A monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein and application thereof. The combination is composed of monoclonal antibodies 1E11 and 5C8, can efficiently and specifically recognize porcine epidemic diarrhea virus NP protein, the amino acid sequences of the complementarity determining regions of the heavy chain and light chain variable regions are clear, are respectively shown as SEQ ID NO. 1-12, ensure high specificity and high affinity of the antibody, the antibody combination can effectively capture and detect the target antigen, reduce the risk of non-specific binding and cross reaction. The combination shows excellent sensitivity and specificity in a colloidal gold immunochromatography detection platform, and provides a stable and reliable biological recognition tool for rapid and accurate detection of porcine epidemic diarrhea virus.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a combination of monoclonal antibodies for detecting porcine epidemic diarrhea virus NP protein and its application. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) belongs to the family Coronaviridae and is a single-stranded positive-sense RNA virus. This virus primarily infects the intestinal epithelial cells of pigs, causing a gastrointestinal infectious disease characterized by acute watery diarrhea, vomiting, dehydration, and severe enteritis. The virus spreads rapidly, and is particularly pathogenic in newborn piglets, accounting for a high mortality rate.

[0003] PEDV virus contains four main structural proteins: spike protein (S), membrane protein (M), envelope protein (E), and nucleocapsid protein (NP). Among these PEDV structural proteins, the nucleocapsid protein is the core packaging material for the viral genomic RNA. It is expressed in large quantities early in viral replication, exhibits high immunogenicity, and is abundant. Furthermore, the NP gene sequence is highly conserved among different PEDV strains, and its protein antigen is stable, thus it is often selected as a target protein for immunoassay.

[0004] In the clinical prevention and control of porcine epidemic diarrhea (PEDV), early screening and diagnosis of infected pigs are crucial for cutting off virus transmission and implementing precise intervention. Currently, PEDV detection methods mainly include nucleic acid testing and immunological testing. While nucleic acid testing has high sensitivity, it also has high requirements for the laboratory environment, equipment, and operators, making it difficult to apply on a large scale in grassroots pig farms or under field conditions. In contrast, immunological testing has a simpler procedure and is more suitable for on-site screening and routine monitoring.

[0005] Immunological products for detecting PEDV viral antigens typically use targeted, specific antibodies as their core component. The performance of these antibodies directly affects the accuracy of the antigen detection results. Polyclonal antibodies often have complex origins, significant batch-to-batch variations, and insufficient specificity, making them unsuitable for high-standard diagnostics. Monoclonal antibodies, on the other hand, offer advantages such as single epitope recognition, high specificity, and good batch-to-batch consistency, thus significantly improving the performance of detection products and making them more popular in immunological testing.

[0006] Colloidal gold immunochromatographic test strips are easy to use, provide intuitive and readable results, and require no complex instruments. They have been widely used in the field detection of many animal diseases. The performance of this type of test product largely depends on the quality of the antibodies used and their pairing combinations. Using monoclonal antibodies with well-defined sequences and stable characteristics can significantly improve the consistency and repeatability of the test products and lay the foundation for large-scale production and quality control.

[0007] Therefore, it is still necessary to screen for PEDV NP monoclonal antibodies that balance detection specificity and sensitivity to meet the market demand for high-performance diagnostic monoclonal antibody raw materials. Summary of the Invention

[0008] (a) Technical problems to be solved

[0009] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a combination of highly specific and highly sensitive monoclonal antibodies for detecting porcine epidemic diarrhea virus (PEDV) NP protein and its application in rapid immunoassay tools. It solves the technical problems of insufficient antibody pairing specificity, high risk of cross-reactivity, and poor batch-to-batch repeatability in existing PEDV antigen detection reagents.

[0010] (II) Technical Solution

[0011] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0012] In a first aspect, this application provides a monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein, said monoclonal antibody combination comprising monoclonal antibody 1E11 and monoclonal antibody 5C8.

[0013] The heavy chain variable region of the monoclonal antibody 1E11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.1-SEQ ID NO.3, respectively.

[0014] The light chain variable region of the monoclonal antibody 1E11 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0015] The heavy chain variable region of the monoclonal antibody 5C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.7-SEQ ID NO.9, respectively.

[0016] The light chain variable region of the monoclonal antibody 5C8 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.10-SEQ ID NO.12, respectively.

[0017] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO. 13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO. 14.

[0018] In some embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO. 15; the amino acid sequence of the light chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO. 16.

[0019] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO. 17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO. 18.

[0020] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO. 19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO. 20.

[0021] Secondly, this application provides the use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting porcine epidemic diarrhea virus NP protein.

[0022] In some embodiments, the tool includes colloidal gold test strips, test strips, reagents, kits, and antibody chips.

[0023] In some embodiments, the colloidal gold test strip or test card uses monoclonal antibody 1E11 as the capture antibody and monoclonal antibody 5C8 as the labeling antibody.

[0024] In some embodiments, the colloidal gold test strip or test card includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

[0025] In some embodiments, the nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 1E11, the control line is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5C8.

[0026] (III) Beneficial Effects

[0027] The monoclonal antibody assemblies for detecting porcine epidemic diarrhea virus (PEDV) nucleocapsid protein (NP protein) provided in this invention consist of monoclonal antibody 1E11 and monoclonal antibody 5C8. The amino acid sequences of the complementarity-determining regions (CDRs) of the heavy and light chain variable regions are clearly defined, as shown in SEQ ID NO. 1–12, ensuring high specificity and high affinity of the antibodies. The antibody assemblies can effectively capture and detect the target antigen, reducing the risk of non-specific binding and cross-reaction. This assemblies exhibit excellent sensitivity and specificity in detection platforms such as colloidal gold immunochromatography. The monoclonal antibody assemblies of this application provide key raw materials for the rapid, accurate, and reliable detection of porcine epidemic diarrhea virus, and provide technical support for establishing a standardized, high-throughput, and low-cost swine disease monitoring system. Attached Figure Description

[0028] 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.

[0029] Figure 1 The SDS-PAGE results for the purification of PEDV-NP protein are shown in the figure.

[0030] Figure 2 The image shows the identification results of the PEDV-NP recombinant protein.

[0031] Figure 3 The image shows the identification results of the purified monoclonal antibody;

[0032] Figure 4 This is a schematic diagram of colloidal gold assembly;

[0033] Figure 5 This is a graph showing the specificity analysis results of the test strip;

[0034] Figure 6 This is a graph showing the sensitivity test results of the test strips;

[0035] Figure 7 This is a graph showing the results of antibody binding identification. Detailed Implementation

[0036] 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.

[0037] This invention utilizes hybridoma technology to successfully prepare monoclonal antibodies capable of specifically recognizing the porcine epidemic diarrhea virus nucleocapsid protein (PEDV-NP). Systematic screening yielded antibody pairs with complementary properties suitable for paired use. These monoclonal antibody pairs are used in the construction of colloidal gold immunochromatographic test strips, suitable for the rapid detection of PEDV-NP protein. Validation has shown that this antibody pairing system possesses good detection sensitivity and specificity, providing a crucial antibody raw material foundation for establishing accurate, reliable, and convenient on-site rapid PEDV detection reagents.

[0038] The detection method described in this application is not intended for disease diagnosis and treatment.

[0039] The porcine epidemic diarrhea virus nucleoprotein in this application includes recombinant porcine epidemic diarrhea virus nucleoprotein (PEDV-NP recombinant protein) and natural porcine epidemic diarrhea virus nucleoprotein.

[0040] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, they should be considered part of the specification.

[0041] Example 1

[0042] 1. Expression of PEDV-NP recombinant protein

[0043] The N gene of PEDV is derived from AAK38660, synthesized by Qingke Biotechnology and cloned into the pET28a vector.

[0044] The nucleotide sequence of the PEDV-N gene is shown in SEQ ID NO.21:

[0045]

[0046] The amino acid sequence is shown in SEQ ID NO.22:

[0047] MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHGDLRYRTRTEGVFWVAKEGAKTEPTNL GVRKASEKPIIPKFSQQLPSVVEIVEPNTPPASRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSQNRGNNQGRGASQNRGGNNNNNNKSRNQSNNRNQSNDRGGVTSRDD LVAAVKDALKSLGIGENPDRHKQQQKPKQEKSDNSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAA LLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKLQRKKEKKNKRETTLQQHEEAIYDDVGAPSDVTHANLEWDTAVDGGDTAVEIINEIFDTGN.

[0048] The recombinant plasmid pET28a-PEDV / NP was transformed into BL21(DE3) competent cells using standard methods and induced to express the gene. Specifically, the transformed bacteria were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours until OD500 reached. 600 The concentration was 0.6-0.9, and the final concentration was 0.1 mM IPTG. The cells were collected after induction at 30℃ and 200 rpm for 4 hours.

[0049] 2. Purification of recombinant proteins

[0050] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. 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 fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, followed by 5-second sonication intervals. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio and loaded onto the chromatography column. The column was washed with buffer A, followed by gradient elution with buffer B. The elution peak of 500 mM imidazole was collected, concentrated to less than 2 ml, and further purified by passing through a loading loop onto a Superdex 75 molecular sieve. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A to obtain the purified protein. The SDS-PAGE electrophoresis results of the purified protein are shown below. Figure 1 Protein concentration was determined using an ultra-micro spectrophotometer and stored at -20°C.

[0051] Figure 1 In the diagram, M represents the protein marker, number 1 represents the supernatant obtained after bacterial induction, number 2 represents the supernatant obtained after centrifugation following bacterial cell lysis, number 3 represents the 100mM imidazole elution buffer, number 4 represents the 500mM imidazole elution buffer, and number 5 represents the target protein collected by molecular sieve.

[0052] The results are as follows Figure 1 As shown, there is a distinct main band near the expected size position (between 43-55 kD), and PEDV-NP with high purity was obtained after purification by Ni column affinity chromatography and molecular sieve, which can be used for subsequent experiments.

[0053] 3. Indirect ELISA was used to identify the activity of the PEDV-NP recombinant protein.

[0054] The recombinant protein PEDV-NP was coated onto an ELISA plate, and its reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was PEDV-NP mouse monoclonal antibody (labeled: Ab) from Luoyang Gushuo Biotechnology. The recombinant protein was first 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, 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 a gradient 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 OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 .

[0055] The reaction of the expressed PEDV-NP recombinant protein with the commercially available PEDV-NP mouse monoclonal antibody was identified by an indirect ELISA method. The PEDV-NP recombinant protein was coated (1 μg / ml). The results showed that the commercially available monoclonal antibody still reacted positively with the PEDV-NP recombinant protein in the dilution range of 10 ng / ml to 100 ng / ml, indicating that the PEDV-NP recombinant protein has good biological activity and can be used for further experiments.

[0056] 4. Screening of PEDV-NP monoclonal antibodies

[0057] 4.1 Mouse Immunization

[0058] Female BALB / c mice were immunized subcutaneously at multiple sites after 6 weeks of age by a mixture of recombinant PEDV-NP protein and an equal volume of Freund's complete adjuvant (total volume 200 μL), at a dose of 20 μg / mouse. At weeks 2 and 4, booster immunizations were administered subcutaneously at multiple sites by a mixture of the same dose and 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-NP protein via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0059] 4.2 Screening of hybridoma cells

[0060] 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 up the well, clones that reacted positively with the PEDV-NP recombinant protein were selected using an indirect ELISA method. Since the immunogen is a prokaryotically expressed recombinant protein containing a His tag, background components needed to be screened to identify specific cell lines targeting the PEDV-NP protein. Positive cells were cloned to a monoclonal state using limiting dilution, and then the cell lines were expanded and cryopreserved.

[0061] 4.3 Screening of positive clones using indirect ELISA method

[0062] PEDV-NP and pET28a-BKV-I / VP1 recombinant proteins were coated in 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 blotted 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 secondary antibody (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 OD450nm value using a microplate reader. Select positive cell lines that react with PEDV-NP recombinant protein but not with the control pET28a-BKV-I / VP1 recombinant protein for subsequent experiments. The screening process is shown in Table 1.

[0063] Table 1. Screening results of monoclonal antibodies

[0064]

[0065] 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.

[0066] 4.4 Purification and Identification of Monoclonal Antibodies

[0067] Centrifuge the ascites fluid at 12000 rpm for 10 minutes, collect 1 ml of the supernatant, add 4 ml of acetate-sodium acetate buffer (0.06 M, pH 4.5), mix well, and slowly add 10 μl of n-octanoic acid while stirring. After the addition is complete, continue stirring for 30 minutes. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the supernatant. Filter the supernatant through defatted cotton, and add saturated ammonium sulfate at a final volume ratio of 50% (V / V) while stirring. After the addition is complete, continue stirring for 30 minutes, and let it precipitate overnight at 2–8°C. Centrifuge at 12000 rpm for 30 minutes at 2–8°C, and collect the precipitate. After the precipitate was completely dissolved in binding buffer (20 mM PB, 150 mM NaCl, pH 7.4), it was filtered through a 0.22 μm filter. The filtered sample was then pumped slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. The column was connected to a protein purification instrument, and the sample was washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled off. Elution was then performed with elution buffer (0.1 M glycine, pH 2.7), and the elution peak was collected. The collected sample was adjusted to neutral with 1 M Tris-HCl at pH 9 and placed in a dialysis bag (MW: 8000-14000). Dialysis was performed at 2-8 °C in 20 mM PBS pH 7.4 solution for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody.

[0068] The purified monoclonal antibody was diluted to 1 μg / ml, and its binding activity with the PEDV-NP recombinant protein and the irrelevant antigen BKV-I / VP1 was detected by indirect ELISA (the BKV-I / VP1 recombinant protein was obtained by the applicant through recombinant expression and purification, see patent CN120623324B). Specific results are shown below. Figure 3 The results showed that the selected monoclonal antibody specifically binds to the PEDV-NP protein and does not react with the irrelevant antigen BKV-I / VP1, and can be used for subsequent testing.

[0069] 5. Colloidal gold pairing of PEDV-NP monoclonal antibodies

[0070] 5.1 Preparation of antibody-colloidal gold labeled complex.

[0071] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes until the colloidal gold particles stabilized, and then cooled to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in gradients of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. The optimal conjugation effect was ultimately found at 5 μL. After mixing, 5 μg of the PEDV-NP monoclonal antibody to be labeled was added, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and incubation at room temperature was continued for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution solution (0.05 M Tris + 0.9% NaCl + 1% BSA + 5% Sucrose + 0.5% N100 + 0.1% Proclin 300, pH 9.2) to obtain the antibody-colloidal gold labeled complex. Store at 4°C protected from light for later use.

[0072] 5.2 Preparation of test strips coated with different monoclonal antibodies of PEDV-NP.

[0073] The 21 selected PEDV-NP monoclonal antibodies were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1 mg / mL with 0.1 M PB + 0.5% NaCl + 2% BSA + 0.5% Trehalose) were sprayed horizontally in a linear pattern using a scribing instrument, with each line containing 0.8 μL / cm, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS (pH 7.4), were then sprayed horizontally in a linear pattern at 6 mm intervals, forming the control line (C line).

[0074] 5.3 Screening of paired monoclonal antibodies

[0075] Nitrocellulose membranes streaked with different monoclonal antibodies against PEDV-NP were individually paired with different colloidal gold-labeled monoclonal antibodies. PEDV-NP protein was diluted to 20 ng / mL for detection, while BKV-I / VP1 recombinant protein was diluted to 20 ng / mL as a negative antigen for detection. The screening process and results are shown in Table 2 and a continuation of Table 2. Table 2 shows the combinations that showed the deepest color development for PEDV-NP protein and did not react with the control protein. Therefore, the optimal pairing for detecting recombinant PEDV-NP protein was determined to be 1E11 streaking and 5C8 gold labeling.

[0076] Table 2: Results of screening paired monoclonal antibodies using PEDV-NP recombinant protein 1

[0077]

[0078] Table 2 (Continued): Results of screening paired monoclonal antibodies using PEDV-NP recombinant protein 2

[0079]

[0080] In the table, - indicates negative, meaning no color develops; + / ++ / +++ indicates positive, meaning a color reaction occurs. The more + signs there are, the darker the color, indicating a stronger positive reaction.

[0081] 6. Preparation and assembly of colloidal gold test strips

[0082] Preparation of gold-labeled pads: Using a 6mm×300mm glass fiber membrane, the prepared colloidal gold-labeled antibody was evenly dropped onto the glass fiber at a rate of 1200ul / strip, allowed to air dry naturally, and then dried at 37℃ for 2 hours for later use.

[0083] See Figure 4 , Figure 4 This diagram illustrates the assembly of the colloidal gold test strip. A 60mm × 300mm PVC backing plate serves as the support, upon which are attached a sample pad, a gold pad (also known as a gold-labeled pad), an NC membrane (nitrocellulose membrane), and an absorbent pad. The nitrocellulose membrane is coated with two lines: a detection line (T line, monoclonal antibody 1E11 scratch) and a control line (C line, goat anti-mouse IgG). The strip is labeled with 5C8 gold and dried at 37℃ for 12 hours before use. The assembled plate is then cut into 4mm strips using a strip cutter. These strips are then wrapped with colloidal gold plastic casings, exposing the sample pad at the sample application well and the control and detection lines at the result observation wells. The colloidal gold test strip assembly is now complete.

[0084] 7. Test strip specificity test

[0085] Recombinant protein samples: PEDV-NP recombinant protein, TGEV-NP recombinant protein (the applicant expressed full-length TGEV NP using E. coli, with the amino acid sequence derived from NP_058428.1), PDCoV-NP recombinant protein (the applicant expressed full-length PDCoV NP using E. coli, with the gene sourced from GenBank: MF095123.1), and PEDV-S recombinant protein (nearshore, DRA252, PEDV-CV777 strain) were diluted to 1 μg / mL with sample diluent for detection.

[0086] Vaccine samples: Porcine circovirus type 2 inactivated vaccine (WH strain), porcine dyscoronavirus inactivated vaccine (CHN-HN-2014 strain), and porcine reproductive and respiratory syndrome live vaccine (R98 strain). After dissolving the above vaccines, they were diluted 1:1 with sample diluent.

[0087] Add 80 μL of the diluted sample to the sample well of the test strip. Simultaneously, add another 80 μL of the diluted solution to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid. See below for test results. Figure 5 .

[0088] The test strip can detect PEDV-NP recombinant protein very well, and there is no cross-reactivity with other recombinant proteins and vaccine strains, indicating that the test strip has good specificity.

[0089] 8. Sensitivity test of test strips

[0090] The PEDV-NP recombinant protein was diluted at concentrations of 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL before detection. Figure 6 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 0.5 ng / mL, while the blank dilution, i.e. the sample dilution (0.05 M Tris + 0.9% NaCl + 1% BSA + 1% Sucrose + 0.5% N100 + 0.1% Proclin 300, pH 9.2) (0 ng / mL), did not show color development, indicating that the limit of detection for PEDV-NP recombinant protein on the test strip is 0.5 ng / mL.

[0091] 9. Identification of the binding activity of paired antibodies to antigens

[0092] Based on the selection of paired antibodies using colloidal gold, the selected paired monoclonal antibodies and other murine-derived unrelated monoclonal antibodies were serially diluted (concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, respectively) using the aforementioned indirect ELISA method to evaluate their binding activity with PEDV-NP recombinant protein. Results are shown below. Figure 7 , Figure 7 Ctrl represents the mouse monoclonal antibody against the negative control PEDV S protein (see monoclonal antibody 4E11 in patent CN120943951B).

[0093] This invention utilizes hybridoma technology to screen and obtain monoclonal antibody pairs capable of specifically recognizing the PEDV-NP protein. These antibody pairs efficiently recognize the PEDV-NP recombinant protein, exhibiting good specificity and sensitivity. This invention applies these monoclonal antibody pairs to an immunoassay platform, constructing rapid test strips or test cards based on colloidal gold immunochromatography technology. These test strips demonstrate high sensitivity to the PEDV-NP recombinant protein and show no cross-reactivity with other proteins, making them suitable for rapid, on-site detection.

[0094] 10. Gene sequence of monoclonal antibodies

[0095] 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 sequence of the variable region of the light and heavy chains of the monoclonal antibody.

[0096] Sequence of the membrane-applied monoclonal antibody 1E11.

[0097] Light chain variable region nucleotide sequence:

[0098] The nucleotide sequence encoding the variable region of the light chain of monoclonal antibody 1E11 is shown in SEQ ID NO.18:

[0099] GATGTTGTGGTGACTCAAACTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATCAGTTCCGATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACGTCCAATCTGGC TTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTGCCACCAGGGTATTACTATACCATTCACGTTCGGCTCGGGGACCAAGCTGGAGCTGAAACGTACGGTG.

[0100] Light chain variable region amino acid sequence:

[0101] The amino acid sequence of the light chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO.14:

[0102] DVVVTQTPTTMAASPGEKITITCSASSSISSDYLHWYQQKPGFSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTIGTMEAEDVATYYCHQGITIPFTFGSGTKLELKRTV.

[0103] Light chain CDR area annotation:

[0104] The amino acid sequence of the light chain variable region CDR-L1 of monoclonal antibody 1E11 is shown in SEQ ID NO.4:

[0105] CDR-L1: SASSSISSDYLH;

[0106] The amino acid sequence of the light chain variable region CDR-L2 of monoclonal antibody 1E11 is shown in SEQ ID NO. 5:

[0107] CDR-L2: RTSNLAS;

[0108] The amino acid sequence of the light chain variable region CDR-L3 of monoclonal antibody 1E11 is shown in SEQ ID NO. 6:

[0109] CDR-L3: HQGITIPFT.

[0110] Heavy chain variable region nucleotide sequence:

[0111] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO.17:

[0112] CAGATGCAGCTCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACTTGCACTGTCTCTGGGTTTTCATTAACCAGATATGGTGTACATTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGCTGGTGGAAACACAAATTATAA TTCGGCTCTCATGTCCAGACTGAGCATCAGCAAAGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGGCATATACTATTGTGCCAATCCTACGATAGTAGCTCCCTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0113] Heavy chain variable region amino acid sequence:

[0114] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO.13:

[0115] QMQLQESGPGLVAPSQSLSITCTVSGFSLTRYGVHWVRQPPGKGLEWLGVIWAGGNTNYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTGIYYCANPTIVAPYGMDYWGQGTSVTVSS.

[0116] Heavy chain CDR region annotation:

[0117] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO. 1:

[0118] CDR-H1: RYGVH;

[0119] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO. 2:

[0120] CDR-H2: VIWAGGNTNYNSALMS;

[0121] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 1E11 is shown in SEQ ID NO. 3:

[0122] CDR-H3: PTIVAPYGMDY.

[0123] The 5C8 sequence of the gold monoclonal antibody.

[0124] Light chain variable region nucleotide sequence:

[0125] The nucleotide sequence encoding the variable region of the 5C8 light chain of the monoclonal antibody is shown in SEQ ID NO.20:

[0126] CAAATTGTTCTCACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAGATAGTAACAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCATGGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATTATACCTCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTG.

[0127] Light chain variable region amino acid sequence:

[0128] The amino acid sequence of the variable region of the 5C8 light chain of the monoclonal antibody is shown in SEQ ID NO.16:

[0129] QIVLTQSPSSLAMSVGQKVTMSCKSSQSLLDSNNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFMGSGSGTDFTLTISSVQAEDLADYFCQQHYTSPYTFGGGTKLEIKRTV.

[0130] Light chain CDR area annotation:

[0131] The amino acid sequence of the complementarity-determining region CDR-L1 of the light chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 10:

[0132] CDR-L1: KSSQSLLDSNNQKNYLA;

[0133] The amino acid sequence of the complementarity-determining region CDR-L2 of the light chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 11:

[0134] CDR-L2: FASTRES;

[0135] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 12:

[0136] CDR-L3: QQHYTSPYT.

[0137] Heavy chain variable region nucleotide sequence:

[0138] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO.19:

[0139] GAGGTGCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGATAAAGCAGAGGCCTGGACATGGCCTTGAGTGGGTTGGAGAGATTTTACCTGGAAGTGGTAATACTAACTA CATTGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGCAAGGATCTATGGTTACGACTGGTACTTCGATGTCTGGGGCGCGGGGACCACGGTCACTGTCTCTGCA.

[0140] Heavy chain variable region amino acid sequence:

[0141] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO.15:

[0142] EVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWIKQRPGHGLEWVGEILPGSGNTNYIEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARIYGYDWYFDVWGAGTTVTVSA.

[0143] Heavy chain CDR region annotation:

[0144] The amino acid sequence of the complementarity-determining region (CDR-H1) of the heavy chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 7:

[0145] CDR-H1: SYWIE;

[0146] The amino acid sequence of the complementarity-determining region (CDR-H2) of the heavy chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 8:

[0147] CDR-H2: EILPGSGNTNYIEKFKG;

[0148] The amino acid sequence of the complementarity-determining region (CDR-H3) of the heavy chain variable region of monoclonal antibody 5C8 is shown in SEQ ID NO. 9:

[0149] CDR-H3: IYGYDWYFDV.

[0150] 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.

[0151] 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. In particular, as long as there is no structural conflict, the various technical features mentioned in the embodiments can be combined in any manner.

Claims

1. A monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 1E11 and monoclonal antibody 5C8. The heavy chain variable region of the monoclonal antibody 1E11 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 1E11 includes 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 5C8 includes 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 5C8 includes 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 NP protein according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 1E11 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting porcine epidemic diarrhea virus NP protein according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5C8 is shown in SEQ ID NO.

20.

6. The use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting porcine epidemic diarrhea virus NP protein.

7. The application according to claim 6, characterized in that, The tools include colloidal gold test strips, test strips, reagents, kits, and antibody chips.

8. The application according to claim 7, characterized in that, The colloidal gold test strip or test card uses monoclonal antibody 1E11 as the capture antibody and monoclonal antibody 5C8 as the labeling antibody.

9. The application according to claim 8, characterized in that, The colloidal gold test strip or test card includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper attached to a backing plate.

10. The application according to claim 9, characterized in that, The nitrocellulose membrane is provided with a detection line and a control line; the detection line is coated with monoclonal antibody 1E11, the control line is coated with goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 5C8.

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