Monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibody and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody 1D3 for detecting a porcine epidemic diarrhea virus antibody and application of the monoclonal antibody 1D3. The CDR sequences of the heavy chain variable region are respectively shown as SEQ ID NO.1-3, and the CDR sequences of the light chain variable region are respectively shown as SEQ ID NO.4-6. The antibody can specifically recognize PEDV / S1 protein, binding of the antibody can be competitively blocked by a PEDV specific neutralizing antibody in serum, and the antibody is suitable for establishing a blocking ELISA method. A detection tool based on the antibody has high specificity and sensitivity, and the blocking rate to PEDV positive serum is higher than 50%. The invention can be used for preparing a detection kit or evaluating the immune effect of a vaccine, and provides an efficient and accurate technical means for PEDV infection diagnosis and immune monitoring.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibodies and its applications. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a digestive tract disease caused by porcine epidemic diarrhea virus (PEDV). It primarily causes vomiting, diarrhea, and even death in piglets. PEDV can be transmitted through the fecal-oral route, breast milk, and respiratory tract, and can infect pigs of all breeds and ages. PEDV infection remains highly prevalent in my country and is classified as a Class II animal disease. Furthermore, the complex and variable genetic evolution of PEDV poses a serious challenge to the development of my country's pig farming industry.

[0003] PEDV belongs to the Coronaviridae family and is a single-stranded, positive-sense, enveloped RNA virus. Its genome encodes 16 non-structural proteins and 4 structural proteins: the surface spike protein (S), membrane protein (M), envelope protein (E), and internal nucleocapsid protein (N). The S protein is composed of two subunits, S1 and S2. The S1 subunit is responsible for binding to the host receptor. The protein contains the virus's main neutralizing epitope, which can stimulate the body to produce neutralizers. It is often used as a target antigen for epidemiological studies and serological diagnosis of PED. Currently, there is no specific drug for treating PEDV infection. Vaccination remains an effective way to control PEDV, but the significant genetic variation of the virus poses a major challenge to the effectiveness of current vaccines. Therefore, establishing and applying rapid and effective detection methods and conducting routine PEDV screening are crucial for controlling porcine epidemic diarrhea. Molecular diagnostic methods such as qRT-PCR determine whether pigs carry the virus by detecting viral nucleic acid. However, these methods require specialized equipment, are expensive, and carry the risk of nucleic acid contamination. Immunological methods such as ELISA detect antibodies such as IgG and IgA in serum, milk, or saliva, which can be used for infection diagnosis or to assess antibody levels after immunization. Neutralization tests are also used clinically to evaluate the protective effect of antibodies. However, these methods still have problems such as poor detection accuracy or cumbersome operation. Therefore, more and more accurate detection methods are needed to achieve precise diagnosis and effective control of PEDV. Studies have shown that specific antibodies can be detected in various types of samples, including serum, milk, and saliva, after PEDV infection. The S protein-specific IgG level curve can be detected as early as 7 days after infection, reaches its peak at 14 days, and can be maintained for up to 6 months. Therefore, the detection of S protein antibodies can effectively improve the accuracy of PEDV infection diagnosis and antibody level evaluation.

[0004] Blocking ELISA is a commonly used immunological detection technique widely applied in infectious disease diagnosis and vaccine efficacy evaluation. Commercially available kits for detecting antibodies against diseases such as African swine fever (ASF) and classical swine fever (CSF) are available for clinical use. The monoclonal antibodies required in blocking methods are typically specific antibodies against key viral proteins, thus greatly reducing cross-reactivity. Compared to indirect methods and other antibody detection methods, it exhibits stronger resistance to matrix interference and provides more accurate results. In blocking ELISA, highly biologically active coating antigens and specific blocking monoclonal antibodies are crucial for ensuring accuracy, with the correct folding and modification of the antigen being extremely important. PEDV / S proteins are transmembrane glycoproteins, and prokaryotic expression systems cannot perform post-translational modifications, failing to accurately represent the antigen's conformation and resulting in poor antigenicity. Eukaryotic expression systems, however, can produce proteins with near-native conformations, leading to more accurate detection and making them more suitable for evaluating vaccine efficacy and serological diagnosis of PEDV infection. Summary of the Invention

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus (PEDV) antibodies and its application, which solves the shortcomings of existing detection methods in terms of specificity, accuracy or ease of operation, and realizes the technical problem of efficient and accurate assessment of PEDV antibody levels in swine herds.

[0006] To achieve the above objectives, the main technical solutions adopted by the present invention include: In a first aspect, this application provides a monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus antibodies. The heavy chain variable region of monoclonal antibody 1D3 includes three complementarity-determining regions, and the amino acid sequences of the complementarity-determining regions are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of monoclonal antibody 1D3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0007] The binding of monoclonal antibody 1D3 to the PEDV / S1 protein can be competitively blocked by PEDV-specific antibodies (such as antibodies produced after infection or immunization) in animal serum samples that can bind to the S1 protein.

[0008] A monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus (PEDV) antibodies, more specifically, a monoclonal antibody 1D3 for blocking ELISA detection of PEDV antibodies, or a monoclonal antibody 1D3 targeting PEDV.

[0009] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1D3 is shown in SEQ ID NO.7.

[0010] In a further embodiment, the amino acid sequence of the variable region of the light chain of monoclonal antibody 1D3 is shown in SEQ ID NO.8.

[0011] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D3 is shown in SEQ ID NO. 9.

[0012] In a further embodiment, the nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1D3 is shown in SEQ ID NO. 10.

[0013] Secondly, this application provides the use of the above-mentioned monoclonal antibody 1D3 in the preparation of a medicament for the prevention or treatment of porcine epidemic diarrhea.

[0014] Thirdly, this application provides the use of the above-mentioned monoclonal antibody 1D3 in the preparation of a tool for detecting porcine epidemic diarrhea virus antibodies.

[0015] In a further embodiment, the tool includes a reagent, a kit, or a test strip.

[0016] In a further embodiment, the kit includes a blocking ELISA kit that uses a blocking ELISA method to detect porcine epidemic diarrhea virus antibodies.

[0017] Fourthly, this application provides the application of the aforementioned monoclonal antibody 1D3 in evaluating the immunization efficacy of porcine epidemic diarrhea virus vaccine.

[0018] Beneficial effects: The monoclonal antibody 1D3 provided by this invention for detecting porcine epidemic diarrhea virus (PEDV) antibodies exhibits high specificity and binding activity, specifically recognizing key epitopes of the PEDV / S1 protein. Based on its unique complementarity-determining regions (CDRs, SEQ ID NO. 1-6), this antibody effectively binds to the PEDV / S1 protein, and this binding can be competitively blocked by PEDV-specific antibodies in animal serum. Therefore, it is particularly suitable for establishing a blocking ELISA detection method. Experimental results show that the blocking ELISA kit based on monoclonal antibody 1D3 has a high blocking rate (>50%) against PEDV antibody-positive serum, while showing no cross-reactivity (blocking rate <10%) against negative serum and positive serum from other swine diseases such as classical swine fever, porcine reproductive and respiratory syndrome (PRRS), and transmissible gastroenteritis (TGEV). It demonstrates excellent specificity, sensitivity, and repeatability, accurately detecting PEDV antibody levels in serum samples, providing an efficient and reliable tool for evaluating vaccine immunization efficacy and serological diagnosis of PEDV infection. Attached Figure Description

[0019] Figure 1 The results of blue-white screening; Figure 2 This is a diagram showing the morphological observation results of Sf9 cells after transfection. Figure 3 Image of PAGE electrophoresis results of denaturation of PEDV / S1 recombinant protein (12%). Figure 4 Image of PAGE electrophoresis results for PEDV / S1 recombinant protein (8%). Figure 5 The image shows the ELISA identification results of the PEDV / S1 recombinant protein. Figure 6 Figure showing the results of identifying the binding activity of different monoclonal antibodies with PEDV / S1 and PEDV / S recombinant proteins; Figure 7 This is a diagram illustrating the binding activity of the monoclonal antibody 1D3.

[0020] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0021] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0022] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0023] This invention selected the gene sequence of the S1 subunit of the PEDV / S protein and used an insect-baculovirus expression system to obtain an expression vector and recombinant baculovirus capable of efficiently secreting and expressing the S1 protein. The resulting high-purity, highly active trimeric S1 protein was then purified. Simultaneously, specific monoclonal antibodies were screened using hybridoma technology, and based on these, a blocking ELISA method for detecting PEDV / S1 protein antibodies was established. This method can specifically detect PEDV-positive serum and shows no cross-contamination with serum positive for classical swine fever virus antibodies, porcine reproductive and respiratory syndrome virus antibodies, and transmissible gastroenteritis virus antibodies. This provides important reference value for the development of diagnostic reagents for PEDV antibody detection.

[0024] Specifically, it is used to detect porcine epidemic diarrhea virus antibodies via ELISA.

[0025] This application uses a blocking ELISA method to detect the level of PEDV-specific antibodies in animal serum in vitro. The antibody level is assessed by calculating the blocking rate of monoclonal antibody 1D3 to PEDV / S1 protein. The method does not involve the diagnosis or treatment of diseases.

[0026] This application relates to the use of monoclonal antibody 1D3 in evaluating the immunization efficacy of a porcine epidemic diarrhea virus vaccine, including the detection of antibody levels or blocking rates in animal serum.

[0027] The PEDV / S1 protein in this application includes the S1 protein naturally present in porcine epidemic diarrhea virus, as well as the recombinant S1 protein expressed through gene recombination technology.

[0028] This application provides a blocking ELISA kit for detecting porcine epidemic diarrhea virus (PEDV) antibodies. The kit includes: a solid-phase carrier coated with PEDV / S1 recombinant protein as an antigen; an enzyme-labeled monoclonal antibody 1D3 as a detection antibody for binding to the PEDV / S1 recombinant protein; positive and negative controls to provide detection benchmarks for PEDV antibody positivity and negativity, respectively; a washing buffer to wash away unbound components; and a chromogenic solution and a stop solution to generate and terminate the chromogenic reaction. The binding of monoclonal antibody 1D3 to the PEDV / S1 recombinant protein can be blocked by serum containing PEDV antibodies, thereby enabling the detection of PEDV antibodies in the sample.

[0029] Example 1: 1. Source of PEDV / S recombinant protein The PEDV-CH-SBC / 2013 strain, i.e. the G2-a type S1 protein, was selected. This protein is a recombinant protein containing the S1 region expressed using an insect-baculovirus expression system. It contains a His tag, has a trimer structure, and has been identified as being able to react with positive monoclonal antibodies, demonstrating good biological activity.

[0030] 1.1 Construction of baculovirus expression vector for PEDV / S1 protein: The pFastBac-l baculovirus expression vector was modified to promote the secretory expression of the target protein. A GP67 secretion signal peptide sequence was added to the N-terminus of the pFastBac-l vector, enabling the protein to be secreted into the cell culture supernatant for easy purification. Furthermore, since the natural PEDV S protein has a trimeric structure, a Foldon sequence derived from the C-terminal domain of T4 phage fibrin (GSS) was added to the C-terminus of the vector to enable S1 to form a near-native trimeric conformation. A truncated mutant (containing 27 aa, conventionally 33 aa) was used to enhance expression and improve stability. A 6×His tag was also added to facilitate efficient purification of the target protein later. The sequence details are as follows: The nucleotide sequence of the GP67 secretion signal peptide (SEQ ID NO.11) is: ATGCTACTAGTAAATCAGTCACACCAAGGCTTCAATAAGGAACACACAAGCAAGATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCCATTCTGCCTTTGCGGCGGATCCCGGG.

[0031] Amino acid sequence (shown in SEQ ID NO.12): MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPG.

[0032] The PEDV gene references the S1 protein gene sequence of strain PEDV-CH-SBC / 2013 (Accession: KC787542), and its nucleotide sequence is shown in SEQ ID NO.13:

[0033] The amino acid sequence is (shown in SEQ ID NO. 14): LPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLTIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDTSVILAEGSIVLHTALGTNFSFVCSNSSDPHLATFAIPLGAIQVPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSNSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGF。

[0034] Foldon sequence: The nucleotide sequence (shown in SEQ ID NO. 15): GGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTG。

[0035] Amino acid sequence (shown in SEQ ID NO.16): GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0036] Linker: Nucleotide sequence (SEQ ID NO.17): GGCAGCAGC.

[0037] Amino acid sequence (SEQ ID NO.18): GSS.

[0038] The sequence SEQ ID NO.17 contains fewer than 10 specifically defined nucleotides. Unintentionally skipped sequences must contain at least 10 specifically defined nucleotides. Therefore, it is marked as an intentionally skipped sequence in the sequence listing and is recorded as 000. The sequence SEQ ID NO.18 contains fewer than 4 specifically defined amino acids. Unintentionally skipped sequences must contain at least 4 specifically defined amino acids. Therefore, it is marked as an intentionally skipped sequence in the sequence listing and is recorded as 000.

[0039] His tag: Nucleotide sequence (shown in SEQ ID NO.19): CATCATCATCATCATCATTAA.

[0040] Amino acid sequence (shown in SEQ ID NO.20): HHHHHH*. The last TAA in the nucleotide is a stop codon that does not encode an amino acid. It is indicated by * and the sequence listing does not display the "*" sign.

[0041] Complete sequence (shown in SEQ ID NO.21):

[0042] The constructed recombinant plasmid pFastBac-PEDV / S1 was sequenced and identified. After confirming that the fragment insertion was correct, downstream experiments were carried out.

[0043] 1.2 Obtaining Recombinant Rod Particles Recombinant plasmid pFastBac-PEDV / S1 was transposed onto DH10Bac competent cells, and blue-white screening was performed to obtain recombinant bamboo plasmid Bacmid-PEDV / S1. Specifically, 1 ng of recombinant plasmid pFastBac-PEDV / S1 was added to 50 μl of DH10Bac competent cells, gently mixed, and placed on ice for 30 min. Then, it was quickly transferred to a 42°C water bath for heat shock for 60 s, immediately placed on ice for 2 min, and then 900 μl of antibiotic-free LB medium was added. The cells were then incubated at 37°C with shaking at 200 rpm for 4 h. Transformed competent cells were diluted 10-fold with antibiotic-free LB medium and evenly spread onto solid LB agar plates containing triple antibodies (kanamycin 50ug / ml, gentamicin 7ug / ml, tetracycline 10ug / ml, X-Gal 100ug / ml, IPTG 40ug / ml). The plates were incubated upside down in a dark incubator at 37°C. After two days, colonies were observed, and independent, relatively large white colonies were selected for identification. See also... Figure 1 The results showed that after transforming DH10Bac cells with pFastBac-PEDV / S1 and spreading them on solid LB culture dishes containing triple antibodies, multiple white colonies appeared after culturing. The white colonies indicate that transposition has been successfully completed, meaning that the target gene has been integrated from the pFastBac plasmid into the Bacmid. These white colonies contain the recombinant baculovirus genome, and subsequent white colonies will be selected for identification.

[0044] 1.3 Identification of recombinant rod-like particles Bacmid-PEDV / S1 White, independent colonies were picked and placed in liquid LB medium containing triple antibodies, and incubated overnight at 37°C with shaking at 200 rpm. Using the bacterial culture as a template, amplification and sequencing were performed using universal primers M13F and S1 gene-specific primers.

[0045] The primer sequences were: M13-F (SEQ ID NO. 22): GTTTCCCAGTCACGAC; S1 gene-specific reverse primer (SEQ ID NO. 23): GGCCGGCACCAGCTGGAAACCAGGCAACTCCCTAGTA; the PCR amplification program was: 98℃ for 5 min; 98℃ for 20 s, 55℃ for 30 s, 72℃ for 70 s, 30 cycles; 72℃ for 5 min. The PCR amplification products were sent for sequencing, and after successful identification, bacteria were inoculated and bacterial granules were extracted.

[0046] 1.4 Extraction of Recombinant Rod Particles Take 40 μL of the bacterial suspension that tested positive for bacterial count and add it to 4 mL of LB medium (containing 7 μg / mL gentamicin, 100 μg / mL kanamycin, and 10 μg / mL tetracycline). Incubate overnight at 37°C and 200 rpm. Take 4 mL of the overnight bacterial suspension and centrifuge at 12,000 rpm for 2 min to collect the bacterial cells, discarding the supernatant. Add 1 mL of Solution I (25 mM Tris-Cl, 10 mM EDTA, 50 mM glucose, 100 μg / mL RNase A, pH 8.0) to the centrifuge tube, mix the bacterial pellet, and transfer the suspension to a new 4 mL centrifuge tube. Add 1 mL of Solution II (0.2 M NaOH, 10 g / L SDS) to the centrifuge tube, gently invert 6-10 times to fully lyse the bacterial cells until the liquid becomes clear and viscous. Add 1 mL of Solution III (60 mL of 5 M potassium acetate, 11.5 mL of glacial acetic acid, and 28.5 mL of deionized water) to the centrifuge tube. Gently invert the EP tube 6-10 times. A white flocculent precipitate will appear in the tube. Centrifuge at 12,000 rpm for 10 min at room temperature. After centrifugation, transfer the clear supernatant to a 4 mL centrifuge tube. Slowly add an equal volume of pre-chilled isopropanol, invert to mix, and place on ice for 10 min. Centrifuge at 12,000 rpm for 15 min and discard the supernatant. Add 2 mL of pre-chilled 70% ethanol to wash the precipitate. Centrifuge at 12,000 rpm for 10 min and discard the supernatant. Repeat the washing process. Air-dry the precipitate in a biosafety cabinet, dissolve it in 80 μL of enzyme-free water, and store at -20°C.

[0047] 1.5 Obtaining recombinant baculovirus Recombinant baculovirus was obtained by transfecting Sf9 cells (ovarian cells of *Ardisia crenata*) with the correctly identified recombinant baculovirus Bacmid-PEDV / S1. Sf9 cells in good condition were pre-treated with 0.5*10... 6The total amount of cells was seeded into 6-well cell culture plates. 1 μg of sterile recombinant Bacmid-PEDV / S1 was added to 100 μL of serum-free insect cell culture medium (MSF1) and gently mixed. Simultaneously, 6-8 μL of the transfection reagent Cellfectin (Thermo, 10362100) was added to 100 μL of serum-free insect cell culture medium and mixed thoroughly. The recombinant Bacmid and transfection reagent were then thoroughly mixed and incubated at room temperature for 30 min. The cells in the wells were washed with serum-free culture medium. 800 μL of serum-free culture medium was added to the recombinant Bacmid-transfection reagent mixture, and the mixture was thoroughly mixed before being added to the 6-well plates. A control containing only the transfection reagent and a normal cell control were also included. The plates were incubated at 27°C for 5 h. The culture medium was discarded, and 2 ml of serum-free culture medium was added to each well. The plates were then incubated at 27°C in the dark, and cell changes were observed. When cells in the transfection wells become larger, rounder, and fuse (approximately 5-7 days), while the cells in the transfection reagent control wells and cell control wells remain normal, the infection is considered successful. The cell culture supernatant is then harvested; this is the P1 generation recombinant virus solution. See also... Figure 2 The left image shows the morphology of diseased sf9 cells, while the right image shows the morphology of normal control sf9 cells. In the left image, cells show significant enlargement and rounding, with some cells beginning to fuse and form large cell clumps—a typical characteristic of diseased cells. In the right image, cells are uniform in shape, spindle-shaped or elliptical, and grow tightly adherently to the cell wall, showing no signs of disease. The comparison between the two images clearly demonstrates the impact of successful recombinant baculovirus transfection on Sf9 cells, confirming the successful acquisition and expression of recombinant baculovirus.

[0048] Cell culture supernatant was collected from 6-well plates by centrifugation. The supernatant from transfection and control wells was concentrated using a 30kD ultrafiltration concentrator. The concentrated supernatant was then diluted 100-fold and coated at 50µl / well onto 96-well microplates (coating buffer: carbonate buffer: 1.59g sodium carbonate, 2.93g sodium bicarbonate, diluted to 1L pure water, pH 9.6). Coating was performed overnight at 4°C. The coating buffer was discarded the next day, and the plates were blocked at 37°C for 2 hours with 3% sucrose + 2% BSA. The plates were then washed once with PBST (PBS containing 0.05% Tween-20) and blotted dry. After blocking, serially diluted PEDV / S protein monoclonal antibody (Guangzhou Qianxun Biotechnology) at 10µg / ml, 1µg / ml, and 100ng / ml concentrations were added at 50µl / well, along with PBS as a control. Incubate at 37°C for 30 minutes. After thorough washing, add 50 μL / well of HRP-labeled goat anti-mouse IgG (5000-fold diluted with PBS). Incubate at 37°C for 30 minutes. After thorough washing, add 50 μL / well of TMB chromogenic solution (Beijing Meikewande). Incubate at room temperature for 10 minutes. Terminate the chromogenic reaction with stop solution and read the OD450nm value using a microplate reader.

[0049] As shown in Table 1, the concentrated supernatant from the transfection wells was diluted 100-fold and coated onto an ELISA plate. It showed a significant positive reaction with the commercial monoclonal antibody. In contrast, the antibody did not react with the concentrated supernatant from the control wells when coated onto the ELISA plate. This indicates that the target protein is secreted and expressed in the supernatant of the transfected cell culture. The harvested supernatant can be used as P1 generation virulence for further infection passage.

[0050] Table 1: Identification of proteins expressed in the supernatant of transfection wells

[0051] Add 200 μL of P1 generation baculovirus to the surface of Sf9 cells (70% density) in a T25 culture flask. Incubate at 27°C until approximately 90% of cells show cytopathic effects. Collect the supernatant; this is the P2 generation recombinant baculovirus. Continue passage to obtain high-titer P3 generation recombinant baculovirus. At this stage, the virulence has a high and relatively stable titer. Add the P3 generation baculovirus virulence at a 1:500 ratio to the culture medium of suspended Sf9 cells, at a cell density of approximately 1 x 10⁻⁶ cells / year. 5 / ml, placed in a shaker at 27℃ and 120rpm for suspension culture until more than 90% of the cells show lesions, then centrifuged to collect the supernatant for protein purification.

[0052] 2. Purification of recombinant proteins Because the expressed recombinant protein carries a histidine tag, it was purified using a TED nickel affinity chromatography column (Solarbio). Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0; Buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The collected culture supernatant was then centrifuged at 12000 rpm for 10 min, filtered through a 0.45 μm filter, diluted 1:1 with buffer A, and slowly loaded onto the column. After loading, the column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purified protein was subjected to 12% SDS-polyacrylamide gel electrophoresis and 8% non-denaturing polyacrylamide gel electrophoresis, respectively. Coomassie brilliant blue staining was used to observe the size and purity of the target protein. The electrophoresis results of the purified protein are shown below. Figure 3 , Figure 4 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C. Figure 3 M: Protein Marker; R: PEDV / S1 protein electrophoresis results at 12% denaturing gel. Figure 4 M: Protein Marker; NR: Results of PEDV / S1 recombinant protein electrophoresis on 8% non-denaturing gel.

[0053] like Figure 3 As shown, under denaturing conditions, the PEDV / S1 protein exhibits a distinct main band around 95 kDa with high purity, slightly larger than the expected size of 88 kDa. This is likely due to glycosylation modification of the PEDV / S1 protein, which may migrate during electrophoresis, causing the band to be slightly larger than expected. Under non-denaturing conditions, the protein is not damaged by reducing agents such as SDS and mercaptoethanol, and maintains its native conformation. Figure 4 As shown, there is a distinct main band around 270 kDa, consistent with the expected size of the trimeric protein, indicating that the expressed protein has a trimeric conformation.

[0054] 3. Indirect ELISA to identify the activity of PEDV / S1 recombinant protein Purified PEDV / S1 recombinant protein was coated onto microplates, and its reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was commercially available PEDV S monoclonal antibody (Guangzhou Qianxun Biotechnology). The recombinant protein was first coated into microplates using a coating buffer (carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at a concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20) and patted dry. The monoclonal antibody was diluted with PBS in gradients of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of the antigen-coated microplate. PBS was used as a negative control. The plate was incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plate was washed four times with PBST. After drying, 50 μL / well of HRP-labeled goat anti-mouse IgG (5000-fold diluted with PBS) was added. The plate was incubated at 37°C for 30 min, washed four more times, and dried. 50 μL / well of TMB chromogenic buffer (Beijing Meike Wande) was added, and the plate was incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution was added to terminate the reaction. The OD450 nm value was measured using a microplate reader. The results are as follows: Figure 5 . Figure 5 The results showed that the purified PEDV / S1 recombinant protein could specifically react with positive monoclonal antibodies, indicating that the recombinant protein has good biological activity and can be used as a coating or immunogenic antigen for monoclonal antibody screening.

[0055] 4. Screening of PEDV / S protein monoclonal antibodies Immunization in mice.

[0056] Purified PEDV / S1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (total volume 200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. At weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites with the same dose mixed with an equal volume of Freund's incomplete adjuvant. At week 6, mice were immunized by direct injection of insulin (5 μg / mouse) into the spleen. Seven days after the final immunization, mouse serum was collected to detect antibody titers. Mice with high titers were selected for a booster immunization of 20 μg of recombinant PEDV / S1 protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0057] 5. Screening of hybridoma cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for both PEDV / S1 recombinant protein and PEDV / S recombinant protein (nearshore, DRA252, PEDV-CV777 strains) were selected by indirect ELISA. Since the immunogen contained a His tag, background components needed to be screened to identify specific cell lines targeting PEDV / S1 recombinant protein. Positive cells were cloned to monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0058] 6. Screening positive clones using indirect ELISA method PEDV / S1 recombinant protein, PEDV / S recombinant protein, and CMV / gB recombinant protein (nearshore, DRA263, containing a His tag) were coated into microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at a concentration of 1 μg / mL, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 150 μL of 3% sucrose + 2% BSA per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. 50 μL of cell culture supernatant was added, and the plates were incubated at 37°C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, blot dry, and add 50 μL / well of HRP-labeled goat anti-mouse IgG (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, blot dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. The results are shown in Table 2. Positive cell lines that reacted with PEDV / S1 and PEDV / S recombinant proteins but not with the control recombinant protein were selected for subsequent experiments.

[0059] Table 2: Screening results of monoclonal antibodies

[0060] The recombinant PEDV / S1 protein is of the PEDV G2 genotype, while the PEDV / S recombinant protein from nearshore organisms is of the GI genotype. The two have certain amino acid similarities. Using these two recombinant proteins as coating antigens, 20 cross-reactive monoclonal antibodies that react with both were obtained, laying the foundation for subsequent screening of blocking monoclonal antibodies.

[0061] After the selected hybridoma cell lines were expanded and cultured, 0.2 ml (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were used to collect ascites fluid approximately 10 days later, when the mice’s abdomens were noticeably swollen.

[0062] 7. Purification of monoclonal antibodies Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, dilute it 10-fold with binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), filter it through a 0.22 μm filter, and pump the filtered sample slowly into a Protein L purification column equilibrated with binding buffer using a peristaltic pump. Connect the column to a protein purification instrument, wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens, then elute with elution buffer (0.1 M glycine, pH 2.7), collect the elution peak, adjust the collected sample to neutral with 1 M Tris-HCl (pH 9), and transfer it to a dialysis bag (MW: 8000-14000). Dialyze the sample in 20 mM PBS (pH 7.4) at 2-8 °C for 16 hours. Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody.

[0063] The purified monoclonal antibody was diluted to 1 μg / ml with PBS, and its binding activity with PEDV / S1, PEDV / S recombinant protein, and irrelevant antigen CMV / gB was further detected by indirect ELISA. Results are as follows: Figure 6 As shown, by Figure 6 It can be seen that the purified monoclonal antibody has significant specific binding (OD) to both PEDV / S1 and PEDV / S recombinant proteins. 450 The nm value is high, and the binding rate with the irrelevant control antigen CMV / gB is extremely low (OD value is close to the background).

[0064] 8. Screening of blocking monoclonal antibodies To improve the efficiency of screening for blocking monoclonal antibodies, 15 monoclonal antibody strains with high binding titers were selected for downstream experiments.

[0065] HRP-labeled monoclonal antibody The selected antibody was diluted to a final concentration of 2 mg / mL using carbonate coupling buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6). 2 mg of HRP was dissolved in 0.5 mL of ultrapure water and thoroughly mixed with 0.5 mL of 0.06 M sodium periodate solution. Then, 1 mg (0.5 mL) of the diluted antibody solution was added to the matching tube containing HRP, and the mixture was pipetted and incubated at room temperature for 1 h, with regular mixing during incubation. The labeling reaction was terminated by adding 50 μL of 5 mg / mL sodium borohydride and mixing for 15 min. Finally, the labeled antibody was dialyzed overnight in 0.01 M PBS, pH 7.4 buffer. Glycerol was added at a 1:1 volume ratio, and the mixture was aliquoted and stored at -20 °C.

[0066] To assess the binding activity of HRP-labeled monoclonal antibodies, a direct ELISA method was used. A monoclonal antibody exhibiting strong reactivity with PEDV / S1 protein was selected for subsequent blocking antibody testing. PEDV / S1 recombinant protein (1 μg / ml, 50 μL / well) was coated into microplates. HRP-labeled antibody was diluted 1000-fold with PBS and added directly to the coated plate. The plate was incubated at 37°C for 30 minutes, the liquid was discarded, the plate was washed four times, and the plate was dried. 50 μL of TMB chromogenic buffer was added per well, and the plate was incubated at room temperature for 10 minutes. Finally, 50 μL of TMB stop solution was added to terminate the reaction. OD was measured using a microplate reader. 450 The nm values ​​are shown in Table 3. Ten HRP-labeled antibodies with read values ​​exceeding 1.0 were selected for subsequent blocking tests.

[0067] Table 3: Results of direct reaction between HRP-labeled antibody and antigen

[0068] 9. Establishment of the blocking ELISA method PEDV / S1 recombinant protein was coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water, pH 9.6), with a coating concentration of 1 μg / mL, 50 μL / well, and incubated overnight at 4 °C. The next day, the coating buffer was discarded, and the plates were blocked with PBS (pH 7.4) containing 3% sucrose + 2% BSA, 150 μL per well, and incubated at 37 °C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. PEDV-positive and SPF-negative swine serum were diluted 5-fold with PBS and added to coated plates at 50 μL / well. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed 4 times with PBST. After drying, 50 μL / well of the HRP-labeled monoclonal antibody (diluted 1000-fold with PBS) was added, and the plates were incubated at 37°C for 30 min. The plates were washed 4 times again, and after drying, 50 μL / well of TMB chromogenic buffer (Beijing Mecowand) was added, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Mecowand) was added to stop the reaction, and the OD450nm value was measured using a microplate reader. The PEDV-positive swine serum was obtained from PEDV-infected convalescent pigs confirmed by RT-qPCR, with a neutralizing antibody titer ≥ 1:128. The SPF-negative swine serum was obtained from healthy pigs from an SPF pig farm, and PEDV-negative results were confirmed by RT-qPCR and neutralization assay.

[0069] The HRP-PEDV / S1 monoclonal antibody with the highest percentage of inhibition (PI) was selected for condition optimization. The inhibition rate was calculated as: PI = (SPF serum OD value - positive serum OD value) / SPF serum OD value × 100%. The results are shown in Table 4.

[0070] Table 4: Results of Blocking Antibody Screening

[0071] The results in Table 4 show that HRP-1D3 has the best blocking effect, and the ELISA method using 1D3 for blocking is optimized.

[0072] 10. Optimization of the blocking ELISA method The PEDV / S1 recombinant protein was diluted with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was blocked with 150 μL of 3% sucrose + 2% BSA at 37 °C for 2 hours. The blocking buffer was discarded, and the plate was washed once with PBST wash buffer (PBS containing 0.05% Tween-20, pH 7.4) and patted dry. PEDV-positive and SPF-positive swine serum were diluted 5-fold with PBS and added to ELISA plates at 50 μL / well. The plates were incubated at 37°C for 30 min, washed 4 times with PBST wash buffer, and then HRP-1D3 diluted 1000, 2000, and 3000 times with PBS at 50 μL / well were added. The plates were incubated at 37°C for 35 min, washed 4 times again, and then dried. TMB chromogenic buffer was added at 50 μL / well, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (Beijing Meike Wande, 1001SA) was added to terminate the reaction. OD was measured using an ELISA reader. 450 nm value. Select the condition with the largest PI value for the next test. The screening process is shown in Table 5.

[0073] Table 5: Results of Tests for Optimal Reaction Conditions for Blocking ELISA

[0074] According to the data in Table 5, the highest blocking rate of HRP-1D3 was observed when the PEDV / S1 recombinant protein coating concentration was 1 μg / ml and the HRP-1D3 was diluted 2000 times.

[0075] 11. Blocking ELISA detection of porcine serum Based on the selected blocking ELISA reaction conditions, the following serum samples were further tested: 6 PEDV antibody-positive swine serum samples (confirmed as PEDV antibody positive by neutralization test), 3 PEDV antibody-negative swine serum samples (confirmed as PEDV antibody negative by neutralization test), 1 classical swine fever antibody-positive serum sample (confirmed as PEDV antibody negative by neutralization test, and positive for classical swine fever antibody by commercial kit), 1 porcine reproductive and respiratory syndrome (PRRS) antibody-positive serum sample (confirmed as PEDV antibody negative by neutralization test, and positive for PRRS antibody by commercial kit), 1 porcine transmissible gastroenteritis virus (TGEV) antibody-positive serum sample (confirmed as PEDV antibody negative by neutralization test, and positive for TGEV antibody by commercial kit), 1 SPF swine serum sample, and a PBS control. All serum samples were diluted 5-fold. The test results are shown in Table 6.

[0076] Table 6: Detection of porcine serum by blocking ELISA

[0077] Six PEDV antibody-positive swine serum samples and three PEDV antibody-negative swine serum samples were all derived from different swine serum samples identified by the laboratory of China Agricultural University. The differences in their blocking effects reflect the true biological variations among different individuals, with different infection histories, or immune states. According to the data in Table 6, the blocking rate of the six PEDV antibody-positive swine serum samples was all higher than 50%, showing a significant blocking response. In contrast, the blocking rates of the PEDV antibody-negative swine serum, classical swine fever antibody-positive serum, porcine reproductive and respiratory syndrome virus (PRRSV) antibody-positive serum, and porcine transmissible gastroenteritis virus (TGEV) antibody-positive serum were all lower than 10%, showing no significant blocking effect. This indicates that the blocking ELISA method has good specificity.

[0078] This invention can be used to prepare test kits or evaluate the immunization effect of vaccines, providing an efficient and accurate technical means for the diagnosis and immune monitoring of PEDV infection.

[0079] 12. Identification of the binding activity of blocking monoclonal antibodies Following the aforementioned indirect ELISA method, the blocking monoclonal antibody 1D3 was serially diluted at 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Another murine-derived unrelated monoclonal antibody, PRRSV-N monoclonal antibody from Jinnuobaitai, was used as a negative control. The binding activity of the antibody to the PEDV / S1 recombinant protein was measured. Results are as follows... Figure 7 The results showed that 1D3 still reacted positively with PEDV / S1 at a dilution of 1 ng / ml, indicating high antibody activity.

[0080] 13. Variable region gene sequence of monoclonal antibodies Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0081] Monoclonal antibody 1D3 sequence: The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 1D3 is shown in SEQ ID NO.10: GAGACCACCGTGACCCAGAGCCCCAGCTACCTGGCCGCCAGCCCCGGCGAGACCATCACCATCAACTGCAGGGCCAGCAAGAGCATCGGCAAGTACCTGGCCTGGTTCCAGGAGAAGCCCGGCAAGACCAACAAGCTGCTGATCTACAGCGGCAGGTGTGGAGAGC TGGGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCATGTACTACTGCAGGGGCCACATCCAGTTCAGCAACCCCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0082] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1D3 is shown in SEQ ID NO. 8: ETTVTQSPSYLAASPGETITINCRASKSIGKYLAWFQEKPGKTNKLLIYSGRVESWGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCRGHIQFSNPFGGGTKLEIKRTV.

[0083] Light chain CDR area annotation: The sequence of the complementarity-determining region CDR-L1 of the monoclonal antibody 1D3 is shown in SEQ ID NO.4: RASKSIGKYLA; The sequence of the complementarity-determining region CDR-L2 of the monoclonal antibody 1D3 is shown in SEQ ID NO.5: SGRVESW; The sequence of the complementarity-determining region CDR-L3 of the monoclonal antibody 1D3 is shown in SEQ ID NO.6: RGHIQFSNP.

[0084] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1D3 is shown in SEQ ID NO.9: GAGGTGAAGCTGGAGGAGTCTGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGCTCTGTCACTGGCGACTCCATCACCAGTGATTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAATACATGGGATACATAAGTTACAGCGGTAGCACTTACTACAATCCATC TTCCAGCAGTCGAATCTCCATCACTCGCGACACATCCAAGAACCAGTTCTACCTGCAGTTGAATTCTGTGACCACTGAGGACACAGCCACATATTACTGTGCAAGATCTCAACAACTCTACTATCATTACGCCGTGAAGGACTTTGACTTCTGGGGCCCAGGCACCACTCTCACAGTCTCCTCA.

[0085] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1D3 is shown in SEQ ID NO.7: EVKLEESGPSLVKPSQTLSLTCSVTGDSITSDYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLSSRISITRDTSKNQFYLQLNSVTTEDTATYYCARSQQLYYHYAVKDFDFWGPGTTLTVSS.

[0086] Heavy chain CDR region annotation: The sequence of the complementarity-determining region CDR-H1 of the monoclonal antibody 1D3 is shown in SEQ ID NO.1: SDYWN; The sequence of the complementarity-determining region CDR-H2 of the monoclonal antibody 1D3 is shown in SEQ ID NO.2: YISYSGSTYYNPSLSS; The sequence of the complementarity-determining region CDR-H3 of the monoclonal antibody 1D3 is shown in SEQ ID NO.3: SQQLYYHYAVKDFDF.

[0087] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0088] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody 1D3 for detecting porcine epidemic diarrhea virus, characterized in that, The heavy chain variable region of the monoclonal antibody 1D3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 1D3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

2. The monoclonal antibody 1D3 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D3 is shown in SEQ ID NO.

7.

3. The monoclonal antibody 1D3 according to claim 2, characterized in that, The amino acid sequence of the variable region of the light chain of the monoclonal antibody 1D3 is shown in SEQ ID NO.

8.

4. The monoclonal antibody 1D3 according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1D3 is shown in SEQ ID NO.

9.

5. The monoclonal antibody 1D3 according to claim 4, characterized in that, The nucleotide sequence encoding the variable region of the light chain of the monoclonal antibody 1D3 is shown in SEQ ID NO.

10.

6. Use of the monoclonal antibody 1D3 according to claim 1 in the preparation of a medicament for the prevention or treatment of porcine epidemic diarrhea.

7. The use of the monoclonal antibody 1D3 according to claim 1 in the preparation of a tool for detecting antibodies against porcine epidemic diarrhea virus.

8. The application according to claim 7, characterized in that, The tools mentioned include reagents, kits, or test strips.

9. The application according to claim 8, characterized in that, The kit includes a blocking ELISA kit, which uses a blocking ELISA method to detect porcine epidemic diarrhea virus antibodies.

10. The application of the monoclonal antibody 1D3 according to claim 1 in evaluating the immunization efficacy of a porcine epidemic diarrhea virus vaccine.

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