A monoclonal antibody ensemble for detecting porcine epidemic diarrhea virus S protein and its application

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a monoclonal antibody combination for detecting porcine epidemic diarrhea virus (PEDV) S protein and its application. The combination comprises monoclonal antibodies 4E11 and 1F3. The heavy chain CDR1-3 (SEQ ID NO. 1-3) and light chain CDR1-3 (SEQ ID NO. 4-6) of 4E11, and the heavy chain CDR1-3 (SEQ ID NO. 7-9) and light chain CDR1-3 (SEQ ID NO. 10-12) of 1F3, together impart high specificity and affinity. Detection methods such as double-antibody sandwich ELISA constructed based on this combination, using 4E11 as the coating antibody and 1F3 as the labeling antibody, have advantages such as high sensitivity, strong specificity, and broad spectrum, and can be used for in vitro detection of PEDV S protein, biological sample analysis, and quality control of biological products.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a combination of monoclonal antibodies for detecting the S protein of porcine epidemic diarrhea virus and its application. 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] Porcine epidemic diarrhea virus (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 one of the most crucial functional proteins of PEDV, acting as the "key" for viral invasion of host cells and playing a vital role in the virus's infectivity and pathogenicity. It consists of two subunits, S1 and S2. The S1 subunit is responsible for binding to host receptors and contains the virus's main neutralizing epitopes, stimulating the body to produce neutralizing agents, making it an important target for vaccine development and diagnostic method design. The S gene is also one of the most variable regions in the PEDV genome. Based on differences in the S gene nucleotide sequence, PEDV strains are mainly divided into two genotypes: G1 and G2. G1 strains, such as the classic CV777 strain, have relatively weak pathogenicity. G2 strains are variants; these strains have mutated S proteins, leading to significantly enhanced transmission speed and pathogenicity. Currently, both PEDV G1 and G2 types are widely prevalent in my country, posing a very serious challenge to the country's pig farming industry.

[0004] Currently, there are no specific drugs for treating PEDV infection, and vaccination remains the most effective way to control PEDV. Due to the central role of the S protein in the PEDV viral particle, it has become a key focus in PEDV vaccine design. Domestic PEDV vaccines are mainly traditional inactivated vaccines and attenuated vaccines. Inactivated vaccines use intact viral particles, inactivating the virus through physical or chemical methods while preserving its antigenicity. Attenuated vaccines reduce viral virulence through cell passage or genetic engineering. In addition, there are subunit vaccines prepared by genetically engineering the expression of the PEDV S protein or its main antigenic domains (such as S1 or RBD). The core of all these vaccines is the S protein, highlighting its importance as a key target in vaccine design.

[0005] Existing monoclonal antibodies and detection reagents developed based on a single strain of the PEDV S protein often only show good reactivity to homologous strains, making it difficult to simultaneously cover different genotypes of circulating strains such as G1 and G2, leading to reduced detection sensitivity or false negatives. Currently, there is a lack of monoclonal antibodies and corresponding detection methods capable of broadly recognizing the S protein of multiple circulating PEDV strains, which restricts the accurate diagnosis of PEDV and the standardized quality control of vaccine production. Summary of the Invention

[0006] Given that existing monoclonal antibodies and detection reagents are unable to effectively identify both G1 and G2 types of porcine epidemic diarrhea virus (PEDV) simultaneously, this invention provides a monoclonal antibody combination for detecting the S protein of PEDV and its application, which can simultaneously identify the G1 and G2 types of PEDV S protein. This solves the technical problems of existing detection methods, such as the inability to broadly cover major circulating strains, the tendency to miss detections, and insufficient sensitivity.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] A monoclonal antibody ensemble for detecting the S protein of porcine epidemic diarrhea virus, comprising monoclonal antibody 4E11 and monoclonal antibody 1F3;

[0009] The heavy chain variable region of monoclonal antibody 4E11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0010] The light chain variable region of monoclonal antibody 4E11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0011] The heavy chain variable region of monoclonal antibody 1F3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0012] The light chain variable region of monoclonal antibody 1F3 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0013] In a further embodiment, the monoclonal antibody combination is capable of recognizing the S protein of G1 porcine epidemic diarrhea virus and / or the S protein of G2 porcine epidemic diarrhea virus.

[0014] In other words, the monoclonal antibody combination can cross-recognize the S protein of G1 and G2 porcine epidemic diarrhea virus.

[0015] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.14.

[0016] In a further embodiment, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.16.

[0017] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO. 18.

[0018] In a further embodiment, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO. 20.

[0019] Secondly, the present invention provides the application of the combination of the above-mentioned monoclonal antibodies in the preparation of a tool for detecting the S protein of porcine epidemic diarrhea virus.

[0020] In a further embodiment, the tools include reagents, kits, test strips, and antibody chips.

[0021] In a further embodiment, the kit includes a double-antibody sandwich ELISA kit.

[0022] In a further embodiment, the ELISA kit uses monoclonal antibody 4E11 as the coating antibody and monoclonal antibody 1F3 as the labeling antibody.

[0023] Thirdly, the above-mentioned combination of monoclonal antibodies is used in the quality control of porcine epidemic diarrhea virus vaccines. The combination of monoclonal antibodies can detect the content of S protein and / or antigen activity in the vaccine.

[0024] Beneficial effects:

[0025] This invention provides a monoclonal antibody combination for detecting porcine epidemic diarrhea virus (PEDV) S protein, comprising highly specific and complementary monoclonal antibodies 4E11 and 1F3. Both antibodies in this combination target the S1 subunit of the PEDV S protein, and their complementarity-determining regions (CDRs) are clearly characterized: the heavy chain CDR1-CDR3 (SEQ ID NO. 1-3) and light chain CDR1-CDR3 (SEQ ID NO. 4-6) of 4E11, and the heavy chain CDR1-CDR3 (SEQ ID NO. 7-9) and light chain CDR1-CDR3 (SEQ ID NO. 10-12) of 1F3, collectively determine their high specificity and high affinity. This antibody combination can effectively recognize G1 and G2 type PEDV S protein, solving the problems of poor cross-reactivity, insufficient sensitivity, and false negatives caused by viral antigen variation in existing detection technologies. The detection methods (such as double-antibody sandwich ELISA) and related detection reagents and kits developed based on this combination have the advantages of high sensitivity, strong specificity, good broad spectrum and good reproducibility. They can be used for in vitro detection of PEDV S protein, analysis of target antigens in biological samples, quality control of viral antigen standards, and research and development and quality evaluation of related immunological products, providing efficient and reliable tools for PEDV-related research and standardized production of biological products. Attached Figure Description

[0026] 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.

[0027] Figure 1 The results of blue-white screening;

[0028] Figure 2 This is a diagram showing the morphological observation results of Sf9 cells after transfection.

[0029] Figure 3 Image of PAGE electrophoresis results for denaturing PEDV / S1 recombinant protein (12%).

[0030] Figure 4 Image showing the non-denaturing PAGE electrophoresis results of PEDV / S1 recombinant protein (8%).

[0031] Figure 5 The image shows the ELISA identification results of the PEDV / S1 recombinant protein.

[0032] Figure 6Figure showing the results of identifying the binding activity of different monoclonal antibodies with PEDV / S1 and PEDV / S recombinant proteins;

[0033] Figure 7 The specificity and sensitivity results of the double-antibody sandwich ELISA for PEDV / S1 recombinant protein and PEDV / S recombinant protein are shown in the figure.

[0034] Figure 8 This is a diagram illustrating the activity of paired monoclonal antibodies. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] This invention uses the full-length S protein of the G1-a type PEDV-CV777 strain and the S1 protein of the G2-a type PEDV-CH / SBC / 2013 strain as co-screening antigens. Since the G2-a type antigen contains only the S1 region and lacks the S2 subunit, and the selected monoclonal antibodies 4E11 and 1F3 can specifically bind to it, this indicates that the antigenic epitope recognized by this antibody is located in the S1 subunit of the S protein.

[0038] The porcine epidemic diarrhea virus (PEDV) S protein includes both G1 and G2 types. This means that a monoclonal antibody combination for detecting the PEDV S protein can be used to simultaneously detect both G1 and G2 types, providing broad-spectrum detection of currently prevalent major PEDV genotypes. Of course, using the monoclonal antibody combination 4E11 and 1F3 of this application to detect either the G1 or G2 type PEDV S protein individually is also within the scope of protection of this application.

[0039] Both monoclonal antibodies 4E11 and 1F3 specifically recognize the S1 subunit of the porcine epidemic diarrhea virus (PEDV) S protein.

[0040] The PEDV / S recombinant protein is the full-length S recombinant protein of type G1-a PEDV in this paper, namely the full-length S protein of type G1 porcine epidemic diarrhea virus.

[0041] The PEDV / S1 recombinant protein is the G2-a type PEDV S1 recombinant protein described in this paper, which is the S1 subunit of the S protein of G2 type porcine epidemic diarrhea virus.

[0042] The PEDV / S1 and PEDV / S mixed recombinant protein is a mixture of PEDV / S recombinant protein and PEDV / S1 recombinant protein.

[0043] The PEDV S protein is the spike protein of porcine epidemic diarrhea virus, which includes both natural and recombinant proteins. It encompasses the full-length S protein and its subunits (such as S1 and S2) of all genotypes (e.g., G1 and G2).

[0044] Example 1:

[0045] 1. Construction of PEDV / S1 recombinant protein

[0046] To obtain cross-reactive monoclonal antibodies that can simultaneously recognize G1 and G2 porcine epidemic diarrhea virus, this application utilizes the high amino acid sequence homology of the S proteins of both viruses and employs a combined immunization and screening strategy. Immunization is performed with G2 PEDV / S1 recombinant protein, followed by parallel screening using PEDV / S1 and G1 PEDV / S recombinant protein, thereby efficiently obtaining monoclonal antibodies with broad-spectrum cross-reactivity. For the G1 type, PEDV-CV777 strain, representing the S protein of the G1-a type, was selected. This protein is a full-length trimer S protein expressed in mammalian cells, containing a His tag, and is a nearshore biological product (DRA-252), denoted as PEDV / S. For the G2 type, PEDV-CH / SBC / 2013 strain, representing the S1 protein of the G2-a type, denoted as PEDV / S1, was selected. This protein is a recombinant protein containing the S1 region expressed using an insect-baculovirus expression system, containing a His tag, and has a trimer structure. It was identified as reacting with positive monoclonal antibodies and exhibits good biological activity (its construction method is detailed below). Comparison of the amino acid sequences of the S1 proteins of the two strains revealed approximately 91% amino acid similarity, indicating high homology, allowing for the screening of cross-reactive monoclonal antibodies.

[0047] 1.1 Method for constructing a baculovirus expression vector for PEDV / S1 recombinant protein:

[0048] 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:

[0049] The nucleotide sequence of the GP67 secretion signal peptide (SEQ ID NO.21) is: ATGCTACTAGTAAATCAGTCACACCAAGGCTTCAATAAGGAACACACAAGCAAGATGGTAAGCGCTATTGTTTTATATGTGCTTTTGGCGGCGGCGGCGCCATTCTGCCTTTGCGGCGGATCCCGGG.

[0050] Amino acid sequence (shown as SEQ ID NO.22): MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAADPG.

[0051] The PEDV gene references the S1 protein gene sequence of PEDV / CH-SBC / 2013 strain (Accession: KC787542), and its nucleotide sequence is shown in SEQ ID NO.23:

[0052]

[0053] The amino acid sequence is (shown in SEQ ID NO.24):

[0054] LPQDVTRCSANTNFRRFFSKFNVQAPAVVVLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFVSHIRGGHGFEIGISQEPFDPSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIPAHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQYVYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLTIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDTSVILAEGSIVLHTALGTNFSFVCSNSSDPHLATFAIPLGAIQVPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCSQVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSNSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGF。

[0055] Foldon sequence:

[0056] The nucleotide sequence (shown in SEQ ID NO.25): GGTTACATCCCGGAAGCTCCGCGTGACGGTCAGGCTTACGTTCGTAAAGACGGTGAATGGGTTCTGCTGTCTACCTTCCTG。

[0057] Amino acid sequence (SEQ ID NO.26): GYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0058] Linker:

[0059] Nucleotide sequence (SEQ ID NO.27): GGCAGCAGC.

[0060] Amino acid sequence (SEQ ID NO.28): GSS.

[0061] The sequence SEQ ID NO.27 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 labeled as 000. The sequence SEQ ID NO.28 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 labeled as 000.

[0062] His tag:

[0063] Nucleotide sequence (SEQ ID NO.29): CATCATCATCATCATCATTAA.

[0064] Amino acid sequence (shown in SEQ ID NO.30): 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.

[0065] Complete sequence (shown in SEQ ID NO.31):

[0066]

[0067] The constructed recombinant plasmid pFastBac-PEDV / S1 was sequenced and identified. After confirming that the fragment insertion was correct, downstream experiments were carried out.

[0068] 1.2 Obtaining recombinant rod particles.

[0069] 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.

[0070] 1.3 Identification of recombinant rod-shaped granules Bacmid-PEDV / S1

[0071] 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.

[0072] The primer sequences were: M13-F (SEQ ID NO. 32): GTTTCCCAGTCACGAC; S1 gene-specific reverse primer (SEQ ID NO. 33): 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.

[0073] 1.4 Extraction of Recombinant Rod Particles

[0074] 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.

[0075] 1.5 Obtaining recombinant baculovirus

[0076] 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.

[0077] 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 patted dry. After blocking, serially diluted PEDV / S protein monoclonal antibody (Guangzhou Qianxun Biotechnology) at 10µg / ml, 1µg / ml, and 100ng / ml levels 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.

[0078] 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.

[0079] Table 1: Identification of proteins expressed in the supernatant of transfection wells

[0080]

[0081] 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.

[0082] 2. Purification of PEDV / S1 recombinant protein

[0083] 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 recombinant 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.

[0084] 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.

[0085] 3. Indirect ELISA to identify the activity of PEDV / S1 recombinant protein

[0086] 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.

[0087] 4. Immunity in mice

[0088] 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 with 20 μg of PEDV-CH / SBC / 2013 strain S1 recombinant protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0089] 5. Screening of hybridoma cells

[0090] 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 and PEDV / S recombinant proteins (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 the PEDV / S recombinant protein. Positive cells were cloned to monoclonal status using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0091] Positive clones were screened using indirect ELISA.

[0092] PEDV / S1 recombinant protein, PEDV / S recombinant protein, and CMV / gB recombinant protein (nearshore protein, 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, 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, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (Solepro, diluted 5000 times with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer. Incubate at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (Beijing Meikewande, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with PEDV / S1 recombinant protein and PEDV / S recombinant protein but not with control recombinant protein were selected for subsequent experiments. The screening process is shown in Table 2.

[0093] Table 2. Screening results of monoclonal antibodies.

[0094]

[0095] 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.

[0096] 6. Purification of monoclonal antibodies

[0097] 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.

[0098] 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).

[0099] 7. Establishment and optimization of conditions for double-antibody sandwich ELISA

[0100] To improve the efficiency of pairing screening, 15 monoclonal antibodies with high binding titers (OD15) were selected. 450 Downstream experiments were conducted using nm values ​​greater than 1.0.

[0101] 7.1 HRP-labeled monoclonal antibodies:

[0102] 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, 0.5 mL of the diluted 1 mg antibody solution was added to the matching tube containing HRP, and the mixture was pipetted and incubated at room temperature for 1 hour, 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.

[0103] 7.2 Establishment of the double-antibody sandwich method:

[0104] The purified monoclonal antibody was diluted to a concentration of 1 μg / mL with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water, pH 9.6) and added to the microplate at 50 μL / well. The plate was coated overnight at 4 °C. The coating buffer was discarded the next day, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20). The plate was patted dry and blocked with 3% sucrose and 2% BSA at 150 μL / well. The plate was incubated at 37 °C for 2 h, the blocking buffer was discarded, and the plate was patted dry. The test antigens PEDV / S1 and PEDV / S mixed recombinant protein and control antigen protein (CMV / gB) were diluted to 100 ng / ml with PBS and added to an ELISA plate at 50 μL / well. The plate was incubated at 37°C for 35 min, washed four times with PBST, and blotted dry. Then, 50 μL / well of enzyme-labeled monoclonal antibody diluted 1000 times with PBS was added and incubated at 37°C for 35 min. The plate was washed four more times, blotted dry, and then 50 μL / well of TMB chromogenic buffer was added. 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 an ELISA reader. The screening process is shown in Table 3.

[0105] Table 3: OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value analysis.

[0106]

[0107] The results are shown in Table 3. Using the mixed recombinant protein of PEDV / S1 and PEDV / S as the positive antigen (i.e., PEDV in Table 3) and CMV / gB protein as the negative antigen, a sandwich sieve was used for detection. The P / N ratio was calculated, and the monoclonal antibody combination with the highest P / N ratio (highest positive detection value and low negative detection value) was selected as the optimal pairing. Monoclonal antibody 4E11 as the coating antibody and monoclonal antibody 1F3 as the labeling antibody showed the highest P / N ratio when detecting the mixed recombinant protein of PEDV / S1 and PEDV / S.

[0108] 7.3 Optimization of the Double Antibody Sandwich ELISA Method

[0109] The purified monoclonal antibody 4E11 was diluted with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L 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 plates were blocked with 3% sucrose and 2% BSA at 150 μL / well. The plates were incubated at 37°C for 2 h, and the blocking buffer was discarded. The test antigen (PEDV / S1 and PEDV / S mixed recombinant protein) and control antigen (CMV / gB) were diluted with PBS at 100 ng / mL and added to the microplate at 50 μL / well. The plates were incubated at 37°C for 35 min, washed 4 times with PBST, and then coated with PBS. HRP-labeled monoclonal antibody 1F3 diluted 1000, 2000, and 3000 times, 50 μL / well, was incubated at 37°C for 35 min. After washing the plate four times and patting dry, 50 μL / well of TMB chromogenic buffer 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, and the OD450nm value was measured using a microplate reader. The pairing condition with the highest P / N value was selected for sensitivity and specificity testing. The screening process is shown in Table 4.

[0110] Table 4: Results of optimization of dual-antibody ELISA conditions.

[0111]

[0112] In the table, PEDV is a mixed recombinant protein of PEDV / S1 and PEDV / S.

[0113] Table 4 shows that the combination with monoclonal antibody 4E11 at 1 ug / ml and HRP-labeled monoclonal antibody 1F3 diluted 2000 times resulted in the highest P / N value and was considered the optimal combination.

[0114] 7.4. Specificity and sensitivity analysis of double-antibody sandwich ELISA for detecting PEDV / S1 recombinant protein and PEDV / S recombinant protein.

[0115] After determining the optimal reaction conditions, including the coating monoclonal antibody 4E11 at a concentration of 1 μg / mL and the HRP-labeled monoclonal antibody 1F3 diluted 2000-fold, and following the above detection steps, PEDV / S1 and PEDV / S recombinant proteins were serially diluted with PBS buffer to obtain concentrations of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. Simultaneously, 50 μL of CMV / gB, PEDV / NP (full-length NP protein expressed in *E. coli*, GenBank: AAK38660), and TGEV / NP (full-length NP protein expressed in *E. coli*, NP_058428.1) recombinant proteins were added to each well at the same concentration for detection, to determine the sensitivity and specificity of the detection system for recombinant proteins. Figure 7 It can be seen that the double-antibody sandwich ELISA composed of this group of paired antibodies still showed a weak positive reaction when the PEDV / S1 recombinant protein and the PEDV / S recombinant protein were diluted to 100 pg / ml, and did not react with irrelevant antigens, demonstrating good sensitivity and specificity.

[0116] 8. Identification of binding activity of paired monoclonal antibodies

[0117] Following the aforementioned indirect ELISA method, the paired monoclonal antibodies were serially diluted to 10 μg / ml, 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml, and 100 pg / ml. Using another murine-derived unrelated monoclonal antibody, PRRSV-N monoclonal antibody from Jin Nuo Bai Tai, as a negative control, the binding activity of the antibodies to the PEDV / S1 recombinant protein was measured. Results are as follows... Figure 8 The results showed that 1F3 and 4E11 still reacted positively with PEDV / S1 at a dilution of 10 ng / ml, indicating high antibody activity.

[0118] Figure 8 In the table, 1F3, 4E11, and PRRSV-N represent monoclonal antibodies 1F3, 4E11, and PRRSV-N, respectively. Since all positive clones were confirmed during the screening phase to simultaneously recognize both the full-length PEDV / S1 and PEDV / S proteins, and the S1 region contains the major antigenic epitope, using PEDV / S1 as the representative antigen for coating the serially diluted monoclonal antibodies is sufficient to reflect the antibody binding activity.

[0119] 9. Light and heavy chain variable region sequences of paired monoclonal antibodies

[0120] 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.

[0121] This invention utilizes hybridoma technology to obtain a monoclonal antibody that can react simultaneously with the S protein of both G1-a and G2-a PEDV viruses, and successfully applied it to the double-antibody sandwich ELISA method. This method has excellent detection sensitivity for the recombinant PEDV-CV777 strain trimeric S protein and the PEDV-CH / SBC / 2013 strain S1 trimeric protein, and has important reference value for the diagnosis of PEDV infection and the quality control of vaccines.

[0122] Coated monoclonal antibody 4E11:

[0123] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.18:

[0124] GACATCGTGATGAGCCAGACCACCGCCACCCTGAGCGTGACCCCCGGCGACAGCGTGAGCCTGAGCTGCAGGGCCAGCCAGAGCGAGCAACACCCTGCACTGGTACCAGCAGAAGAGCCACGAGAGCCCCAGGCTGCTGATCAAGCTGATGCAGCAGAGGCAC AGCGGCATCCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAGCATCAACAGCGTGGAGACCGAGGACTTCGGCATGTACTTCTGCAGCCAGGGCGTGTCTGGGAGTGGCCCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0125] The amino acid sequence of the light chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.14:

[0126] DIVMSQTTATLSVTPGDSVSLSCRASQSESNTLHWYQQKSHESPRLLIKLMQQRHSGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCSQGVFWEWPFGGGTKLEIKRTV.

[0127] CDR area annotation:

[0128] The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 4E11 is shown in SEQ ID NO.4: RASQSESNTLH;

[0129] The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 4E11 is shown in SEQ ID NO.5: LMQQRHS;

[0130] The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 4E11 is shown in SEQ ID NO.6: SQGVFWEWP.

[0131] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.17:

[0132] CAGGTGCAGCTGCAGCAGTCTGGGCCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCTAGCTACACATTCACTGAATATGCTATGCACTGGGTGAGGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAATTATTAGTACTTACTATGGTAATACTAAC TACAACCAGAAGTTTAAGGACAAGGCCACAATGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAACTTGCCAGATTGACATCTGAGGATTCTGCCGTCTATTACTGTGCAAGATCGGGGAGGTACGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACTGTCTCTGCA.

[0133] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4E11 is shown in SEQ ID NO.13:

[0134] QVQLQQSGPELVRPGVSVKISCKGSSYTFTEYAMHWVRQSHAKSLEWIGIISTYYGNTNYNQKFKDKATMTVDKSSSTAYMELARLTSEDSAVYYCARSGRYGAMDYWGQGTSVTVSA.

[0135] CDR area annotation:

[0136] The amino acid sequence of the heavy chain variable region CDR-H1 of monoclonal antibody 4E11 is shown in SEQ ID NO.1: EYAMH;

[0137] The amino acid sequence of the heavy chain variable region CDR-H2 of monoclonal antibody 4E11 is shown in SEQ ID NO.2: IISTYYGNTNYNQKFKD;

[0138] The amino acid sequence of the heavy chain variable region CDR-H3 of monoclonal antibody 4E11 is shown in SEQ ID NO.3: SGRYGAMDY.

[0139] Labeled monoclonal antibody 1F3:

[0140] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.20:

[0141] GAGATCCAGATGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCAAGGCCAGGCAGAGCTTCGACTACCACAGCGAGAGCGTGATGAACTGGTACCAGCAGAAGCCCGGCCAGCCCCCCCAAGCTGCTGATCTTCCCCGGCCAGAAC ATCAGCGCCGGCATCCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCTGGAGGAGGAGGACGCCGCCACCTACTACTGCACCAGCAGCATGTGGCTGACCTTCAGCTTCGGCAGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0142] The amino acid sequence of the light chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO. 16:

[0143] EIQMTQSPASLAVSLGQRATISCKARQSFDYHSESVMNWYQQKPGQPPKLLIFPGQNISAGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCTSSMWLTFSFGGSGTKLEIKRTV.

[0144] CDR area annotation:

[0145] The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 1F3 is shown in SEQ ID NO.10: KARQSFDYHSESVMN;

[0146] The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 1F3 is shown in SEQ ID NO.11: PGQNISA;

[0147] The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 1F3 is shown in SEQ ID NO.12: TSMWLTFS.

[0148] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO. 19:

[0149] CAGGTGCAGCTGCACCAGTCTGGACCTGGCCTCGTGAAAACCTTCTCAGTCTCTGTCTCTCACCTGCTTTGTCACTGGCTACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAATGGTTGGGCTACATAAGCTACGCCGGTCGCAATAACTACAA CCCATCTCTCAACAATCGAATCTCCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGACTCAGCTACATATTACTGTGCAAGAGGAATACTTCGCTGGTGGCTACTCGTTTCCTTACTGGGGCCAAGGGACTCTGGTCACAGTCTCCTCA.

[0150] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO. 15:

[0151] QVQLHQSGPGLVKPSQSLSLTCFVTGYSITSGYYWNWIRQFPGNKLEWLGYISYAGRNNYNPSLNNRISITRDTSKNQFFLKLNSVTTEDSATYYCAREEYFAGGYSFPYWGQGTLVTVSS.

[0152] CDR area annotation:

[0153] The CDR-H1 amino acid sequence of the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.7: SGYYWN;

[0154] The CDR-H2 amino acid sequence of the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.8: YISYAGRNNYNPSLNN;

[0155] The CDR-H3 amino acid sequence of the heavy chain variable region of monoclonal antibody 1F3 is shown in SEQ ID NO.9: EEYFAGGYSFPY.

[0156] 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.

[0157] 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 combination for detecting porcine epidemic diarrhea virus S protein, the monoclonal antibody combination comprising monoclonal antibody 4E11 and monoclonal antibody 1F3; the heavy chain variable region of the monoclonal antibody 4E11 comprises 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 4E11 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4-SEQ ID NO. 6 respectively; the heavy chain variable region of the monoclonal antibody 1F3 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7-SEQ ID NO. 9 respectively; the light chain variable region of the monoclonal antibody 1F3 comprises three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 10-SEQ ID NO. 12 respectively.

2. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus S protein according to claim 1, characterized by, the monoclonal antibody combination can recognize G1 porcine epidemic diarrhea virus S protein and / or G2 porcine epidemic diarrhea virus S protein.

3. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus S protein according to claim 1, characterized by, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 4E11 is shown in SEQ ID NO. 13;the amino acid sequence of the light chain variable region of the monoclonal antibody 4E11 is shown in SEQ ID NO.

14.

4. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus S protein according to claim 1, characterized by, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO. 15;the amino acid sequence of the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.

16.

5. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus S protein according to claim 1, characterized by, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4E11 is shown in SEQ ID NO. 17;the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4E11 is shown in SEQ ID NO.

18.

6. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus S protein according to claim 1, characterized by, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO. 19;the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1F3 is shown in SEQ ID NO.

20. 7.Use of the monoclonal antibody combination of claim 1 in the preparation of a tool for detecting porcine epidemic diarrhea virus S protein.

8. Use according to claim 7, characterized in that, the tool comprises reagents, kits, test strips and antibody chips.

9. Use according to claim 8, characterized in that, the kit comprises a double antibody sandwich ELISA kit;the ELISA kit uses monoclonal antibody 4E11 as a coating antibody and monoclonal antibody 1F3 as a labeled antibody. 10.Use of the monoclonal antibody combination of claim 1 in the quality control of porcine epidemic diarrhea virus vaccine.

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