Monoclonal antibody for resisting porcine epidemic diarrhea virus and application thereof

Abstract

The invention discloses an anti-porcine epidemic diarrhea virus monoclonal antibody and application thereof. The anti-porcine epidemic diarrhea virus monoclonal antibody comprises a monoclonal antibody 2D14 or an antigen binding fragment thereof, and a monoclonal antibody 8F17 or an antigen binding fragment thereof. The two monoclonal antibodies can be combined with PEDV fusion protein with high affinity, have no cross reaction with other common porcine enterovirus antigens, are good in specificity, can construct a stable double-antibody sandwich immunochromatography system, are simple and convenient to operate, high in detection sensitivity, low in background and rapid in reaction, can develop color within 10-15 minutes, and can be applied to detection of porcine enteroviruses. Clear interpretable signals can be generated for a plurality of subtype samples of PEDV, the result can be interpreted by naked eyes, the detection result is stable, the repeatability is good, and the kit is suitable for on-site rapid detection and clinical application.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to a monoclonal antibody against porcine epidemic diarrhea virus and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a single-stranded positive-sense RNA virus belonging to the family Coronaviridae and the genus Alphacoronavirus. It is a major intestinal pathogen that causes acute diarrhea, vomiting, and dehydration in pigs, and leads to high mortality in suckling piglets.

[0003] The spike protein (S protein) on the viral membrane is a key structural and immune target of PEDV, primarily responsible for binding to host cell receptors (S1 subunit) and mediating viral-cell membrane fusion (S2 subunit). The S protein contains multiple neutralizing antibody sites and fusion-related functional regions, such as the fusion peptide and two heptad repeat regions (HR1, HR2). Recent cryo-electron microscopy revealed significant variations in the prefusion conformation of the PEDV S protein across different subtypes. These variations may lead to immune escape or reduce the sensitivity and specificity of existing neutralizing antibody / antigen detection methods.

[0004] In the detection and control of PEDV, vaccines, antibody ELISA, or antibody / nucleic acid combinations are currently widely used. Although some recombinant S protein fusion proteins have been used for neutralizing antibody induction, serum detection, or competitive ELISA, some problems and challenges remain. On the one hand, PEDV genotypes and subtypes are highly diverse, and the S protein, especially the S1 subunit, frequently undergoes mutations, insertions, or deletions, leading to significant antigenic differences between different strains and reduced recognition ability of existing antibodies for some variant strains. On the other hand, the current ELISA, colloidal gold immunochromatography, and competitive immunoassay methods exhibit significant differences in detection sensitivity and specificity among different strains and clinical samples, with issues such as high detection limits and insufficient quantitative stability. Furthermore, while the S2 subunit and its fusion peptide, as well as the helical repeat regions (HR1, HR2) of the S protein that mediate viral fusion with the host membrane, are highly conserved across different subtypes, they have not yet been fully utilized for antigen design and antibody preparation.

[0005] Therefore, there is an urgent market and technological need for a novel, highly specific monoclonal antibody that recognizes the fusion domain or its conserved structural domain of fusion proteins. This antibody should not only have good binding ability and neutralization potential to recombinant fusion proteins in vitro, but also maintain a low limit of detection (LOD) for multiple PEDV subtypes in colloidal gold rapid detection, and require high sensitivity and specificity. At the same time, its preparation process, sequence analysis, and epitope conservation must be fully validated to ensure its innovation and practicality.

[0006] The nucleocapsid (N) protein of porcine epidemic diarrhea virus (PEDV) is one of the key structural proteins encoded by the viral genome, playing multiple core roles in the viral life cycle and pathogenic mechanism. As a member of the coronavirus family, PEDV mainly causes acute intestinal infectious diseases in pigs, with particularly high mortality rates in newborn piglets, leading to severe dehydration, diarrhea, and other symptoms, causing significant economic losses to the global pig industry. The N protein, as the most abundant protein in the viral particle, is a core component of viral structural assembly and functional regulation.

[0007] The N protein plays a systemic regulatory role in the viral life cycle and host interaction, extending beyond its structural function. The PEDV N protein is a multifunctional protein whose core functions include encapsulating the viral single-stranded positive-sense RNA genome to form a stable helical nucleocapsid structure, providing the structural basis for viral replication and assembly. Simultaneously, the N protein participates in the formation of the viral replication complex, regulates viral genome transcription and translation, and interferes with host immune signaling pathways through interactions with host cell proteins, facilitating viral evasion of host defenses. Furthermore, the PEDV-N protein exhibits strong immunogenicity and conservation, stimulating the body to produce specific antibodies. It is an important target for antigen detection in clinical diagnostics (such as ELISA and colloidal gold assays) and a key candidate antigen for the development of novel vaccines (such as subunit vaccines and nucleic acid vaccines). In addition, epitopes on the N protein may trigger cell-mediated immunity. Therefore, the PEDV N protein has significant application value in diagnostic and immunological research. However, current research on the epitopes of the PEDV N protein is limited.

[0008] Focusing on the molecular mechanism of the N protein from macroscopic PEDV pathogenicity research is not only key to solving the failure of clinical testing, but also a breakthrough in revealing the fatal intestinal immunopathological damage induced by virulent strains in piglets. Summary of the Invention

[0009] To address at least one of the aforementioned problems, this invention provides a specific monoclonal antibody against porcine epidemic diarrhea virus (PEDV) and its uses. The antibody can recognize the conserved domains of PEDV fusion proteins and exhibits stable binding ability to different subtypes and variants, making it suitable for rapid detection systems using double-antibody sandwich colloidal gold immunochromatography.

[0010] To achieve the above objectives, the present invention employs the following technical means: A first aspect of the present invention provides a monoclonal antibody against porcine epidemic diarrhea virus, said monoclonal antibody being monoclonal antibody 2D14 or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising amino acid sequences such as CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 3-5; said light chain variable region comprising amino acid sequences such as CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 8-10.

[0011] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D14 or its antigen-binding fragment is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the monoclonal antibody 2D14 or its antigen-binding fragment is shown in SEQ ID NO: 7.

[0012] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D14 or its antigen-binding fragment is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D14 or its antigen-binding fragment is shown in SEQ ID NO: 6.

[0013] In some embodiments of the present invention, the monoclonal antibody is monoclonal antibody 8F17 or its antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises amino acid sequences such as CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 13-15; the light chain variable region comprises amino acid sequences such as CDR-L1, CDR-L2, and CDR-L3 as shown in SEQ ID NO: 18-20.

[0014] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 8F17 or its antigen-binding fragment is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of the monoclonal antibody 8F17 or its antigen-binding fragment is shown in SEQ ID NO: 17.

[0015] In some embodiments of the present invention, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8F17 or its antigen-binding fragment is shown in SEQ ID NO: 11, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8F17 or its antigen-binding fragment is shown in SEQ ID NO: 16.

[0016] A second aspect of the invention provides the use of monoclonal antibody 2D14 or its antigen-binding fragment, and monoclonal antibody 8F17 or its antigen-binding fragment, in the preparation of an immunochromatographic kit for detecting PEDV in biological samples.

[0017] The heavy chain constant region of monoclonal antibody 2D14 belongs to the IgG1 type, and the light chain constant region belongs to the K (kappa) type. This antibody can specifically bind to the conserved region of the PEDV N protein, and its epitope recognition is not affected by common mutations, exhibiting good cross-subtype recognition ability.

[0018] The heavy chain constant region of monoclonal antibody 8F17 is of type IgG2a, and the light chain constant region is of type K.

[0019] Pairing verification showed that monoclonal antibody 2D14 and monoclonal antibody 8F17 can recognize different epitopes on the fusion protein, and their binding sites are complementary and do not compete for binding, making them suitable for constructing a double-antibody sandwich detection system.

[0020] The "antigen-binding portion" as described in this invention refers to an antigen recognition fragment or analogue capable of retaining the binding properties of the parent antibody, typically containing one or more variable regions or CDR sequences. For example, the antigen-binding fragment may be selected from Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, and single-chain variable fragments (scFv).

[0021] The “Fab fragment” consists of a light chain and a heavy chain CH1 domain and their variable regions; The “Fab’ fragment” contains a light chain and a heavy chain containing VH and CH1 domains and some constant regions, which can form the F(ab’)2 structure through disulfide bonds; The “F(ab')2 segment” consists of two light chains and two heavy chains, which are connected by disulfide bonds between the two heavy chains to form a stable dimer. The “Fv segment” contains only the VH and VL variable regions; "Single-chain Fv (scFv)" is a soluble antigen-binding unit formed by linking VH and VL to the same polypeptide chain through a flexible peptide chain.

[0022] In this invention, monoclonal antibodies 2D14 and 8F17 are the names of hybridoma cell lines that have been screened with a 100% positive rate and stably secrete antibodies.

[0023] A third aspect of the present invention provides a colloidal gold immunochromatographic test kit, the test kit containing a colloidal gold immunochromatographic test strip prepared using the monoclonal antibody 2D14 or its antigen-binding fragment described above and / or the monoclonal antibody 8F17 or its antigen-binding fragment described above.

[0024] The structure of a colloidal gold immunochromatographic test strip includes a base plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane, and an absorbent pad.

[0025] In some embodiments of the present invention, the colloidal gold immunochromatographic test strip comprises a colloidal gold pad prepared by labeling colloidal gold with one of the antibodies, and a detection line prepared by immobilizing another antibody on a nitrocellulose membrane.

[0026] In some embodiments of the present invention, the particle size of the colloidal gold in the colloidal gold label is 40-45 nm.

[0027] In some embodiments of the present invention, antibody 2D14 or its antigen-binding portion is paired with antibody 8F17 or its antigen-binding portion: one antibody is prepared as a colloidal gold pad for detection by colloidal gold labeling; the other antibody is immobilized on a nitrocellulose membrane as a detection line (T line) coated antibody.

[0028] When the PEDV fusion protein is present in the sample, the antigen binds to the gold-labeled antibody to form a complex, which is captured by the T line and forms a red colored band; when the sample is negative, only the C line is colored.

[0029] Beneficial effects of the present invention The monoclonal antibodies 2D14 and 8F17 prepared in this invention can both bind to the PEDV fusion protein with high affinity and show no cross-reactivity with other common porcine enterovirus antigens, exhibiting good specificity. The epitopes bound by the two antibodies do not overlap, allowing for the construction of a stable double-antibody sandwich immunochromatographic system. This system is easy to operate, and the prepared test strips exhibit high sensitivity, low background, and rapid reaction (color development within 10-15 minutes). It produces clear and interpretable signals for multiple PEDV subtypes, with results readily interpretable by the naked eye. The detection results are stable and reproducible, making it suitable for rapid on-site testing and clinical applications.

[0030] This invention achieves broad-spectrum detection and rapid visualization of different PEDV subtypes by selecting conserved epitopes of porcine epidemic diarrhea virus (PEDV) fusion proteins as targets. This technology is significantly innovative in antibody screening, antigen structure analysis, and LFA signal amplification strategies, providing a new technical pathway and productization foundation for the field diagnosis and monitoring of animal-derived coronaviruses. Attached Figure Description

[0031] Figure 1The results of purity verification for porcine epidemic diarrhea virus (PEDV) antigen recombinant expression in Example 1 of this application are shown. Figure 2 The results of monoclonal antibody specificity identification in Example 2 of this application are shown; Figure 3 The results of the sensitivity determination of the PEDV colloidal gold test strip in this application are shown. Detailed Implementation

[0032] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited therein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by ordinary experimentation by those skilled in the art. These equivalents will be included in the claims.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores; all quantities, concentrations, and percentages mentioned in the following examples are calculated based on mass (weight).

[0035] Example 1: Preparation of porcine epidemic diarrhea virus (PEDV) antigen A method for preparing porcine epidemic diarrhea virus (PEDV) nucleocapsid protein (N protein) using an Escherichia coli prokaryotic expression system: The optimized PEDV-N gene sequence was cloned into the prokaryotic expression vector pET-28a(+), and a His tag was introduced at the N-terminus to facilitate purification. The recombinant plasmid was transformed into... E. coli After BL21(DE3) competent cells were cultured in LB medium containing kanamycin until OD2000. 600≈0.6-0.8, add 0.5 mmol / L IPTG for induction, and continue culturing at 18℃ for about 12 h to promote soluble protein expression. After centrifugation, collect the bacterial cells and resuspend them in lysis buffer, then sonicate and centrifuge to remove cell debris. Purify the target protein using Ni-NTA affinity chromatography. The target band in the eluent, analyzed by SDS-PAGE, showed a purity greater than 90%. Figure 1 As shown, the purified protein underwent dialysis to remove impurities and the buffer system was replaced to ensure its stability and structural integrity. Western blot analysis confirmed that the recombinant N protein could be specifically recognized by anti-PEDV antibodies, indicating good immunogenicity. The obtained N protein can be used as an immunogen for monoclonal antibody preparation and the construction of colloidal gold immunochromatographic detection systems, providing an antigenic basis for broad-spectrum detection of multiple PEDV subtypes. Studies have shown that the amino acid homology of the N protein from different PEDV strains (such as the classic strain CV777 and the variant strain AJ1102) exceeds 94%, significantly higher than that of the S protein, indicating a highly conserved sequence. Furthermore, the N protein does not participate in the binding of the virus to host cell receptors and is less susceptible to mutations due to immune selection pressure; therefore, its antigenic epitopes exhibit high stability and cross-subtype recognition characteristics. The recombinant N protein obtained by this method has high expression efficiency, good purity, and structural stability, making it suitable as a key antigen for preparing broad-spectrum anti-PEDV monoclonal antibodies and constructing rapid multi-subtype detection methods.

[0036] Example 2: Preparation of monoclonal antibodies against porcine epidemic diarrhea virus (PEDV) Five healthy female BALB / c mice aged 6–8 weeks were selected and immunized with purified recombinant PEDV N protein as the immunogen. Each mouse was injected with 80 µg of antigen, emulsified with complete Freund's adjuvant. Booster immunizations were then performed on days 10, 20, and 30 using incomplete Freund's adjuvant, with consistent doses each time. Three days after the final immunization, orbital blood was collected to measure serum titer. When the titer exceeded 1:64,000, spleen cells were harvested for hybridoma cell fusion. Mouse spleen cells were aseptically treated and mixed with logarithmically growing mouse myeloma cells SP2 / 0 at a ratio of 4:1. Fusion was performed at 37 °C using 50% PEG-1450, with the fusion reaction completed within 90 s. PEG was gradually diluted with preheated DMEM medium (37 °C). After two centrifugation washes, the fused cells were resuspended in HAT selective medium and cultured in 96-well plates containing feeder cells. Cells were cultured at 37°C and 5% CO2 for 7 days to observe their growth status. The supernatant was then collected for indirect ELISA detection of positive clones. Recombinant PEDV N protein was used as the coating antigen during screening.

[0037] After positive wells were confirmed by indirect ELISA, three consecutive rounds of limiting dilution cloning were performed until the monoclonal positivity rate reached 100%. The obtained hybridoma cell lines that stably secreted antibodies were named 2D14 and 8F17. Subsequently, the hybridoma cells were expanded and cultured, the supernatant was collected, and the monoclonal antibodies were purified by Protein G affinity chromatography. After dialysis and replacement of PBS buffer, the purified product was adjusted to a concentration of 1 mg / mL and stored at -80 °C for later use.

[0038] The preparation process in this embodiment is simple, the cell fusion efficiency is high, and the antibody secretion is stable, which can obtain high-purity, high-affinity anti-PEDV monoclonal antibodies, providing reliable antibody materials for the subsequent establishment of a colloidal gold rapid detection system based on double antibody sandwich.

[0039] Example 3: Identification of PEDV monoclonal antibody specificity To verify the specificity of the prepared antibodies and their ability to recognize different viral subtypes, porcine enteric-associated viruses that share certain structural and antigenic similarities with PEDV were selected as detection targets, including porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), and porcine epidemic diarrhea virus (PEDV). Each viral antigen was diluted to 1×10⁻⁶ with coating buffer. 6 100 μL of sample was added per well to coat a 96-well microplate. After incubation overnight at 4 °C, the plate was washed with PBST and blocked with 1% BSA for 2 h. 2D14 and 8F17 monoclonal antibodies diluted 1:2000 were added as primary antibodies, incubated at room temperature for 1 h, washed, and then HRP-labeled goat anti-mouse IgG (GAM-HRP) diluted 1:10000 was added as secondary antibody. After TMB colorimetric reaction and termination, the absorbance was read at 450 nm. The coating buffer served as a blank control. Results are shown below. Figure 2 .

[0040] The results showed that the two monoclonal antibodies, 2D14 and 8F17, only exhibited a significant reaction (OD) with the PEDV antigen. 450 The OD value was significantly higher than other groups (P<0.01), and no significant cross-reactivity was observed with TGEV, SADS-CoV, PoRV, or other non-coronaviruses, indicating high specificity for PEDV. Furthermore, to further evaluate the antibody's subtype recognition ability, the classic strain CV777, the variant strain AJ1102, and several domestically isolated circulating PEDV-N proteins were used as antigens for indirect ELISA detection. The results showed that both antibodies produced strong reaction signals to the aforementioned PEDV strains from different sources, with OD values ​​significantly higher than other groups (P<0.01). 450The difference in values ​​was not significant (P>0.05), indicating that the obtained monoclonal antibody can simultaneously identify multiple PEDV subtypes and has broad-spectrum detection performance.

[0041] These results demonstrate that the anti-PEDV monoclonal antibody prepared in this invention not only possesses strong species specificity and does not cross-react with other porcine enteroviruses, but also stably recognizes conserved epitopes of the PEDV multi-subtype N protein, exhibiting excellent antigen recognition stability and cross-variant detection capability. This invention, for the first time, systematically verifies monoclonal antibody specificity combined with subtype broad-spectrum evaluation, providing an innovative technological foundation for developing rapid PEDV detection products based on double-antibody sandwich colloidal gold.

[0042] Example 4: Identification of antigenic epitopes of anti-PEDV monoclonal antibody To clarify whether the two obtained monoclonal antibodies, 2D14 and 8F17, recognize different antigenic epitopes of porcine epidemic diarrhea virus (PEDV) N protein, an epitope difference analysis was performed. Epitope validation was conducted using the ELISA Additive Index (AI) method, with recombinant PEDV N protein as the coating antigen. The additive effect between antibodies was assessed by comparing the absorbance changes after individual and combined antibody loading.

[0043] Specifically, the absorbance values ​​(A1, A2) of 2D14 and 8F17 monoclonal antibodies added separately, and the absorbance value (A2) of a mixture of the two in equal proportions, were measured separately. 1+2 ), and according to the formula AI=(A 1+2 The additive index is calculated as (A1) / A2 × 100%. When AI > 10%, it indicates that the two antibodies recognize different antigenic epitopes and have a synergistic binding effect; when AI < 10%, it indicates that the recognition sites of the two antibodies are adjacent or overlap.

[0044] Experimental results showed that the AI ​​values ​​of 2D14 and 8F17 were 28%, indicating that both antibodies could independently bind to the target antigen molecule targeting epitopes at different spatial locations on the PEDV N protein. This result demonstrates that there is no significant competition between the two antibodies during binding to the N protein, allowing them to form a stable double-antibody sandwich structure. This characteristic makes them highly suitable for use in colloidal gold immunochromatography or double-antibody sandwich detection systems, thereby achieving high sensitivity and high specificity for antigen detection.

[0045] Furthermore, by validating the recombinant N protein of different PEDV subtypes (including the classic strain CV777 and the mutant strain AJ1102), the binding ability of the two antibodies remained consistent across all subtypes, further confirming that their recognition sites are located in a conserved region of the N protein. This epitope complementarity not only ensures cross-subtype coverage of the detection but also provides a theoretical basis and technological innovation for constructing a double-antibody sandwich broad-spectrum detection method.

[0046] Example 5: Identification of Types and Subclasses of Anti-PEDV Monoclonal Antibodies To clarify the immunoglobulin types and subtypes of the prepared PEDV monoclonal antibodies, the isotypes of two antibodies, 2D14 and 8F17, were identified. Monoclonal antibody isotypes refer to the differences in the structure of their immunoglobulin heavy and light chain constant regions, including major classes such as IgM, IgG, IgA, IgE, and IgD, and their different subclasses (e.g., IgG1, IgG2a, IgG2b, IgG3, etc.). Different isotypes of antibodies exhibit significant differences in complement activation ability, Fc receptor binding affinity, and in vivo stability, which are important parameters affecting their immune function and the construction of subsequent detection systems.

[0047] The test results showed that both antibodies belonged to the IgG type, with 2D14 being the IgG1 subtype and 8F17 the IgG2a subtype. Both antibodies had a light chain constant region of type κ (Igκ). This subtype distribution indicates that the two antibodies represent different immune response modes: IgG1 antibodies are typically mediated by the Th2 cell pathway and tend to recognize conformation-dependent epitopes; while IgG2a antibodies are more associated with Th1 immune responses, can recognize linear conserved epitopes, and possess strong binding stability and biological activity.

[0048] Structural and functional analysis reveals that the synergistic presence of IgG1 and IgG2a subtypes facilitates multi-site recognition and cross-validation of PEDV antigens, significantly enhancing the sensitivity and anti-interference capability of the double-antibody sandwich detection system. Particularly concerning the PEDV N protein, this region is highly conserved across different viral subtypes (homology > 95%). The two subtype monoclonal antibodies obtained in this study recognize different epitopes, forming complementary antibody pairs. This provides a solid foundation and technological innovation for developing a highly specific and broad-coverage rapid PEDV detection method.

[0049] Example 6: Gene sequencing of hybridoma cells 2D14 and 8F17 Sequencing of the light and heavy chain variable regions of monoclonal antibodies 2D14 and 8F17 was performed by Anhui General Biotechnology.

[0050] The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 2D14 is shown below: GAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTATGTTATGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGTAT TAAGTTCAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAGATCCTCCAGCACAGCCTTCATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAGGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID No.1) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 2D14 is shown below: EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGIKFNEKFKGKATLTSDRSSSTAFMELSSLTSEDSAVYYCAREGFDYWGQGTTLTVSS (SEQ ID No. 2) The CDR region was determined by Kabat analysis of the heavy chain variable region sequence of monoclonal antibody 2D14. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of monoclonal antibody 2D14 are as follows: CDR-H1: SYVMH (SEQ ID No.3) CDR-H2: YINPYNDGIKFNEKFK (SEQ ID No. 4) CDR-H3: YCAREGFD (SEQ ID No.5) The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 2D14 is shown below: GACATTTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTCATAGTCTTTTGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCA ATCTAGAATCTGGGATCCCAGCCAGGTTTGGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAGGTTATGAGGTTCCTCGGACGTTCGGTGGAGGCACCAACCTGGAAATCAAAC (SEQ ID No.6) The amino acid sequence of the variable region of the light chain of monoclonal antibody 2D14 is shown below: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGHSLLNWYQQKPGQPPKLLIYAASNLESGIPARFGGSGSGTDFTLNIHPVEEEDAATYYCQQGYEVPRTFGGGTNLEIK (SEQ ID No. 7) The CDR region of the heavy chain variable region of monoclonal antibody 2D14 was analyzed using Kabat (Kabat). The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 in the light chain variable region of monoclonal antibody 2D14 are as follows: CDR-L1: KASQSVDYDGH (SEQ ID No.8) CDR-L2: GIPARF (SEQ ID No.9) CDR-L3: TYYCQQGY (SEQ ID No.10) The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 8F17 is shown below: GAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGGCTCTCCTGTGCAACTTCTGGGTTCACCTTCACTGATTACTACATGACCTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGTTTTATTAGAAACAAAGCTAATGGTTACACAACAGA CTACAGTGCATCTGTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACACCCTGAGAGCTGAGGACAGTGCCACTTATTGTGCAAGAGATGGGGATGGTTACGAGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG (SEQ IDNo.11) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 8F17 is shown below: EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMTWVRQPPGKALEWLGFIRNKANGYTTDYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCARDGDGYEFAYWGQGTLVTVSA (SEQ ID No. 12) The CDR region of the heavy chain variable region of monoclonal antibody 8F17 was determined by Kabat analysis. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of monoclonal antibody 2D14 are as follows: CDR-H1:DYYMT (SEQ ID No.13) CDR-H2:IRNKANGYTTDYSASV (SEQ ID No.14) CDR-H3: TYYCARDGDGYEFAYW (SEQ ID No.15) The nucleotide sequence encoding the variable region of the heavy chain of monoclonal antibody 8F17 is shown below: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAGTGCCAGCTCAAGTGTAAGTTCCAGCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCCTCCCCCAAACTCTGGATTTATAGCACATCCAACCT GGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTTACCCACCCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAAC (SEQ ID No.16) The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 8F17 is shown below: QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPPTFGSGTKLEIK (SEQ ID No. 17) The CDR region of the heavy chain variable region of monoclonal antibody 8F17 was determined by Kabat analysis. The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region of monoclonal antibody 2D14 are as follows: CDR-H1: SASSSVSSSYL (SEQ ID No.18) CDR-H2: STSNLA (SEQ ID No.19) CDR-H3:CHQWSSYP (SEQ ID No.20) Alignment results of the antibody variable region and complementarity determining region (CDR) sequences revealed significant differences in amino acid composition and spatial conformation between the light chain variable regions (VL) of monoclonal antibodies 2D14 and 8F17. Furthermore, the sequence characteristics of their three CDRs (CDR-L1, CDR-L2, and CDR-L3) also differed. These differences in the variable region and CDRs directly determine the specificity differences in antigen recognition between the two antibodies, placing their binding sites in different epitope regions of the antigen molecule. It is inferred that 2D14 and 8F17 can form independent and non-competitive binding modes during the binding process with the target antigen, exhibiting complementary recognition characteristics. This structural difference not only explains the differences in binding kinetics and affinity between the two monoclonal antibodies but also provides a theoretical basis for constructing a double-antibody sandwich detection system, contributing to the achievement of high sensitivity and high specificity recognition of the target antigen.

[0051] Example 7: Preparation and Detection of Anti-PEDV Monoclonal Antibody Colloidal Gold Immunochromatographic Test Strip This embodiment utilizes two prepared anti-PEDV monoclonal antibodies, 2D14 and 8F17, to construct a double-antibody sandwich colloidal gold immunochromatographic test strip for rapid detection of porcine epidemic diarrhea virus. This detection system leverages the synergistic effect of two monoclonal antibodies recognizing different antigenic epitopes to achieve specific recognition of PEDV antigens.

[0052] First, the screened monoclonal antibody 2D14 was used to prepare a colloidal gold probe, which formed a stable covalent adsorption complex with the surface of 40 nm gold particles to maintain the antibody's biological activity and spatial orientation. After blocking, centrifugation, and reconstitution, the antibody-labeled colloidal gold probe was uniformly sprayed onto a glass fiber pad to form a signal release region and stored under dry conditions. Subsequently, a detection line (T line) and a control line (C line) were sprayed onto a nitrocellulose membrane, where the T line was immobilized with the capture antibody 8F17, and the C line was coated with goat anti-mouse IgG to verify the effectiveness of the detection system. The membrane strips were assembled sequentially with a sample pad, a gold-labeled pad, a nitrocellulose membrane, and an absorbent pad, and then adhered to a PVC base plate. After cutting, drying, and packaging, they were packaged into individual test strips.

[0053] During testing, the sample to be tested (such as feces, intestinal contents, or culture supernatant) is diluted with buffer and then dropped onto the sample pad. The PEDV antigen in the sample binds to the gold-labeled antibody 2D14 to form an immune complex, which migrates along the chromatographic direction and is intercepted by the capture antibody 8F17 at the T line, forming a red band. The appearance of a red band at the C line indicates that the system is functioning normally. The test results can be read within 10 minutes.

[0054] The specific operating procedure is as follows: 1. Preparation of gold standard test strips 1.1 Preparation of Colloidal Gold Solution Add 1000 mL of ultrapure water to an Erlenmeyer flask and heat to boiling on a magnetic stirrer. Then add 4 mL of 10% chloroauric acid (Sigma) and 4.8 mL of 10% trisodium citrate solution. Continue heating to boiling for 5 min. After cooling to room temperature, filter the colloidal gold through a 0.22 μm filter and store at 4 °C for later use.

[0055] 1.2 Labeling of monoclonal antibody 2D14 Take 100 mL of colloidal gold solution and put it into a beaker. Stir and add 0.2 M K2CO3 to adjust the pH of the gold solution to 9.5. After stirring, add 1 mg of purified monoclonal antibody 2D14 and stir at room temperature for 15 min. Add 1 mL of 10% BSA solution and stir at room temperature for 15 min. Centrifuge at 12000 rpm for 10 min. Carefully aspirate and discard the supernatant. Dilute the precipitate to 1 mL with gold labeling dilution buffer (Tris 2.5 g / L, casein 2.5 g / L, 0.03% Proclin 300). This is the labeled monoclonal antibody 2D14-colloidal gold complex (i.e., obtain the colloidal gold probe of monoclonal antibody 2D14).

[0056] 1.3 Mouse IgG markers Take 100 mL of colloidal gold solution and put it into a beaker. Add 0.2 M K2CO3 to adjust the pH of the gold solution to 7.0. After stirring, add 1 mg of mouse IgG (Hangzhou LONGi Biotechnology Co., Ltd., catalog number: AS00901). Stir at room temperature for 15 min, add 1 mL of 10% BSA solution, stir at room temperature for 15 min, centrifuge at 12000 rpm for 10 min, carefully aspirate and discard the supernatant, and dilute the precipitate to 1 mL with gold label dilution buffer (Tris 2.5 g / L, casein 2.5 g / L, 0.03% Proclin 300). This is the labeled mouse IgG colloidal gold complex.

[0057] The above gold-labeled composite was diluted with gold-labeled diluent to an OD of 80-100, and then 0.2 g / ml of sucrose was added. The mixture was then sprayed evenly onto glass fiber at a spray rate of 2.0 μl / cm and dried at 37°C for 8-12 hours to prepare the gold-labeled pad.

[0058] 1.4. Antibody dotting The capture antibody 8F17 was diluted to 1.2 mg / mL using spot dilution buffer (2.9 g / L Na2HPO4·12H2O, 0.282 g / L NaH2PO4·2H2O, 8.775 g / L NaCl, 200 μL Proclin 300) to serve as the test line for the colloidal gold test strip. Goat anti-mouse IgG (Hangzhou LONGi Biotechnology Co., Ltd.) was diluted to 1.5 mg / mL using the same dilution buffer to serve as the control line (C line) for the colloidal gold test strip. The two diluted solutions were then streaked onto a nitrocellulose membrane and dried at 37°C overnight.

[0059] 1.5 Assembly of Sandwich Method Test Strips for Detecting Porcine Epidemic Diarrhea Antigen The gold-labeled pad, coated nitrocellulose membrane (NC membrane), absorbent paper, and sample pad are mounted onto a PVC backing to assemble a porcine epidemic diarrhea virus sandwich test strip. The test strip structure is as follows: the sample pad, gold-labeled pad, NC membrane, and absorbent paper are assembled onto a PVC backing. A portion of the sample pad is overlapped and pressed onto the gold-labeled pad, a portion of the gold-labeled pad is overlapped and pressed onto the NC membrane, and a portion of the absorbent paper is overlapped and pressed onto the NC membrane. The NC membrane is divided into a test area and a control area. The test area has a test line (T line), and the control area has a control line (C line). The test line is close to the gold-labeled pad, and the control line is close to the absorbent paper.

[0060] 2. Preparation of test reagent cards The assembled test strips are cut into strips with a width of 3mm to 4mm using a strip cutter, and then inserted into specially designed plastic cards to form test reagent cards.

[0061] 3. Detection of porcine epidemic diarrhea virus antigen After thoroughly dissolving the sample to be tested (such as feces, intestinal contents, or culture supernatant) in the sample diluent, drop it onto the sample loading point (S). Let it stand at room temperature for 10 minutes before judging the results. The result judgment criteria are as follows: (1) If two bands appear, one in the quality control area and the other in the T detection area, it is positive, indicating that the pig to be tested is infected with porcine epidemic diarrhea virus; (2) A band appears in the quality control area, indicating a negative result; (3) If no band appears on the control line, it indicates that the test strip is damaged. Regardless of whether a band appears on the test line, a new test strip should be replaced and the test should be repeated.

[0062] Example 8: Determination of the detection sensitivity of PEDV colloidal gold test strips To evaluate the detection sensitivity of the prepared double-antibody sandwich colloidal gold immunochromatographic test strip against porcine epidemic diarrhea virus (PEDV), PEDV positive samples were serially diluted with PBST buffer to different viral concentration gradients: 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 TCID 50 / mL. Add 100 μL of diluted sample to the sample application end of a horizontally placed test strip, allow to stand at room temperature for 10 min, and then read the results. The color development of the test line (T line) is used to determine the intensity of the positive signal, and the color development of the control line (C line) is used as the criterion for determining the validity of the detection system. The test results are as follows: Figure 3 As shown.

[0063] The test results showed that when the virus concentration in the sample was 10... 2 TCID 50 At a concentration of 10 mL, a red band is visible on the T line. Below this concentration, the signal gradually weakens until it becomes invisible. Sensitivity testing results indicate that the detection limit of this test strip is 10 mL. 2 TCID 50 / mL. The color intensity is positively correlated with the virus concentration, and the PEDV content in the sample can be semi-quantitatively determined by the intensity of the band color development. The reaction is rapid and the visualization effect is obvious.

[0064] Example 9: Specificity verification of PEDV colloidal gold immunochromatographic test strip To verify the specificity of the test strips, porcine viruses belonging to the same genus as PEDV or prone to cross-reactivity were used as controls, including transmissible gastroenteritis virus (TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), and porcine circovirus type 2 (PCV2). Each virus sample was diluted to 10 μL with PBST buffer. 7 TCID 50 / mL, add 100 μL to the sample wells of the test strip for detection. The result interpretation criteria are: the appearance of a red band on the T line indicates a positive result ("+"), and the appearance of only the C line indicates a negative result ("-").

[0065] The results are shown in Table 1. The test strips showed a clear positive reaction only to PEDV samples, while no specific colorimetric reaction was observed for TGEV, SADS-CoV, PoRV, PRV, and PCV2, consistent with the results of indirect ELISA. These results indicate that the colloidal gold test strips constructed based on 2D14 and 8F17 exhibit high species specificity and do not cross-react with other porcine enteroviruses. Because the recognized epitope is located in a conserved region of the PEDV N protein, this system not only demonstrates high specificity but also stable recognition ability for both classic and variant strains, validating the reliability and innovation of this colloidal gold immunochromatographic method for rapid on-site detection of multiple PEDV subtypes.

[0066] Table 1. Specificity test results of colloidal gold immunochromatographic test strips

[0067] Where "+" indicates that the T line has developed color, indicating that the reagent strip has a specific reaction with the corresponding strain. "T line does not show color, indicating that the reagent strip does not specifically react with the corresponding strain."

[0068] The results show that the double-antibody sandwich colloidal gold test strip in this application has high specificity for PEDV detection and does not cross-react with common porcine enteroviruses such as TGEV, SADS-CoV, PoRV, PRV and PCV2.

[0069] Example 9: Verification of Multi-Subtype Detection of PEDV Colloidal Gold Test Strips To verify the applicability and broad-spectrum detection performance of the constructed double-antibody sandwich colloidal gold immunochromatographic test strip against different subtypes of porcine epidemic diarrhea virus (PEDV), representative strains were selected for validation, including five PEDV isolates with different genetic backgrounds: the classic strain CV777, the variant strain AJ1102, the GD-B strain, the CH / ZMDZY / 11 strain, and the CH / HNZZ / 2017 strain. The viruses were titered to 1×10⁻⁶. 4 TCID 50 / mL, take 100 μL and add it to the sample application end of the test strip. After reacting at room temperature for 10 min, read the result. The color development of the detection line (T line) is used as the positive judgment standard.

[0070] The test results showed that all PEDV subtype samples produced a clear red signal at the T line, while the non-PEDV control samples (TGEV, SADS-CoV, PoRV) showed no color development at the test line. This indicates that the test strip can simultaneously identify and detect different genotypes of PEDV, and the detection system has good cross-subtype recognition capability, capable of simultaneously detecting different PEDV strains such as the classic strain CV777 and the variant strain AJ1102. Signal intensity analysis results showed that the color intensity differences among the subtypes were not significant (P>0.05), indicating that the antigenic epitopes recognized by the monoclonal antibodies 2D14 and 8F17 used in the detection system are highly conserved and unaffected by amino acid differences between viral variants.

[0071] The broad-spectrum recognition performance of this test strip is mainly attributed to the biological characteristics of the N protein as a detection target. The N protein exhibits over 94% amino acid sequence homology across different PEDV subtypes, significantly higher than the S protein (approximately 80%). Furthermore, this protein does not participate in the viral-host receptor binding process, resulting in lower evolutionary pressure and a lower mutation rate. Therefore, antibodies targeting the N protein show stable epitope recognition across different subtypes, demonstrating superior cross-strain detection capabilities. The 2D14 and 8F17 monoclonal antibodies recognize different conserved epitopes of the N protein, forming complementary binding structures in the immunochromatographic system, significantly enhancing antigen capture efficiency. This system combines the high affinity of the antibodies with the rapid visualization advantages of immunochromatography, providing an innovative, convenient, and highly sensitive detection platform for rapid on-site screening and epidemiological surveillance.

[0072] Example 10: Detection and validation of PEDV colloidal gold test strips in clinical samples. To verify the applicability and detection rate of the constructed double-antibody sandwich colloidal gold immunochromatographic test strip in clinical sample testing, we tested 30 negative and positive clinical samples (anal swabs and stools) collected. The test results are shown in Table 2.

[0073] Table 2. Clinical sample validation results of colloidal gold test strips

[0074] The results above show that the sensitivity of the sample detection reaches 100%.

[0075] In summary, the results of this embodiment demonstrate that the colloidal gold immunochromatographic test strip constructed in this invention based on the sandwich principle of anti-PEDV N protein double antibodies can achieve simultaneous detection of multiple PEDV subtypes. It has innovative features of broad-spectrum recognition, rapid response and high specificity, providing a practical and scalable detection platform for field epidemiological monitoring and rapid clinical diagnosis.

Claims

1. A monoclonal antibody against porcine epidemic diarrhea virus, characterized in that, The monoclonal antibody is monoclonal antibody 2D14 or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 with the amino acid sequence as shown in SEQ ID NO: 3-5, and a light chain variable region comprising CDR-L1, CDR-L2, CDR-L3 with the amino acid sequence as shown in SEQ ID NO: 8-10.

2. The monoclonal antibody against porcine epidemic diarrhea virus according to claim 1, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2D14 or an antigen-binding fragment thereof is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the monoclonal antibody 2D14 or an antigen-binding fragment thereof is shown in SEQ ID NO:

7.

3. The monoclonal antibody against porcine epidemic diarrhea virus according to claim 1, characterized by, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2D14 or an antigen-binding fragment thereof is shown in SEQ ID NO: 1, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2D14 or an antigen-binding fragment thereof is shown in SEQ ID NO:

6.

4. The monoclonal antibody against porcine epidemic diarrhea virus according to claim 1, characterized by, The monoclonal antibody is monoclonal antibody 8F17 or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1, CDR-H2, CDR-H3 with the amino acid sequence as shown in SEQ ID NO: 13-15, and a light chain variable region comprising CDR-L1, CDR-L2, CDR-L3 with the amino acid sequence as shown in SEQ ID NO: 18-20.

5. The monoclonal antibody against porcine epidemic diarrhea virus according to claim 4, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 8F17 or an antigen-binding fragment thereof is shown in SEQ ID NO: 12, and the amino acid sequence of the light chain variable region of the monoclonal antibody 8F17 or an antigen-binding fragment thereof is shown in SEQ ID NO:

17.

6. The monoclonal antibody against porcine epidemic diarrhea virus according to claim 4, characterized by, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 8F17 or an antigen-binding fragment thereof is shown in SEQ ID NO: 11, and the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 8F17 or an antigen-binding fragment thereof is shown in SEQ ID NO:

16.

7. Use of monoclonal antibody 2D14 or an antigen-binding fragment thereof, monoclonal antibody 8F17 or an antigen-binding fragment thereof in the preparation of an immunochromatographic kit for detecting PEDV in a biological sample.

8. A colloidal gold immunochromatographic test strip kit, characterized in that: The test kit comprises a colloidal gold immunochromatographic test strip prepared using the monoclonal antibody 2D14 or an antigen-binding fragment thereof of any one of claims 1-3 and / or the monoclonal antibody 8F17 or an antigen-binding fragment thereof of any one of claims 4-6.

9. The colloidal gold immunochromatographic test strip of claim 8, characterized in that: The colloidal gold immunochromatographic test strip comprises a colloidal gold pad prepared using colloidal gold labeling of one of the antibodies, and a detection line prepared using nitrocellulose membrane on which the other antibody is immobilized.

10. The colloidal gold immunochromatographic test strip of claim 9, characterized in that: The particle size of colloidal gold in the colloidal gold labeling is 40-45 nm.

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