Senecavirus a swine-derived fab antibody and its competitive elisa detection method

Abstract

This invention discloses a porcine Fab antibody against Seneca virus A (SVA) and its competitive ELISA detection method. The amino acid sequences of the heavy and light chain variable regions of the neutralizing Fab genetically engineered antibody are shown in SEQ ID No. 4 and SEQ ID No. 7. The antibody exhibits high affinity for SVA. A competitive ELISA detection method was established, determining the optimal SVA particle coating concentration to be 1 μg / mL, the optimal working concentration of the competitive antibody to be 0.5 μg / mL, the optimal dilution ratio of the serum sample to be tested to be 1:10, and the optimal dilution ratio of the enzyme-labeled avidin to be 1:30000. The highest sensitivity and specificity (96.88% and 100%, respectively) were achieved when the inhibition percentage (PI) was 47%, and the antibody did not react with positive sera from common major porcine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, and the inter-assay coefficient of variation ranged from 1.19% to 11.72%, indicating good reproducibility. This method and VNT detection were used to simultaneously detect 224 clinical porcine serum samples, with a concordance rate of 93.75%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a porcine Fab antibody against Seneca virus A and its competitive ELISA detection method. Background Technology

[0002] Senecavirus A (SVA) is the sole member of the genus Senecavirus in the family Picornaviridae. It is the pathogen that causes vesicular lesions in pigs. SVA is a non-enveloped, single-stranded, positive-sense RNA virus with a genome length of approximately 7.3 kb, consisting of a 5′-untranslated region (UTR) (0.66 kb), an open reading frame (ORF) (6.54 kb), and a 3′-UTR (0.07 kb). The SVA ORF encodes a large polyprotein precursor, which is then cleaved by proteases to form four structural proteins (VP4, VP2, VP3, and VP1) and eight non-structural proteins (L, 2A, 2B, 2C, 3A, 3B, 3C, and 3D). Clinical symptoms of SVA infection include vesicles and ulcers on the snore, oral epithelium, and coronary band, leading to lameness. In young piglets, it can cause lethargy, diarrhea, neurological symptoms, and acute death, with mortality rates as high as 40%–80% in piglets aged 0–3 days and 0%–30% in piglets aged 4–7 days. The clinical symptoms of SVA infection are similar to those caused by foot-and-mouth disease, vesicular stomatitis, and other swine diseases, making them difficult to distinguish clinically. Since October 2014, SVA outbreaks have occurred in multiple countries, and the spread has continued to expand, causing significant economic losses to the pig industry. Therefore, establishing rapid and specific diagnostic methods is essential for the prevention and control of SVA infection.

[0003] Currently, the main diagnostic methods for SVA include reverse transcription-PCR (RT-PCR), virus neutralization assay (VNT), indirect fluorescent antibody assay (IFA), and enzyme-linked immunosorbent assay (ELISA). In comparison, ELISA is simple, rapid, and easy to perform. However, the antibodies used in these ELISA methods are mostly murine monoclonal antibodies or rabbit polyclonal antibodies. The preparation of rabbit polyclonal antibodies and murine monoclonal antibodies is not only time-consuming and labor-intensive, requiring animal culling, but the resulting polyclonal and monoclonal antibodies, being of rabbit or murine origin, may recognize epitopes that differ from the antigen epitopes recognized by the animal itself. This necessitates improvements in the specificity and sensitivity of methods based on these antibodies. In recent years, monoclonal antibody preparation technology based on a combination of single-cell technology and flow cytometry (i.e., single B-cell antibody technology) has further optimized the development of antibody preparation technology, making it possible to obtain animal-derived monoclonal antibodies against a specific antigen. The core of this technology involves isolating antigen-specific B cells from immunized or infected animals (tissues or peripheral blood), then amplifying the antibody heavy and light chains from individual antibody-secreting B cells using single-cell PCR technology, and finally expressing them in mammalian cells to obtain biologically active monoclonal antibodies. The antibodies obtained using this technology possess the naturally paired heavy and light chains, which is crucial for antibody specificity and affinity. Furthermore, this method features high genetic diversity, high efficiency, and all-natural origin. This technology has already been applied to the search for neutralizing antibodies against some important human infectious diseases, such as HIV and influenza.

[0004] Fab antibodies are antigen-binding fragments of immunoglobulin IgG, composed of a complete light chain (variable region VL and constant region CL) and a partial heavy chain structure (variable region VH and a constant region fragment CH1). The light and heavy chains are linked by a disulfide bond. Their length is approximately one-third that of full-length antibodies, lacking an Fc fragment, and their small size makes them less prone to steric hindrance. Therefore, compared to full-length IgG, Fab antibodies have advantages such as strong tissue penetration, easier reach to lesions, and low immunogenicity. Furthermore, the absence of an Fc fragment prevents antibody-dependent cytotoxicity or complement-dependent cytotoxicity. No ELISA antibody detection method for SVA has been reported based on the animal-derived antibody obtained using single B cell antibody technology. Summary of the Invention

[0005] The purpose of this invention is to provide a porcine Fab antibody against Seneca virus A and its competitive ELISA detection method.

[0006] This invention provides a Seneca virus A porcine Fab antibody, wherein the amino acid sequence of the heavy chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No. 3; and the amino acid sequence of the light chain of the anti-Seneca virus porcine Fab genetically engineered antibody is shown in SEQ ID No. 6.

[0007] This invention also provides the application of Seneca virus A porcine Fab antibody in the preparation of reagents or kits for detecting Seneca virus.

[0008] This invention also provides a competitive ELISA detection method based on porcine Fab antibodies against Seneca virus A, comprising the following steps:

[0009] (1) Coating: Dilute Seneca virus A antigen with PBS to 1 μg / mL, add 100 μL / well to the microplate, incubate overnight at 4℃, wash the plate 3 times with PBST, and pat dry.

[0010] (2) Blocking: Prepare PBST solution containing 1% BSA and 5% sucrose, 100 μL / well, and block at 37℃ for 1 h;

[0011] (3) Detection: Take the test serum, standard negative serum, and standard positive serum diluted 1:10 and mix them with porcine Fab antibody diluted to 0.5 μg / mL. Transfer the mixture to an ELISA plate at 100 μL / well and incubate at 37℃ for 1 h. Wash the plate 3 times with PBST and pat dry. Add 100 μL / well of enzyme-labeled avidin and incubate at 37℃ for 30 min. Wash the plate 3 times with PBST and pat dry. The enzyme-labeled avidin is streptavidin-HRP, and the dilution ratio of streptavidin-HRP is 1:30000.

[0012] (4) Color development: TMB color development, 100 μL / well, incubate at 37℃ in the dark for 15 min; terminate and read the absorbance at 450 nm;

[0013] (5) Result determination: Calculate the inhibition percentage (PI). Use 47% PI as the cutoff value. When the PI of the serum to be tested is <47%, the test result is negative. When the PI of the serum to be tested is ≥47%, the test result is negative.

[0014] This invention constructs porcine Fab antibodies with naturally paired light and heavy chains, and uses these antibodies to establish a competitive ELISA detection method for SVA. The sensitivity, specificity, repeatability, and concordance rate with the virus neutralization test (VNT) are then determined. The results showed that a Fab genetically engineered antibody neutralizing SVA, named 1M33Fab, was successfully prepared and exhibited high affinity for SVA. The optimal coating concentration of SVA particles for the C-ELISA method was determined to be 1 μg / mL, the optimal working concentration of the competitive antibody Bio-1M33Fab was 0.5 μg / mL, the optimal dilution ratio of the serum sample to be tested was 1:10, and the optimal dilution ratio of the enzyme-labeled avidin was 1:30,000. The highest sensitivity and specificity (96.88% and 100%, respectively) were achieved when the inhibition percentage (PI) was 47%, and the antibody did not react with positive sera from common major swine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, and the inter-assay coefficient of variation ranged from 1.19% to 11.72%, indicating good reproducibility. This method and VNT detection were used to simultaneously test 224 clinical swine serum samples, with a concordance rate of 93.75%. Attached Figure Description

[0015] Figure 1 Polyacrylamide gel electrophoresis of Fab antibodies; M: Protein molecular weight standard; A: Denaturing polyacrylamide gel electrophoresis of Fab antibodies; B: Non-denaturing polyacrylamide gel electrophoresis of Fab antibodies;

[0016] Figure 2 Results of indirect immunofluorescence assay for Fab genetically engineered antibody: A: Results of specific binding of 1M33Fab to BHK-21 cells infected with SVA HN / 11 / 2017 strain. B: Control of uninfected normal BHK-21 cells;

[0017] Figure 3 To detect the reactivity of 1M33Fab antibody with SVA antigen using indirect ELISA;

[0018] Figure 4 For the determination of sensitivity, specificity and critical value;

[0019] Figure 5 For cross-reactivity testing;

[0020] Figure 6 A: Electrophoresis results of PCR amplification of the variable region gene of porcine IgG antibody; B: Electrophoresis results of PCR products of porcine IgG heavy chain variable region; Detailed Implementation

[0021] The present invention will be further described below through specific embodiments.

[0022] Reagents used in this invention:

[0023] Endotoxin-free plasmid extraction kit was purchased from Tiangen Biotech Co., Ltd.; SMM293-TⅡ medium and M293-SUPI feed were purchased from SinB Biological Co., Ltd.; Polyethylenimine Linear (PEI) MW25000 was purchased from Poiyscience Co., Ltd.; Ni-NTA affinity chromatography medium was purchased from Genscript Co., Ltd.; MEM basal medium, trypsin, and penicillin-streptomycin solution were all purchased from Gibco Co., Ltd.; Fetal bovine serum (FBS) was purchased from Biological Industries Co., Ltd.; Long-chain biotin (EZ-Link™ Sulfo-NHS-LC-Biotin) was purchased from Thermo Fisher Scientific Co., Ltd.; Diethyleneimine (BEI), bovine serum albumin (BSA), FITC-labeled rabbit anti-pig IgG antibody, horseradish peroxidase (HRP)-labeled mouse anti-His antibody, streptavidin-HRP, and TMB substrate chromogenic reagent were all purchased from Sigma-Aldrich Co., Ltd.

[0024] Cells, virus strains, and serum samples used in this invention:

[0025] The following serum samples were collected: BHK-21 (a passaged cell line of suckling hamster kidney), HEK293F suspension cells, SVA (HN / 11 / 2017) strain, SVA positive control serum, SVA negative control serum, serum from non-immune healthy pigs (n = 64), serum from SVA positive pigs (n = 61), serum from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (n = 6), porcine circovirus (PCV) (n = 6), porcine epidemic diarrhea virus (PEDV) (n = 6), classical swine fever virus (CSFV) (n = 5), foot-and-mouth disease virus (FMDV) (n = 6), and positive serum from pigs infected with Seneca virus (SVA) (n = 6); field serum samples (n = 224) were prepared, identified, and preserved by the Host Antiviral and Immunobiology Team of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0026] Example 1: Preparation of SVA antigen and porcine Fab genetically engineered antibody

[0027] 1. Preparation of SVA antigen

[0028] To obtain high-concentration, pure SVA (HN / 11 / 2017) particles, the virus was first multiplied in large quantities using NHK-21 cells. After harvesting and repeated freeze-thaw cycles, the virus was inactivated in 0.1 mol / L BEI solution at 30°C for 28 h. After centrifugation to remove cell debris, 8% PEG-6000 and 4% NaCl (volume-to-mass ratio) were added, and the mixture was stirred overnight at 2–8°C. The virus was then collected using a high-speed centrifuge. A 30% sucrose solution (mass ratio) was prepared using PBS. Using a long-needle syringe, 10 mL of the 30% sucrose solution was added to the bottom of the centrifuge tube containing the virus solution. The tube was centrifuged at 36,000 r / min for 3.5 h at 4°C, and the supernatant was discarded. Sucrose solutions of 15%, 25%, 35%, and 45% were prepared. The precipitate was resuspended in PBS buffer and centrifuged using a continuous density gradient centrifugation method at 36,000 r / min for 3.5 h at 4°C. After centrifugation, fractions of 0.5 mL were collected from low to high concentrations for determination. OD 260nm Value, Collection OD 260nm Add 4-5 samples from the peak to a 100kDa ultrafiltration tube, then fill with PBS buffer, centrifuge at 3000 r / min for 10 min at 4℃, remove sucrose using PBS buffer, determine the concentration using the BCA method after sugar removal, add 30% glycerol and store at -80℃.

[0029] SVA virus particles were purified by ultracentrifugation and sucrose continuous density gradient centrifugation, and the concentration was determined to be 500 μg / mL by the BCA method.

[0030] 2. Multicolor flow cytometry sorting of SVA-specific single B cells

[0031] 2.1 Isolation of monocytes and macrophages (PBMCs)

[0032] Healthy 3-month-old pigs were treated with SVA cytotoxicity (HN / 11 / 2017 strain, titer 1×10⁻⁶). 9 TCID 50 Infection was performed via intramuscular injection behind the ear (4 mL / head, 2 mL / side). After 21 days, the pigs were immunized again with an inactivated vaccine prepared from the same strain in the same manner. Peripheral blood was collected from the pigs 28 days post-immunization, and monocyte-macrophages (PBMCs) were isolated from the peripheral blood using lymphocyte separation medium (density = 1.077 g / mL). The specific procedures are as follows:

[0033] (1) Equilibrate the PBS solution (pH 7.4) and lymphocyte separation medium at room temperature, and take 6 mL of lymphocyte separation medium into a 15 mL centrifuge tube;

[0034] (2) Dilute porcine EDTA anticoagulated blood with PBS solution at a ratio of 1:1, take 8 mL of diluted whole blood and slowly add it to the upper layer of lymphocyte separation medium, and centrifuge at 1200×g for 30 min;

[0035] (3) Add the milky white layer containing PBMCs to a 15 mL centrifuge tube containing 1 / 2 volume of cell sorting medium (PBS solution containing 1% BSA and 2 mMEDTANa2), and centrifuge at 600×g for 5 min.

[0036] (4) Discard the supernatant, add 1-2 mL of red blood cell lysis buffer, lyse at room temperature for 1-2 min, add 5 mL of cell sorting buffer, and centrifuge at 250×g for 10 min.

[0037] (5) Discard the supernatant, wash the cells twice with cell sorting solution, centrifuge at 400×g for 5 min, discard the supernatant, add cell sorting solution and disperse into single cells, and the cells obtained are PBMCs.

[0038] 2.2 Flow cytometry staining

[0039] (1) Take 10 8 One enriched PBMC was resuspended in 200 μL of cell sorting medium, and 0.5 μg of biotin-labeled SVA virus particle antigen and 2 μg of mouse anti-pig IgG-FITC fluorescent antibody (MyBioSource, USA) were added and incubated on ice for 25 min. Isotype controls and a reduced-number control were set up using the same cells. Single-positive tubes were also prepared for compensatory adjustment.

[0040] (2) Wash the cells twice with cell sorting solution, centrifuge at 400×g and 4℃ for 5 min.

[0041] (3) Resuspend the cells in 200 μL of cell sorting medium, add 2 μL of Anti-biotin-APC secondary antibody, and incubate on ice for 20 min.

[0042] (4) Wash the cells twice with cell sorting solution, centrifuge at 400×g and 4℃ for 5 min.

[0043] (5) Resuspend the cells in 500 μL of cell sorting solution, place them on ice in the dark, and prepare them for cell sorting.

[0044] 2.3 Sorting of SVA-specific single B cells

[0045] SVA-specific single B cells were sorted using a BD FACSAria IIu flow cytometer. Instrument settings were as follows: nozzle size: 100 μm; sorting mode: single-cell mode; sorting speed: 10,000 cells / second; amplitude: 20 psi; oscillation frequency: 30 kHz. After adjusting these parameters, the Sweat button was turned off, and the liquid delay time was calculated using Accudrop (catalog number: 642412) delay microspheres. The position of the 96-PCR plate in the sorting chamber was adjusted until the sorted cells accurately fell to the center of the wells. After completing all settings, the 96-PCR plate containing 10 μL of lysis buffer was placed in the sorting chamber, and sample loading began. Lymphocytes and monocytes were delineated by squaring, and adherent cells were excluded according to the FSC-A and FSA-H settings. Single cells were delineated diagonally. IgG cells were then further delineated from these. + Cell population, phylogenetic sorting of IgG + SVA + The cell is a single B cell specific to SVA.

[0046] 3. Amplification of the variable region gene of a single B cell-derived IgG antibody

[0047] 3.1 Preparation of single-cell cDNA molecules

[0048] After sorting, add 1 μL of stop solution to each well and incubate at room temperature for 2 min to terminate the reaction. Then, add 4 μL of SuperScriopt VILO mix and 6 μL of LDNase / RNase-free water to each well, mix gently, and centrifuge at 1500 rpm, 4°C for 5 min. Then, place the 96-well PCR plate into a PCR instrument for reverse transcription. The reaction conditions are: 25°C, 5 min; 42°C, 120 min; 85°C, 5 min. The obtained cDNA is stored at -20°C for subsequent nested PCR amplification.

[0049] 3.2 Amplification of the IgG antibody variable region gene

[0050] Using the primers designed and synthesized in Table 1, nested PCR, i.e., two rounds of PCR amplification, was performed to amplify the IgG heavy chain variable region (VH) gene and the κ light chain variable region (VL) gene of a single porcine B cell.

[0051] (1) First round PCR amplification protocol

[0052] First round PCR reaction system:

[0053] The first-round reaction system for amplifying the porcine IgG heavy chain variable region (VH) gene and the κ light chain variable region (VL) gene was prepared according to Table 2, except for the primers and templates.

[0054]

[0055]

[0056] The first round of PCR amplification program was as follows: pre-denaturation at 94℃ for 1 min; then denaturation at 98℃ for 10 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 1 min, for a total of 30 cycles; and finally extension at 72℃ for 10 min.

[0057] (2) Second round PCR amplification protocol

[0058] Second round reaction system:

[0059] Using the first-round amplification products as templates, corresponding upstream and downstream primers were added to amplify the porcine IgG heavy chain variable region (VH) gene and the κ light chain variable region (VL) gene. The components of the second-round PCR reaction system were as shown in Table 3.

[0060]

[0061] The second round of PCR amplification program was as follows: first, pre-denaturation at 94℃ for 1 min; then denaturation at 98℃ for 10 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 1 min, for a total of 35 cycles; finally, extension at 72℃ for another 10 min.

[0062] 3.3 Sequencing of PCR products

[0063] 4 µL of the PCR product from the second round of amplification was subjected to agarose gel electrophoresis to observe the amplification results. The PCR products of the successfully amplified VH and VL genes were then sequenced for DNA analysis, and the sequencing results were compared and analyzed using SnapGene software.

[0064] Nested PCR amplification products were analyzed by agarose gel electrophoresis, and the results are as follows: Figure 6 As shown, a clearly visible band appears in the porcine IgG heavy chain between 450 bp and 600 bp. Figure 6 A); A clear band appears in the variable region of the porcine κ light chain at approximately 500 bp ( Figure 6 B). The sequencing results were compared with the BLAST database, confirming that the sequence obtained by nested PCR amplification was the variable region gene sequence of porcine IgG antibody.

[0065] 4. Construction of porcine Fab-derived genetically engineered antibody expression vector

[0066] A signal peptide sequence “MEFRLNW0VVLFALLQGVQG” was introduced at the front end of the amplified porcine IgG antibody VH gene, and inserted into the CH-pcDNA3.4 vector containing the CH1 constant region of the IgG heavy chain through two restriction enzyme sites, Not I and BbvCI. Similarly, a signal peptide sequence “MRAPMHLLGLLLLWVPGARS” was introduced at the front end of the κ light chain VL gene, and inserted into the CL-pcDNA3.4 vector containing the IgG κ light chain constant region through two restriction enzyme sites, Not I and BbvCI. The heavy chain constant region of the IgG antibody contains a 6×His tag at its end. Sequence synthesis and vector construction were commissioned to Genewiz Biotechnology Co., Ltd. Endotoxin-free plasmids were prepared according to the instructions of the endotoxin-free large-scale plasmid extraction kit, yielding the heavy chain plasmid CH-pcDNA3.4-1M33Fab and the light chain plasmid CL-pcDNA3.4-1M33L.

[0067] 5. Preparation of SVA porcine Fab genetically engineered antibodies

[0068] First, prepare endotoxin-free plasmids according to the instructions of the commercially available endotoxin-free large-scale plasmid extraction kit. Take 20 μg of heavy chain plasmid (CH-pcDNA3.4-1M33Fab) and 20 μg of the corresponding light chain plasmid (CL-pcDNA3.4-1M33L) (heavy and light chains in a 1:1 ratio) and mix them in a 5 mL sterile centrifuge tube. Add 2 mL of DPBS to dilute the plasmid. Take 15 μL of PEI (2 mg / mL) transfection reagent and add it to another new 2 mL centrifuge tube. Add 2 mL of DPBS to dilute the transfection reagent, invert to mix, and let stand at room temperature for 10-15 min. Slowly add this mixture dropwise to a solution with a density of 2×10⁻⁶. 6 HEK293F cells / mL were cultured for 7–9 days. Cell supernatant was collected, filtered through a 0.45 μm filter, and then purified using Ni-NTA packing material. The eluted antibody was dialyzed against PBS at 4°C and pH 8.0, with the dialysate changed every 2 h for a total of three dialyses. The dialyzed antibody was then embedded in PEG-8000 solid particles and concentrated at 4°C, and the concentration was determined. Finally, SDS-PAGE gel chromatography was performed for verification, and the cells were stained with Coomassie Brilliant Blue.

[0069] 20 μg each of the heavy chain plasmid and the corresponding light chain plasmid (heavy chain to light chain ratio 1:1) were co-transfected into HEK293F cells for expression. Nine days after transfection, the cell supernatant was collected, and after protein purification, polyacrylamide gel electrophoresis analysis was performed. The results are as follows: Figure 1As shown, the Fab genetically engineered antibody showed the target band at 24 kDa under denaturing conditions and at 50 kDa under non-denaturing conditions. The results were consistent with expectations, indicating that the porcine Fab antibody molecule was successfully expressed and purified, and it was named 1M33Fab.

[0070] Nucleotide sequence of porcine Fab antibody heavy chain (SEQ ID No. 1):

[0071] ATGGAGTTTCGGCTGAACTGGGTGGTCTTGTTTGCTCTCTTACAAGGTGTCCAGGGTGAGGAGAAGCTGGTGGAGTCTGGGGGAGGCCTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGTCGGCTCTGGATTCACCTTCAGAAGTACCTCCATTAACTGGGTCCGCCAGGCTCCAGGGAA GGGGCTGGAGTGGCTGGCATATATTGGTAGCAGTGGACTTGGCACCGGCTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACTCCCAGAACACGGCCTATCTGCAAATCAACAGCCTGAGAACCGAAGACACGGCCCGCTATTACTGTGCACGCGGTTGCGGTAATAGTGCCG CATGTGCTCTGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGCCCCAAAGACTGCCCCTTCCGTGTACCCACTGGCTCCTTGCGGAAGGGATACTAGCGGCCCAAATGTCGCTCTGGGCTGTCTGGCCAGCAGCTACTTCCCAGAGCCAGTGACAATGACATGGAACAGCGGAGCT CTGACATCCGGCGTGCACACTTTTCCAAGCGTGCTGCAACCAAGCGGACTGTACTCTCTGAGCTCCATGGTGACAGTCCCAGCCAGCTCCCTCTCCAGCAAGAGCTACACATGCAACGTGAATCACCCAGCCACTACAACTAAGGTCGACAAGAGGGTGGGCGGACACCACCACCACCCATATTGA

[0072] The amino acid sequence of the porcine Fab antibody heavy chain (SEQ ID No. 3):

[0073] MEFRLNWVVLFALLQGVQGEEKLVESGGGLVQPGGSLRLSCVGSGFTFRSTSINWVRQAPGKGLEWLAYIGSSGLGTGYADSVVKKKGFTISRDNSQNTAYLQINSLRTEDTARYYCARGCGNSAACALDLWGPGVEVVVSSAPKTAPSVYPLAPCGRDTSGPNVALGCLASSYFPEPVTMTWNSGALTSGVHTFPSVLQPSGLYSLSSMVTVPASSLSSKSYTCNVNHPATTTKVDKRVGGHHHHHH

[0074] Nucleotide sequence of the light chain of the porcine-derived Fab antibody (SEQ ID No. 5):

[0075] ATGAGGGCCCCCATGCACCTCCTTGGCCTCCTGCTGCTCTGGGTCCCA-GGTGCCAGGAGTGCCATCCAGCTGACCCAGTCTCCAGCCTCCCTGGCTGCATCTCTCGGAGACACGGTCTCCATCACTTGCCGGGCCAGTCAGAGTGTTAGCAGTAATTTAGACTGGTATCAACAACAACCAGGGAAGGCTCCTAAACTCTTGATCTATTCAGTTTCCACTTTGCAAAGTGGGGTCCCATTCCGGTTCAAGGGCAGTGGATCTGGCACCGATTTCACCCTCACCATCAGTGGCCTGCAGGCTGAAGATGTTGCAACTTATTACTGTCACCAGCATAACAGTACACCGTATGGTTTCGGCGCGGGGACCAAGCTGGAGCTCAAACGGGCTGATGCCAAGCCATCCGTCTTCATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGGTGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAAGTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCCACGTCACAGTACCTAAGTCATAATTTATATTCCTGTGAGGTCACCCACAAGACCCTGGCCTCCCCTCTGGTCACAAGCTTCAACAGGAACGAGTGTGAGGCTTAG

[0076] Amino acid sequence of porcine-derived Fab antibody light chain (SEQ ID No. 6):

[0077] MRAPMHLLGLLLLWVPGARSAIQLTQSPASLAASLGDTVSITCRASQSVSSNLDWYQQQPGKAPKLLIYSVSTLQSGVPFRFKGSGSGTDFTLTISGLQAEDVATYYCHQHNSTPYGF GAGTKLELKRADAKPSVFIFPPSKEQLATPTVSVVCLINNFFPREISVKWKVDGVVQSSGHPDSVTEQDSKDSTYSLSSTLSLPTSQYLSHNLYSCEVTHKTLASPLVTSFNRNECEA

[0078] Example 2: Activity detection of SVA porcine Fab genetically engineered antibodies

[0079] 1. Indirect immunofluorescence assay (IFA)

[0080] The SVA HN / 11 / 2017 strain was inoculated into a monolayer of BHK-21 cells and incubated at 37°C for 8 h. The cells were then fixed at room temperature for 20 min using a 1:1 mixture of methanol and acetone as the fixative. After washing three times with PBS, the cells were permeabilized with 0.1% Triton X-100 for 10 min. After washing three times with PBS, blocking buffer (1% BSA + 0.1% Triton X-100) was added for blocking. After washing three times with PBS, purified Fab antibody was added and the cells were incubated at 37°C for 1 h. After washing three times with PBS, FITC-labeled rabbit anti-pig IgG antibody (diluted 1:400) was added as a secondary antibody and the cells were incubated at 37°C for 1 h. The secondary antibody was discarded, and the cells were washed five times with PBS. Fluorescence signals were observed and photographed under a fluorescence microscope in the dark.

[0081] IFA test results are as follows Figure 2 As shown, the Fab genetically engineered antibody 1M33Fab specifically binds to BHK-21 cells infected with the SVA HN / 11 / 2017 strain, exhibiting obvious green fluorescence, while uninfected control cells show no visible green fluorescence. This result indicates the successful acquisition of a porcine monoclonal genetically engineered antibody that specifically reacts with SVA.

[0082] 2. Indirect ELISA

[0083] The purified SVA (HN / 11 / 2017 strain) antigen was diluted to 2 μg / mL with PBS and coated onto an ELISA plate overnight at 4°C. The plate was then blocked with 1% BSA at 37°C for 1 h and dried. The antibody to be tested was serially diluted and added to the ELISA plate, incubated at 37°C for 1 h. HRP-labeled mouse anti-His antibody was added and incubated at 37°C for 1 h. TMB chromogenic buffer was added and incubated at room temperature for 10 min. The chromogenic process was terminated by adding stop solution, and the absorbance at 450 nm was measured using an ELISA reader. OD 450nm Calculate the average value of the negative control group, and then multiply the average value by 2.1 to obtain the Cut-off (CO) value. Based on the Cut-off value, wells with values ​​greater than this value are judged as positive wells, and wells with values ​​less than this value are judged as negative wells.

[0084] Indirect ELISA was used to determine whether the antibody specifically bound to the antigen. The results were as follows: Figure 3 As shown, antibody 1M33Fab can specifically bind to SVA antigen, and the binding of antibody to SVA antigen is positively correlated with antibody concentration.

[0085] 3. Virus neutralization experiment

[0086] Virus neutralization experiments were performed on the Fab genetically engineered antibodies screened using the SVA HN / 11 / 2017 strain. First, the antibody was serially diluted 2-fold and added to a 96-well plate at a rate of 50 μL / well, typically with two replicates. Then, it was added to a plate containing 50 μL of 100 TCID50. 50 The SVA venom was mixed and incubated at 37°C for 1 hour. Simultaneously, samples containing 0.1, 1, 10, and 100 TCID molecules were prepared. 50 The control wells (without antibody) were then filled with 100 μL of a solution containing 5 × 10⁻⁶ antibodies. 4 Complete culture medium for BHK-21 cells was prepared, along with a normal cell control. The 96-well cell culture plates were incubated at 37°C with 5% CO2 for 72 h to observe the cytopathic effect. The minimum antibody concentration (IC50) required to induce cytopathic effects in 50% of cells was used. 50 (Indicates the antibody's ability to neutralize the virus, in μg / mL.) This makes IC50... 50 A concentration of 50 μg / mL is used as the critical value for neutralization. ≥50 μg / mL is defined as non-neutralizing activity, and <50 μg / mL is defined as having neutralizing activity.

[0087] A microviral neutralization assay was performed on BHK21 cells to detect the activity of this antibody in neutralizing the SVA HN / 11 / 2017 strain. The results showed that the porcine Fab genetically engineered antibody 1M33Fab possessed the ability to neutralize the HN / 11 / 2017 strain, qualifying it as an SVA neutralizing antibody, with an IC50 value of [missing information]. 50 It is 1.26 μg / mL.

[0088] 4. Western Blot (WB) assay

[0089] SDS-PAGE electrophoresis was performed on an SVA HN / 11 / 2017 strain antigen containing approximately 1 μg. The separated protein bands were then transferred to a nitrocellulose membrane and blocked with TBST buffer containing 5% skim milk for 2 h. After washing, the antibody was diluted to the working concentration (5 μg / mL) with TBST buffer containing 5% skim milk and incubated at room temperature for 2 h. After washing the membrane with TBST, HRP-labeled anti-His tag antibody (1:4000) was added and incubated at room temperature for 1 h. After washing the membrane with TBST, ECL chemiluminescent substrate was added for exposure imaging.

[0090] Viral particles purified from SVA HN / 11 / 2017 strain were used as antigens for SDS-PAGE electrophoresis and then transferred to a membrane. Western blot results showed that no reaction band appeared in the porcine Fab genetically engineered antibody, while a clearly visible reaction band appeared in the SVA positive serum, indicating that the obtained Fab genetically engineered antibody does not recognize linear epitopes but recognizes conformational epitopes.

[0091] Example 3: Establishment and Optimization of Competitive ELISA Detection Method

[0092] 1. Biotin-labeled competitive antibodies

[0093] Bring biotin to room temperature beforehand, then briefly centrifuge. Add 180 μL of enzyme-free water to 0.1 mg of biotin, dissolve completely, and briefly centrifuge again. Dilute the purified antibody to 100 μg / mL, add 60 μL of biotin solution per mL, mix well by pipetting, and incubate on ice for 4 h. Transfer the biotin-labeled antibody to a dialysis bag and dialyze using PBS solution, changing the PBS solution every 2 h for a total of four dialysis cycles to remove free biotin molecules. Store at 4 ℃ for later use.

[0094] 2. Establishment of competitive ELISA detection methods

[0095] The purified SVA antigen was diluted to the optimal coating concentration with PBS, and 100 μL / well was added to the ELISA plate. The plate was incubated overnight at 4°C, washed three times with PBST, and then blotted dry. A PBST solution containing 1% BSA and 5% sucrose was prepared, and 100 μL / well was added to the plate. The plate was blocked at 37°C for 1 hour. 60 μL of diluted serum (test serum, standard negative serum, and standard positive serum) and 60 μL of competitive antibody diluted to the optimal working concentration were added to the serum dilution plate. After mixing, the solution was transferred to the ELISA plate, 100 μL / well, and incubated at 37°C for 1 hour. The plate was washed three times with PBST and then blotted dry. Enzyme-labeled avidin was added, 100 μL / well, and incubated at 37°C for 30 minutes. The plate was washed three times with PBST and then blotted dry. TMB color development was performed, 100 μL / well, and incubated at 37°C in the dark for 15 minutes. The incubation was terminated and the reading was taken at 450°C. The absorbance value at nm is expressed as the suppression percentage (PI), where PI = [1 - (absorbance value of the sample / absorbance value of the blank control) × 100%].

[0096] 3. Optimization of competing ELISA detection methods

[0097] 3.1 Determination of the optimal working concentration of competitive antibody and the optimal serum dilution

[0098] After purification, SVA (HN / 11 / 2017 strain) antigen was diluted to 0.5 μg / mL with PBS for plating. Biotin-labeled antibody was diluted with PBS to 2, 1, 0.5, and 0.25 μg / mL. Negative and positive control sera were serially diluted from 1:5 to 1:640. The absorbance at 450 nm was measured using an ELISA reader, and the N / P ratio was calculated (N: negative control). OD 450nm Value, P: positive control OD 450nm The optimal value is when the N / P value is maximized.

[0099] Measured by matrix titration OD 450nm The N / P values ​​are shown in Table 4. The N / P value is highest when the concentration of the competitive antibody Bio-1M33Fab is 0.5 μg / mL and the serum dilution ratio is 1:10. Therefore, the optimal working concentration of Bio-1M33Fab is determined to be 0.5 μg / mL and the optimal serum dilution ratio is 1:10.

[0100]

[0101] 3.2 Determination of the optimal coating concentration of SVA and the optimal dilution ratio of streptavidin-HRP

[0102] SVA antigen coating concentrations of 2, 1, 0.5, and 0.25 μg / mL were selected. Streptavidin-HRP was serially diluted at 1:15,000, 1:20,000, 1:25,000, 1:30,000, 1:35,000, 1:40,000, and 1:45,000. Negative and positive control sera were diluted to the optimal dilution. Measurements were performed using an ELISA reader. OD 450nm The value is calculated, and the N / P value is determined. The optimal value is when the N / P value is maximized.

[0103] The optimal coating concentration of SVA and the optimal dilution ratio of streptavidin-HRP were determined using matrix titration. OD 450nm The values ​​and N / P values ​​are shown in Table 5. The N / P value is the highest when the SVA coating concentration is 1 μg / mL and the streptavidin-HRP is diluted 1:30,000. Therefore, the optimal SVA coating concentration is determined to be 1 μg / mL and the optimal streptavidin-HRP dilution ratio is 1:30,000.

[0104]

[0105] 4. Determination of sensitivity, specificity, and critical values

[0106] This method was used to test 64 non-immune healthy pig serum samples and 61 SVA-positive pig serum samples. The PI value was calculated, and the sensitivity, specificity, and critical value of the method were determined by ROC curve analysis.

[0107] This method was used to test 64 non-immune healthy swine serum samples and 61 SVA-positive swine serum samples. The PI value was calculated, and ROC curve analysis was performed to determine the sensitivity, specificity, and cutoff value. The results are as follows: Figure 4 As shown in the figure, the sensitivity and specificity were highest at a cutoff value of 47%, reaching 96.88% and 100%, respectively. The area under the ROC curve was 0.9992, with P < 0.0001. Therefore, the cutoff value for PI was set at 47%. That is, serum samples with PI < 47% were considered negative, while serum samples with PI ≥ 47% were considered positive.

[0108] 5. Cross-reactivity test

[0109] This method was used to detect positive sera from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (n=6), porcine circovirus (PCV) (n=6), porcine epidemic diarrhea virus (PEDV) (n=6), classical swine fever virus (CSFV) (n=5), foot-and-mouth disease virus (FMDV) (n=6), and Seneca virus (SVA). The test results were statistically analyzed to determine if cross-reactivity existed.

[0110] To evaluate the cross-reactivity of this method, positive sera from pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) (n=6), porcine circovirus (PCV) (n=6), porcine epidemic diarrhea virus (PEDV) (n=6), classical swine fever virus (CSFV) (n=5), and foot-and-mouth disease virus (FMDV) (n=5) were tested. The results are as follows: Figure 5 As shown, all sera except for SVA-positive sera were negative, with PI values ​​below 47%, indicating that this method is specific for detecting SVA-specific antibodies and has no cross-reactivity with sera infected with other viruses.

[0111] 6. Repeatability test

[0112] Intra-assay repeatability: Eight serum samples were tested using this method, with three replicate tests performed at different time points using C-ELISA plates prepared in the same batch. Inter-assay repeatability: The same eight serum samples were tested three times at the same time point using C-ELISA plates prepared in different batches. The intra-assay and inter-assay coefficients of variation of this method were evaluated.

[0113] To evaluate the reproducibility of this method, C-ELISA reaction plates coated with the same and different batches were used, and eight serum samples were reproducibly tested. Intra-batch and inter-batch coefficients of variation (CV) were calculated. The results are shown in Table 6. The intra-batch CV ranged from 1.12% to 7.34%, and the inter-batch CV ranged from 1.19% to 11.72%, indicating that this method has high reproducibility.

[0114]

[0115] 7. Comparison of ELISA and VNT field sample detection results

[0116] This method was used to test 224 field samples, and the results of the two methods were compared and the concordance rate was calculated.

[0117] To verify the applicability of this method, 224 clinical swine serum samples collected from Gansu Province in 2024 were tested using C-ELISA and VNT. As shown in Table 7, 182 serum samples were negative for VNT and 42 were positive, while 172 serum samples were negative for C-ELISA and 52 were positive. Twelve serum samples were positive for C-ELISA but negative for VNT, and two were negative for C-ELISA but positive for VNT. The results indicate that the concordance rate between C-ELISA and VNT is approximately 93.75% (210 / 224).

[0118]

[0119] A porcine Fab-derived SVA-specific antibody, 1M33Fab, was obtained by co-transfecting HEK293F cells with heavy chain plasmids and corresponding light chain plasmids. To evaluate the reactivity of the porcine Fab-derived antibody 1M33Fab to SVA, indirect ELISA, immunofluorescence assay (IFA), virus neutralization assay (VNT), and Western blotting were performed. Indirect ELISA results showed that antibody 1M33Fab responded to purified SVA antigen in a dose-dependent manner. IFA results showed that antibody 1M33Fab specifically reacted with SVA antigen in SVA-infected BHK-21 cells, while Western blotting results showed that the antibody did not react with SVA antigen, indicating that this antibody recognizes the conformational epitope of the viral capsid. Furthermore, an in vitro neutralization assay was performed, showing that this antibody can effectively neutralize SVA in vitro, with an IC50 value of [missing value]. 50 The optimal concentration of SVA particles was 1.26 μg / mL. A C-ELISA method was then established using 1M33Fab. The optimal coating concentration of SVA particles, the optimal working concentration of the competitive antibody Bio-1M33Fab, and the optimal dilution ratio of the serum samples to be tested were determined to be 1:10 and 1:30,000, respectively. The highest sensitivity and specificity (96.88% and 100%, respectively) were achieved when the inhibition percentage (PI) was 47%, and the method did not react with positive sera from common major swine viral diseases. The intra-assay coefficient of variation ranged from 1.12% to 7.34%, and the inter-assay coefficient of variation ranged from 1.19% to 11.72%, indicating good reproducibility. To further evaluate the performance of this method, 224 clinical swine serum samples collected in Gansu Province in 2024 were tested using C-ELISA, and the results were compared with VNT results. The concordance rate between C-ELISA and VNT was approximately 93.7% (210 / 224), indicating strong consistency in clinical diagnostic performance between the two. Therefore, the C-ELISA developed in this study is comparable to VNT for SVA antibody detection and can replace VNT for large-scale screening because it is faster, simpler, and less expensive to perform.

[0120] This invention relates to the establishment and application of a competitive ELISA detection method for SVA based on Fab antibodies, providing strong technical support for the epidemiological monitoring and prevention of SVA.

Claims

1. A porcine Fab antibody against Seneca virus A, characterized in that: The amino acid sequence of the heavy chain of the porcine Fab antibody against Seneca virus A is shown in SEQ ID No. 3; the amino acid sequence of the light chain of the porcine Fab antibody against Seneca virus A is shown in SEQ ID No.

6.

2. The application of the Seneca virus A porcine Fab antibody according to claim 1 in the preparation of reagents or kits for detecting Seneca virus.

Images