAPTVNLFPPSSEELGTNK.

3. The use of the broad-spectrum monoclonal antibody pOTB-1 according to claim 1 or 2 in the preparation of a competitive ELISA detection reagent or kit for type A or type O foot-and-mouth disease virus.

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