Prussian blue nano test strip for detecting African horse pestivirus antibody as well as preparation method and application of Prussian blue nano test strip

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Prussian blue nano test strip for detecting an African horse pestivirus antibody as well as a preparation method and application of the Prussian blue nano test strip. A sample pad, a combination pad, a chromatography detection membrane and a water absorption pad are sequentially fixed on the supporting bottom plate; the chromatography detection membrane is provided with a detection line and a quality control line, the detection line is coated with staphylococcus A protein, and the quality control line is coated with a monoclonal antibody of African horse pestivirus VP7 recombinant protein; and the combination pad is coated with an African horse pestivirus VP7 delta N1-129 recombinant protein fragment coupled with Prussian blue nanoparticles. According to the invention, Prussian blue nano is used as a coupling tracer, and the specific reaction of an antigen and an antibody is fully utilized to detect the African horse pestivirus antibody. After the conjugate is combined with a corresponding antibody, a result can be clearly observed, and the conjugate is good in stability, high in sensitivity and strong in specificity, has a clinical application function and huge development potential, and is an extremely ideal antibody test strip detection material.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Prussian blue nano-test strip for detecting African horse sickness virus antibodies, its preparation method, and its application. Background Technology

[0002] African horse sickness (AHS) is a highly fatal infectious disease caused by the African horsesickness virus (AHSV).

[0003] The VP7 protein is a major structural protein of the viral core, possessing multiple key functions. VP7 plays a crucial role in viral core assembly, forming the viral core structure on the scaffold provided by the secondary core protein VP3, laying the foundation for viral replication and assembly. Unlike the VP7 protein of bluetongue virus (BTV), the VP7 protein of AHSV exhibits a unique self-assembly capability, forming insoluble hexagonal crystal particles. This crystallization characteristic may affect the assembly efficiency and replication rate of the viral core. VP7 is also one of the major immunogenic proteins of AHSV, containing B-cell and T-cell epitopes, and is highly conserved among various orbiviruses, making it an important candidate protein for vaccine development. The crystallization characteristic of VP7 may affect viral replication efficiency and pathogenicity by limiting its availability in viral core assembly, and is associated with cytopathological changes caused by AHSV infection. Overall, the VP7 protein plays an important role in viral structure, immunogenicity, replication, and pathogenicity.

[0004] African horse sickness is a highly contagious disease that poses a significant threat to equines. While laboratory diagnostic methods such as real-time quantitative RT-PCR and ELISA (enzyme-linked immunosorbent assay) are accurate, their widespread application at the grassroots level is limited by equipment and the availability of qualified personnel. Therefore, developing rapid detection methods suitable for frontline use is crucial.

[0005] Therefore, this invention develops an antibody test strip based on Prussian blue nanoparticle material, which has the advantages of simple operation, speed, and high sensitivity, and can meet the needs of field testing. Summary of the Invention

[0006] This invention provides a Prussian blue nanoparticle test strip for detecting African horse sickness virus (ASV), comprising: a sample pad, a conjugate pad, a chromatographic detection membrane, and an absorbent pad sequentially fixed on a supporting base plate; the chromatographic detection membrane has a detection line and a control line, the detection line being coated with Staphylococcus aureus protein A, and the control line being coated with a monoclonal antibody against recombinant ASV VP7 protein; the conjugate pad is coated with ASV VP7 ΔN conjugate protein coupled with Prussian blue nanoparticles. 1-129Recombinant protein fragments;

[0007] Among them, the African horse sickness virus VP7 ΔN 1-129 The amino acid sequence of the recombinant protein fragment is: GAVEVQQSGRYYVPQGRTRGGYINSNIAEVCMDAGAAGQVNALLAPRRGDAVMIYFVWRPLRIFCDPQGASLESAPGTFVTVDGVNVAAGDVVAWNTIAPVNVGNPGARRSILQFEVLWYTSLDRSLDTVPELAPTLTRCYAYVSPTWHALRAVIFQQMNMQPINPPIFPPTERNEIVAYLLVASLADVYAALRPDFRMNGVVAPVGQINRALVL.

[0008] As a preferred embodiment of the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to the present invention: the monoclonal antibody of the recombinant protein VP7 of African horse sickness virus has the antigenic epitope PPIFPP.

[0009] As a preferred embodiment of the Prussian blue nanopattern test strip for detecting African horse sickness virus antibodies according to the present invention: the monoclonal antibody of the recombinant protein VP7 of African horse sickness virus is secreted by hybridoma cells with accession number GDMCC No:67154.

[0010] As a preferred embodiment of the Prussian blue nanoparticle test strip for detecting African horse sickness virus antibodies according to the present invention: the Prussian blue nanoparticles are coupled with African horse sickness virus VP7 ΔN 1-129 Recombinant protein fragments, including African horse sickness virus VP7 ΔN 1-129 The amount of recombinant protein fragment on the binding pad is 1-3 μg / cm 2 The amount of Prussian blue nanoparticles on the conjugate pad was 3-9 μg / cm. 2 .

[0011] As a preferred embodiment of the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to the present invention, the amount of Staphylococcus A protein coated on the detection line is 1.5-2.5 μg / cm.

[0012] As a preferred embodiment of the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to the present invention, the amount of monoclonal antibody against the recombinant VP7 protein of African horse sickness virus coated on the control line is 1-1.2 μg / cm.

[0013] As a preferred embodiment of the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to the present invention: the material of the chromatographic detection membrane includes a nitrocellulose membrane; the material of the conjugate pad includes glass cellulose.

[0014] The present invention also provides a method for preparing the Prussian blue nano test strip for detecting African horse sickness virus antibodies, characterized in that: an absorbent pad is fixed on one side of the chromatographic detection membrane with overlapping edges, a conjugate pad is fixed on the other side of the chromatographic detection membrane with overlapping edges, and a sample pad is fixed on the conjugate pad on the side away from the absorbent pad with overlapping edges.

[0015] Among them, the Prussian blue nanoparticles are coupled with African horse sickness virus VP7 ΔN 1-129 The preparation method of the recombinant protein fragment includes: adding a suspension of Prussian blue nanoparticles to a 2-(N-morpholino)ethanesulfonic acid solution, centrifuging, resuspending the precipitate in a 2-(N-morpholino)ethanesulfonic acid solution, dispersing evenly, adding carbodiimide and N-hydroxysuccinimide, incubating, centrifuging, resuspending in a borate-borax buffer solution at pH 8.0, and adding African horse sickness virus VP7 ΔN. 1-129 The recombinant protein fragment was reacted at 4°C, blocked with BSA, centrifuged, and resuspended in resuspending buffer to obtain Prussian blue nanoparticle-conjugated African horse sickness virus VP7 ΔN. 1-129 Recombinant protein fragment solution;

[0016] The process of adding a resuspension solution and resuspending the virus yields Prussian blue nanoparticles coupled with African horse sickness virus VP7 ΔN. 1-129 Recombinant protein fragment solution, containing African horse sickness virus VP7 ΔN 1-129 The concentration of the recombinant protein fragment was 0.025-0.075 μg / μL, and the concentration of the Prussian blue nanoparticles was 0.08-0.26 μg / μL.

[0017] As a preferred embodiment of the preparation method of the Prussian blue nanoparticle test strip for African horse sickness virus antibody detection according to the present invention, the preparation method of the Prussian blue nanoparticles includes: dissolving citric acid or hyaluronic acid in water to obtain solution A, dividing solution A into two portions for later use; adding a soluble ferrous salt to one portion of solution A to obtain solution B; adding potassium ferricyanide to the other portion of solution A to obtain solution C; heating solution B and adding solution C dropwise under high-speed stirring to obtain Prussian blue nanoparticles; the average particle size of the Prussian blue nanoparticles is 70-80 nm.

[0018] This invention also provides the application of the aforementioned Prussian blue nanotest strip for detecting African horse sickness virus antibodies in the preparation of a kit for detecting African horse sickness virus antibodies, characterized in that: it further includes a sample diluent, the sample diluent being formulated as follows: a 20-30 mM HEPES solution containing, by mass percentage, 0.5-1% BSA, 0.05-0.1% sodium ascorbate, 2-4% trehalose, 2-4% sucrose, 5-8% glycerol, and 0.5-1 M betaine.

[0019] The beneficial effects of this invention are as follows: This invention uses Prussian blue nanoparticles as a conjugated tracer, fully utilizing the specific reaction between antigen and antibody to detect African horse sickness virus antibodies. After the conjugated compound binds to the corresponding antibody, the results can be clearly observed. It exhibits good stability, high sensitivity, and strong specificity, making it highly clinically applicable and possessing enormous development potential. It is an ideal material for antibody test strips.

[0020] This invention selects AHSV-VP7 ΔN 1-129 A Prussian blue nano-immunochromatographic test strip for the rapid detection of African horse sickness virus (ASV) antibodies was developed using recombinant protein as an antigen. The VP7 protein exhibits good immunogenicity, inducing the host to produce specific antibodies early after infection, and antibody levels can be maintained for several weeks. The VP7 protein is highly conserved in the viral structure, its antigenic epitope is stable, and it can bind efficiently to specific antibodies, thereby improving the specificity and sensitivity of the detection. Furthermore, the VP7 protein-based detection method can be combined with advanced immunochromatographic technology to achieve rapid and convenient on-site testing, avoiding false negative results, and is particularly suitable for rapid screening at the grassroots level and in the field. Therefore, the VP7 protein is an ideal target for the detection of ASV antibodies and has broad application prospects.

[0021] This invention utilizes a monoclonal antibody made from recombinant AHSV-VP7 protein on the control line. In immunochromatographic assays, the high-quality monoclonal antibody on the control line enhances the detection capability of the test strip, reduces background noise, and makes the test results more accurate and stable, thereby improving the detection sensitivity of the test line. Furthermore, the novel VP7 protein monoclonal antibody targeting the epitope produced in this invention provides biomolecular material support for further in-depth research.

[0022] This invention successfully obtained a monoclonal antibody mAb 2E9 that specifically recognizes the AHSV VP7 protein. After identifying its epitopes, it was demonstrated that the antibody can be used for the detection, diagnosis, and study of the viral mechanism of AHSV. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0024] Figure 1 A schematic diagram of the reaction principle of the Prussian blue nanoantibody test strip for detecting African horse sickness virus.

[0025] Figure 2 For purified VP7 ΔN 1-129 Diagram of recombinant protein and monoclonal antibody mAb strain 2E9.

[0026] Figure 3 The image shows the results of Western blot (WB) verification of alpaca positive serum and VP7 FL (full-length) protein.

[0027] Figure 4 Figure showing the truncation of the VP7 FL protein in African horse sickness and the results of monoclonal antibody epitope identification. Detailed Implementation

[0028] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0029] This invention screens and obtains the preferred fragment VP7 ΔN of the African horse sickness VP7 protein. 1-129 (Recombinant protein with amino acids 1-129 missing from the N-terminus). This includes the African horse sickness virus VP7 ΔN. 1-129 The amino acid sequence of the recombinant protein fragment is: GAVEVQQSGRYYVPQGRTRGGYINSNIAEVCMDAGAAGQVNALLAPRRGDAVMIYFVWRPLRIFCDPQGASLESAPGTFVTVDGVNVAAGDVVAWNTIAPVNVGNPGARRSILQFEVLWYTSLDRSLDTVPELAPTLTRCYAYVSPTWHALRAVIFQQMNMQPINPPIFPPTERNEIVAYLLVASLADVYAALRPDFRMNGVVAPVGQINRALVL

[0030] The monoclonal antibody of this invention was secreted by hybridoma cells with accession number GDMCC No:67154, deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 23, 2025, at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its epitope (antigenic determinant) is: 886-903bp: CCGCCAATTTTTCCACCG (296-301 aa: PPIFPP).

[0031] Example 1:

[0032] AHSV-VP7 FL, VP7 ΔN 1-129 VP7 ΔC 264-345 Preparation of recombinant proteins and production of African horse sickness virus positive serum (alpaca positive serum):

[0033] 1. Materials:

[0034] 1.1 Main instruments: Low-temperature high-speed centrifuge, purchased from Thermo Fisher Scientific (China) Co., Ltd., LEGENDMICRO 17 R; Pure water system, purchased from Sichuan Youpu Ultrapure Technology Co., Ltd., μPH-II-5 T; Ultrasonic signal generator, purchased from Nanjing Xianou Instrument Manufacturing Co., Ltd., XO-650 D;

[0035] 1.2 Reagents: Disodium hydrogen phosphate dihydrate, sodium chloride, glycerol, imidazole, etc., were purchased from Guangzhou Pharmaceutical Group; HiTrapQFF and 6×His-tag Ni affinity chromatography columns were purchased from General Electric. 250 kDa pre-stained protein marker and a rapid 12.5% ​​PAGE gel preparation kit were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; 180 kDa pre-stained protein marker, BCA protein concentration assay kit, and high-sensitivity ECL chemiluminescence detection kit were purchased from Nanjing Novizan Medical Technology Co., Ltd.; protein desalting centrifuge tubes were purchased from Bebo Biotechnology Co., Ltd.; polyvinylidene fluoride (PVDF) membranes were purchased from PALL; other reagents were domestically produced analytical grade.

[0036] 2 methods:

[0037] 2.1 Induction of expression of three recombinant VP7 proteins:

[0038] Targeting the AHSV VP7 gene, VP7 FL and VP7 ΔN were designed by comparing and analyzing the VP7 antigen region using software such as TMHMM-2.0 and IEDB Analysis Resource. 1-129 VP7 ΔC 264-345 Three recombinant protein sequences were used simultaneously with the pET-28a vector to synthesize the target plasmids VP7FL-pET-28a and VP7ΔN. 1-129 -pET-28a,VP7ΔC 264-345 -pET-28a and glycerol bacteria VP7-pET-28a-BL21(DE3), VP7 ΔN 1-129 -pET-28a-BL21 (DE3), VP7 ΔC 264-345 -pET-28a-BL21(DE3) should be stored at -80 ℃.

[0039] Three types of VP7 recombinant protein Escherichia coli BL21(DE3) samples were cryopreserved and streaked onto LB agar containing kanamycin, then incubated at 37 °C for 12 h. Single colonies from the solid medium were picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 °C with shaking at 200 r / min until the bacterial growth reached OD. 600 Stop when the growth rate is approximately 0.6, and use it as seed for further cultivation.

[0040] The three revived VP7 recombinant protein strains were inoculated at a 1:100 ratio into 1 L LB medium (containing kanamycin) and cultured at 37°C and 220 rpm until OD500. 600nm =0.8-0.9, after cooling to 16 ℃ for 1 h, add IPTG inducer with a final concentration of 0.2 mM, and induce culture for 16 h.

[0041] 2.2 Purification and Identification of AHSV-VP7 Recombinant Protein

[0042] 2.2.1 Preparation of relevant reagents

[0043] Buffer 1: 0.01M PBS solution containing 2 M urea, 20 mM PB, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, pH 7.4;

[0044] Buffer 2: 0.01M PBS solution of 8M urea, 20 mM PB, 500 mM NaCl, 20 mM imidazole, 1 mM DTT, pH 7.4;

[0045] Different concentrations of buffer: 30 mM imidazole, 8 M urea, 20 mM PB, 500 mM NaCl, 0.01 M PBS solution at pH 7.4 (for other imidazole elution buffers, only the imidazole concentration was changed while the other components remained unchanged).

[0046] Buffer 4: 5.80 g disodium hydrogen phosphate dodecahydrate and 0.59 g sodium dihydrogen phosphate dihydrate, dissolved and diluted to 100 mL of pure water, pH 7.4;

[0047] Buffer 5 at different concentrations: Protein refolding-elution buffers containing 10%, 20%, 30%, 40%, 50%, and 100% sodium chloride were prepared by adding different amounts of sodium chloride to Buffer 4, with a solution pH of 7.4.

[0048] 2.2.2 AHSV-VP7 protein purification

[0049] Purification steps (operated entirely on ice)

[0050] (1) After collecting the bacterial cells by high-speed centrifugation, 20 mL of PBS was added to each 1 g of bacterial cells (wet weight) for resuspension, and 1 mMMPMSF was added to the final concentration. The bacterial cells were then sonicated for 30 min to break them up.

[0051] (2) Centrifuge at 12000 xg at 4 ℃ for 45 min, and collect the broken precipitate;

[0052] (3) Resuspend the broken precipitate in 40 mL Buffer 1, sonicate for 3 min, and repeat washing 2-3 times;

[0053] (4) Centrifuge at 12000 xg at 4 ℃ for 45 min, discard the supernatant and collect the precipitate;

[0054] (5) Each 1 g of the crushed precipitate (wet weight) was resuspended in 40 mL of Buffer 2, sonicated for 20 min, and the supernatant was collected by high-speed centrifugation and filtered through a 0.22 μm filter;

[0055] (6) Remove the nickel column and rinse it with 10 column volumes of pure water to remove ethanol (all the following steps are carried out in a chromatography cabinet at 4 ℃).

[0056] (7) Equilibrate the nickel column with 10 mM imidazole buffer 3 and rinse with 10 column volumes of buffer.

[0057] (8) Dissolve the supernatant and load it onto the column, collect the flow-through liquid, and cycle the column 3 times.

[0058] (9) Elute the column sequentially with 10 column volumes of 10 mM, 20 mM, 40 mM, 80 mM, 100 mM, 150 mM, 200 mM and 300 mM imidazole Buffer 3, and collect the eluent.

[0059] (10) Elute the Ni column with 20 column volumes of 500 mM imidazole Buffer 3 and collect the eluent. Then wash the column with 10 column volumes of pure water, equilibrate with 3 column volumes of 20% ethanol, seal the column and store at 2-8 °C.

[0060] Protein refolding (all steps are performed on ice)

[0061] (1) Select a high-purity eluent from the Ni column purification eluent for the next step, and dilute it 15-60 times with buffer 4. Measure the corresponding volume of buffer 4 into a beaker, and slowly add Ni eluent dropwise while stirring. After mixing, let it stand at 4 ℃ for 1 h.

[0062] (2) After standing, filter with filter paper or a 0.45 μm filter.

[0063] (3) Remove column Q and rinse it with pure water to remove the ethanol.

[0064] (6) Balance column Q with a buffer 4 that is 10 times the column volume.

[0065] (7) Load the refolded protein solution onto the column, collect the flow-through liquid, and cycle the column 3 times.

[0066] (8) 0-50% Buffer 5 linear elution, collect the eluent.

[0067] After elution, wash the column sequentially with 100% Buffer 5 buffer and 10 column volumes of pure water, then equilibrate with 3 column volumes of 20% ethanol, seal the column, and store at 2-8°C.

[0068] 2.2.3 AHSV-VP7 ΔN 1-129 Protein desalting

[0069] Before use, the protein desalting inner tube was rinsed with 1 mL of 0.1 M NaOH and 1 mL of pure water, respectively. The rinsed inner tube was inserted into the matching centrifuge tube, and 500 μL of the obtained recombinant protein sample was added and the tube was capped. The capped centrifuge tube was then placed in the rotor, with the white filter membrane of the inner tube and the cap connecting strap facing the center of the rotor, and leveled. Centrifugation was performed at 4 °C (10,000 ×g) for 30 min. The volume of liquid in the inner tube was then replenished to 500 μL using 0.01 M PBS, and centrifugation was performed again at 4 °C (10,000 ×g) for 30 min, repeated three times. Finally, the inner tube was removed and inverted into another clean microcentrifuge tube, with the open cap facing the center of the rotor. Centrifugation was performed at 4 °C (9,000 ×g) for 2 min. The concentrated sample was transferred from the inner tube to the collection tube, and after measuring the concentration, it was diluted to 1 mg / mL. 80 μL of the processed recombinant protein was prepared for SDS-PAGE electrophoresis. See details below. Figure 2 .

[0070] 2.3 Preparation of alpaca positive serum targeting the VP7 protein of African horse sickness virus

[0071] 2.3.1 Immunogen Preparation

[0072] In this example, the recombinant VP7 protein of African horse sickness virus was used as the immunogen. The VP7 FL protein was prepared and purified according to Example 1 of this invention, with a purity greater than 90% as determined by SDS-PAGE electrophoresis. The VP7 protein was diluted to a working concentration of 1 mg / mL using 0.01 M PBS (pH 7.4). An equal volume of the protein solution was thoroughly emulsified with Freund's adjuvant. Full Freund's adjuvant was used for the initial immunization, and incomplete Freund's adjuvant was used for subsequent booster immunizations.

[0073] 2.3.2 Animal Immunization

[0074] One-year-old, healthy male alpacas were selected as the immunization animals. The immunization schedule is as follows:

[0075] First immunization: The above-mentioned emulsified antigen containing complete Freund's adjuvant was injected subcutaneously at multiple points in the neck of the alpaca. The dosage was 500 μg per alpaca (the total amount of 1 mL antigen and 1 mL adjuvant emulsified).

[0076] Booster immunizations: Two booster immunizations were administered on days 21 and 42 following the initial immunization. The method was the same as for the initial immunization, but with the use of incomplete Freund's adjuvant for emulsification. The dose for each injection was 500 μg of VP7 protein.

[0077] 2.3.3 Serum Collection and Preparation

[0078] On day 10 post-immunization, 100 mL of whole blood was collected from the jugular vein of alpacas. The whole blood was incubated at 37 °C for 1 hour, then transferred to a 4 °C refrigerator and allowed to stand overnight to allow the blood clots to fully shrink. After centrifugation at 8,000 xg for 15 minutes, the supernatant was collected to obtain crude alpaca positive serum. Western blotting was used for verification (see details). Figure 3 The serum was then sterilized by filtration through a 0.22 μm filter membrane, aliquoted, and stored at -80 °C.

[0079] Example 2:

[0080] Preparation and epitope identification of AHSV-VP7 recombinant protein monoclonal antibody:

[0081] 1. Materials

[0082] 250 kDa pre-stained protein marker and a rapid 12.5% ​​PAGE gel preparation kit were purchased from Shanghai Yamei Biomedical Technology Co., Ltd.; 180 kDa pre-stained protein marker, BCA protein concentration assay kit, and high-sensitivity ECL chemiluminescence detection kit were purchased from Nanjing Novizan Medical Technology Co., Ltd.; polyvinylidene fluoride (PVDF) membrane was purchased from PALL; HRP-labeled rabbit anti-mouse IgG, FITC-labeled goat anti-mouse IgG, and His monoclonal antibody were purchased from Proteintech; TMB substrate and stop solution were purchased from Beyotime Biotechnology Co., Ltd.; RPMI-1640 medium and fetal bovine serum were purchased from Gibco; Freund's complete adjuvant, Freund's incomplete adjuvant, HAT and HT medium were purchased from Sigma; other reagents were domestically produced analytical grade.

[0083] 2. Immunogen Preparation

[0084] The recombinant protein of African horse sickness virus (AHSV) VP7 FL purified by the method described in Example 1 was used as an immunogen.

[0085] 2.1 Animal Immunity and Cell Fusion

[0086] (1) Experimental animals and grouping: Six-week-old female BALB / c mice were randomly divided into an immune group and a negative control group, with 5 mice in each group.

[0087] (2) Immunization procedure: The immunization group received at least three antigen injections according to the predetermined immunization procedure. The negative control group received an equal volume of sterile PBS at the same time points.

[0088] (3) Potency assessment: Seven days after the third immunization, mouse blood was collected, serum was separated, and the specific antibody titer against the target antigen in the serum was determined by indirect ELISA.

[0089] (4) Shock immunization: Mice with serum antibody titers of not less than 1:10,000 and the highest titers were selected. Three days before the planned cell fusion, 100 μg of recombinant protein antigen was injected intraperitoneally for shock immunization.

[0090] 2.2 Hybridoma Screening and Subcloning

[0091] (1) Feeder cell preparation: One day before cell fusion, mouse peritoneal macrophages were collected and their density was adjusted to 1×10⁻⁶. 5 / mL, inoculated into 96-well plates.

[0092] (2) Cell fusion: Mice that have been enhanced with immunization were sacrificed, spleen cell suspension was prepared aseptically, and mixed with SP2 / 0 cells at a ratio of 5:1. Chemical fusion was carried out using the PEG4000 method.

[0093] (3) Selective culture: The fused cells were resuspended in selective medium containing HAT and seeded in 96-well plates containing feeder cells. Initially, HAT medium was used for half-medium replacement, and then switched to HT medium.

[0094] (4) Screening and cloning: After culturing for about 14 days, the antibody titer in the cell supernatant was detected by indirect ELISA. The positive wells were subcloned using limiting dilution at least 2-3 times until the antibody positivity rate reached 100%, thereby obtaining a stable monoclonal hybridoma cell line.

[0095] (5) Cell line expansion and preservation: The identified positive hybridoma cell lines were expanded and gradually adapted to low serum or serum-free culture media to facilitate antibody production. The cell line that could stably secrete antibodies was named monoclonal antibody mAb2E9 and frozen in liquid nitrogen.

[0096] 2.3 Monoclonal antibody purification

[0097] (1) Hybridoma cell culture: RPMI-1640 complete medium containing 20% ​​fetal bovine serum was used to revive and culture hybridoma cells at 37 ℃ and 5% CO2. When the cell confluence reached 80%-90%, the cells were passaged.

[0098] (2) Animal sensitization and inoculation: 1-2 weeks before inoculation, 10-week-old SPF-grade BALB / c mice were sensitized by intraperitoneal injection of 0.5 mL of sterile liquid paraffin. Hybridoma cells in the logarithmic growth phase were collected, resuspended, and their density adjusted to 5 × 10⁻⁶. 6 1 × 10⁻⁶ cells / mL, at a concentration of 1 × 10⁻⁶ cells / mL per mouse 6 A dose of 1 cell was administered via intraperitoneal injection.

[0099] (3) Ascites fluid collection and treatment: Closely observe the mice from the 7th day after inoculation. When the abdomen of the mice is obviously distended, collect the ascites fluid by abdominal puncture. Centrifuge the collected ascites fluid at 12,000 ×g for 10 minutes, take the supernatant and store it at -80 ℃.

[0100] (4) Monoclonal antibodies in ascites fluid were purified using a Protein G affinity chromatography column, eluted with glycine-hydrochloric acid buffer at pH 2.7, and immediately neutralized with Tris-HCl buffer (pH 9.0). The purified antibodies were identified and aliquoted and stored at -80 °C.

[0101] 2.4 Epitope Identification

[0102] A series of truncated fragments were expressed and purified based on AHSV VP7 FL protein, such as Figure 4As shown in A in the figure. After SDS-PAGE electrophoresis and membrane transfer, Western blotting analysis was performed using monoclonal antibody mAb 2E9 as the primary antibody. The results showed that monoclonal antibody mAb 2E9 specifically bound only to the truncated fragment containing amino acid sequence 296-301. See Figure A for details. Figure 4 Therefore, the epitope recognized by monoclonal antibody mAb 2E9 is located in the amino acid region from amino acid position 296 to 301 of the VP7 protein. This embodiment successfully obtained a monoclonal antibody mAb 2E9 that specifically recognizes the AHSV VP7 protein, and after identifying its epitope, it was demonstrated that this antibody can be used for the detection, diagnosis, and study of the viral mechanism of AHSV.

[0103] Example 3:

[0104] Preparation of African horse sickness virus antibody test strips

[0105] 1. Experimental materials:

[0106] Sodium borohydride, sodium citrate, sodium chloride, arginine, trehalose, 1M pH 6.8 Tris-HCl, 1.5M pH 8.0 Tris-HCl, 1.5M pH 7.8 HEPES, borax, boric acid, 1.5M pH 8.8 Tris-HCl, Tween-20, and Triton X-100 were all purchased from Nanjing Shoude Biotechnology Co., Ltd.; BSA, casein, 2-(N-morpholino)ethanesulfonic acid (MES), carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich; L-proline, sodium ascorbate, carboxymethyl chitosan, and betaine reagent were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.; SB08 sample pads, Millipore 135 chromatography membranes, G1 binding pads, H-2 absorbent pads, and J-B6... PVC support base plates were purchased from Shanghai Jiening Biotechnology Co., Ltd.; the African horse sickness blocking ELISA antibody detection kit was purchased from INGENASA, Spain; ferrous chloride and potassium ferricyanide were generously provided by Southeast University. All other reagents were domestically produced analytical grade.

[0107] Main instruments: Multifunctional microplate reader, ENSPIRE 2300, purchased from PerkinElmer, USA; Low-temperature high-speed centrifuge, LEGEND MICRO 17 R, purchased from Thermo Fisher Scientific (China) Co., Ltd.; Vacuum drying oven DFZ-6051, purchased from Shanghai Yiheng Instrument Co., Ltd.; Ultrasonic cleaner KQ2200E, purchased from Kunshan Ultrasonic Instrument Co., Ltd.; XYZ spot spray system, XYZ 3050, purchased from Baidao Trading (Shanghai) Co., Ltd.; Paper cutter, purchased from Deli Group Co., Ltd.; Transmission electron microscope from FEI, USA.

[0108] Biological materials: three recombinant VP7 proteins prepared in Example 1 and alpaca positive serum; VP7 protein monoclonal antibody (2E9) prepared in Example 2; SPA (staphylococcal A protein), purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0109] 2 methods:

[0110] 2.1 Preparation of Prussian Blue Nanoparticles (PBNPs)

[0111] (1) Dissolve 200 mg of citric acid in 40 mL of ultrapure water;

[0112] (2) Take 20 mL of the solution from step (1) and add 3 mg of ferrous chloride;

[0113] (3) Take another 20 mL of the solution from step (1) and add 6.5 mg of potassium ferricyanide;

[0114] (4) Heat the solution in step (2) to 60 °C, and add step (3) dropwise to the solution in step (2) while stirring, and react for 30 minutes.

[0115] (5) Dialyze the blue solution obtained in step (4) to a molecular weight cutoff of 8000-12000. After overnight, ultrafilter and centrifuge 3 times to obtain Prussian blue nanoparticles. The nanoparticles are observed by transmission electron microscopy and their particle size is 70-80 nm.

[0116] 2.2 AHSV-VP7 recombinant protein Prussian blue nanoconjugate

[0117] 2.2.1 Selection of Buffer Solution

[0118] This invention uses Tris-HCl (pH 7.4, 8.0, 8.8), PBS, citrate-sodium citrate buffer, borate-borax buffer (a mixture of 0.02 M boric acid and 0.005 M borax solutions in different proportions), and 2-(N-morpholino)ethanesulfonic acid (MES) solution for the coupling of Prussian blue nanoparticles. The pH used is 6.0, a condition widely adaptable to the nanomaterials, for activation experiments. Comparative experiments with different buffer solutions revealed that, except for the borate-borax buffer solution, the stability of Prussian blue nanoparticles during protein coupling was significantly worse in other solutions than in the borate-borax buffer solution. Therefore, in this invention, the borate-borax buffer solution is used as the coupling buffer solution after activation.

[0119] 2.2.2 Determination of the optimal coupling pH

[0120] This invention, through experimental comparison, found that boric acid-borax buffer solution is more conducive to the binding of tracer materials to proteins. Therefore, this experiment uses boric acid-borax buffer solution as the coupling buffer system. Different pH values ​​were set according to the ratio of boric acid-borax buffer solution to seek the optimal coupling conditions. This invention selected boric acid-borax buffer solutions with different pH values ​​of 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.7, and 9.0 to couple Prussian blue nanoparticle materials.

[0121] Through practical coupling experiments, it was observed that when the pH is above 8.0 or below 7.6, the coupling solution cannot maintain stability, exhibiting varying degrees of discoloration and aggregation. In the coupling of Prussian blue nanoparticles, a higher pH value can regulate the surface charge of the nanomaterials or antibodies, thereby enhancing their stability and reducing aggregation. Simultaneously, a higher pH value helps improve the binding efficiency of antibodies to antigens or other conjugates, thus enhancing the sensitivity of the test strip. Furthermore, a higher pH value also makes the color development of the detection line (T line) and control line (C line) clearer, enhancing the readability of the test results. Therefore, in practical coupling experiments, this invention uses VP7 FL and VP7 ΔN... 1-129 The recombinant protein was coupled at pH 8.0. VP7ΔC 264-345 The optimal coupling condition for the recombinant protein was pH 7.8.

[0122] Based on the determination of the special conditions in this invention as described above, the final coupling method adopted in this invention is as follows:

[0123] Take 3 μL of a 17 mg / mL Prussian blue nanoparticle suspension (solvent: 0.1 M KCl) and add 200 μL of a 20 mM, pH 6.0 2-(N-morpholino)ethanesulfonic acid solution. Centrifuge at 8,500 × g for 15 min, discard the supernatant, and resuspend the precipitate in 200 μL of a 20 mM, pH 6 2-(N-morpholino)ethanesulfonic acid solution. Sonicate for 2 min to disperse the precipitate evenly. Then add 10 μL of 10 mg / mL carbodiimide and 20 μL of 10 mg / mL N-hydroxysuccinimide, and incubate at 37 °C, 200 r / min on a shaker for 30 min. Centrifuge at 8,500 × g for 15 min at room temperature, discard the supernatant, and resuspend in 200 μL of a 0.02 M, pH 8.0 borate-borax buffer solution. Slowly add 15 μg of the African horse sickness virus VP7 recombinant protein fragment. The mixture was incubated at 4 °C and 60 r / min for 12 h on a shaker; 30 μL of 10 wt% BSA was added, and the mixture was sealed at 4 °C and 60 r / min on a shaker for 2 h; then centrifuged at 9,200 ×g for 20 min at room temperature, the supernatant was removed, and 200 μL of resuspension was added for resuspension and storage.

[0124] The Prussian blue nano-conjugated resuspension was prepared at a concentration of 25 μL / cm³. 2 The standard is added dropwise to the prepared conjugate pad and placed in a vacuum drying oven at 37 ℃ for 6 hours to dry.

[0125] 2.3 Preparation of Prussian Blue Nanotest Strip Sample Diluent

[0126] This invention proposes a suitable sample diluent formulation for this test strip. Experiments were conducted using different types of sample diluents prepared according to Table 1, and repeated tests were performed. It was found that the C11 formulation enabled the test strip to stably detect the target sample. Therefore, this invention selects the following sample diluent: a resuspension containing 0.5% BSA, 0.05% sodium ascorbate, 2% trehalose, 2% sucrose, 5% glycerol, and 0.5 M betaine in 20 mM HEPES (pH 7.8) to dilute the collected blood or serum samples.

[0127] Table 1. Sample dilutions with different components

[0128]

[0129] 2.4 Preparation of Prussian Blue Nanotest Strips

[0130] (1) Preparation of sample pad

[0131] The sample pads were cut into strips of 1.7 cm × 30 cm, soaked in the sample pad treatment solution for 1 h, and then dried in a vacuum drying oven at 37 ℃ for 12 h. They were then stored at 4 ℃ for further drying. The sample pad treatment solution consisted of 0.5% polyvinyl alcohol (PVA) 17-88, 0.5% Tween-20, 2% PEG2000, and 1% BSA, dissolved in 0.01 M PBS.

[0132] (2) Preparation of the binding pad

[0133] The conjugate pads were cut into strips of 0.5 cm × 30 cm, soaked in conjugate pad treatment solution for 30 min, then placed in a vacuum drying oven at 37 ℃ for 5 h, and stored at 4 ℃ for drying. The coupled crosslinked material was then used at a concentration of 25 μL / cm². 2 Add to conjugate pads and vacuum dry at 37 °C for 12 h. The conjugate pad treatment solution consisted of 0.5% trehalose, 0.75% sucrose, 1.5% BSA, and 0.5% Tween-20, dissolved in 0.01M PBS.

[0134] (3) Preparation of absorbent pad

[0135] Cut the absorbent pad into strips of 1.7 cm × 30 cm and store them in a dry environment.

[0136] (4) Selection of the optimal detection line and related conditions for chromatography detection membrane:

[0137] A 2.5 cm × 30 cm membrane was adhered to a PVC support substrate and preheated at 37 ℃ for 5 min. A gold sputtering spectrometer was used to scribing lines. Staphylococcal protein A was diluted to 2 mg / mL with 0.01 M PBS and sprayed at 1 μL / cm onto the chromatographic detection membrane as the detection line (T line). 3% methanol was added to improve reaction sensitivity. African horse sickness VP7 monoclonal antibody 2E9 was diluted to 1 mg / mL with 0.01 M PBS and sprayed at 1 μL / cm onto the chromatographic detection membrane as the control line (C line). The two lines were 0.5 cm apart. After spraying, the membrane was placed at 37 ℃ for 6 h, dried under vacuum, and then stored at 4 ℃.

[0138] (5) Assembly of test strips

[0139] Fix the absorbent pad onto the chromatographic detection membrane, overlapping it by approximately 2 mm. Then, attach the dried conjugate pad, overlapping it by approximately 2 mm onto the chromatographic detection membrane. Next, attach the sample pad, overlapping it by approximately 2 mm onto the conjugate pad. Cut the PVC support plate with the sample pad, conjugate pad, chromatographic detection membrane, and absorbent pad attached into 4 mm wide strips, and snap them onto the plastic clips to obtain the test strip.

[0140] Example 4:

[0141] AHSV-VP7 ΔN 1-129 Applications of Prussian Blue Nano Test Strips

[0142] 1. Experimental Materials

[0143] Reagents and Instruments

[0144] African horse sickness blocking ELISA antibody detection kit, purchased from INGENASA, Spain.

[0145] 1.2 Biomaterials

[0146] Equine influenza virus positive serum, equine nasal pneumonia virus positive serum, equine arteritis virus positive serum; African swine fever virus (ASFV) standard positive serum, purchased from the China Institute of Veterinary Drug Control; equine clinical serum sourced from Yunnan, Inner Mongolia, Shanghai, Qingdao and other places.

[0147] Detection principle of Prussian blue nano test strips:

[0148] The Prussian blue nanoparticle test strip consists of five physical parts: a sample pad, a conjugate pad, a chromatographic detection membrane, an absorbent pad, and a support base. The conjugate pad is coated with a protein or antibody that binds to the target. The chromatographic detection membrane has one or more test lines (T lines) and control lines (C lines) containing the intercepting protein or antibody. In the immunochromatographic test strip, after the liquid containing the detection target is added to the sample pad, it flows laterally in the order of sample pad, conjugate pad, chromatographic detection membrane, and absorbent pad due to capillary action. The detection target binds to the Prussian blue nanoparticle conjugate on the conjugate pad and is intercepted by the test line and control line, resulting in the observable band. See details... Figure 1 .

[0149] 2. Instructions for using Prussian blue nano test strips and interpretation of results

[0150] 2.1 Instructions for use of Prussian blue nano test strips

[0151] (1) Bring the test strip and the sample to be tested to room temperature (25 °C).

[0152] (2) Dilute the serum to be tested with the diluent at a ratio of 1:100 and mix by repeatedly inverting the tube.

[0153] (3) Reaction: Open the aluminum foil bag, take out the test strip, take 50 μL (one drop) of the diluted sample to be tested, add it to the reaction well, and wait for observation.

[0154] (4) Observation of results: Observe the results in 5-20 minutes, and the results should not exceed 30 minutes.

[0155] 2.2 Determination Method for Prussian Blue Nano Test Strips

[0156] (1) Positive (+): Blue bands appear on both the control line (C) and the test line (T), indicating that the sample contains AHSV antibody.

[0157] (2) Negative (-): The control line (C) is blue and the test line (T) is not colored, indicating that the sample does not contain AHSV antibody or the antibody content is lower than the detection threshold.

[0158] (3) Invalid: White board (i.e., the control line does not show color), indicating that the operation process is incorrect or the test strip has expired and needs to be tested again.

[0159] 3. Prussian Blue Nano Test Strip Sensitivity Test

[0160] Place the test strip flat on a table with the sample wells and detection window facing upwards. Add 50 μL of each of the sensitive samples obtained by diluting the alpaca positive serum of this invention to 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400 times to the reaction wells, let stand horizontally, and start timing after adding the samples. Simultaneously, use the sample dilution solution provided with the test strip and PBS (0.1 mol / L, pH 7.4) as controls. Perform two replicates for each dilution. Use a concentration of 10... 3 Monoclonal antibodies against the recombinant VP7 protein (μg / mL) were added to healthy, negative equine serum at a specific ratio to test the sensitivity of the test strips. Simultaneously, a positive control from a purchased African horse sickness blocking ELISA antibody detection kit was serially diluted 2-fold to compare the sensitivity with existing methods.

[0161] The test results show that this test strip has high sensitivity and can stably detect alpaca positive serum diluted 1:25600. The test results are shown in Table 2. The detection limit of this test strip for VP7 recombinant protein antibody reaches 25 ng / mL, as shown in Table 3. The positive control in the African horse sickness blocking ELISA antibody detection kit can stably detect 2... 7 Positive standards in the multi-fold diluted test kit are shown in Table 4.

[0162] Table 2. Sensitivity results of Prussian blue nano strips for testing positive serum.

[0163]

[0164] Table 3. Sensitivity results of monoclonal antibody tests using Prussian blue nano strips.

[0165]

[0166] Table 4. Sensitivity results of the positive control for the Prussian blue nano strip test kit.

[0167]

[0168] 4. Specificity test of Prussian blue nano strips

[0169] Place the test strip flat on the table with the sample well and detection window facing upwards. Add 50 μL of a 1:100 dilution of specific serum sample to the reaction well, allow it to stand horizontally, and observe after sample addition. Use the sample diluent and PBS (0.1 mol / L, pH 7.4) provided with the test strip as controls. Perform two replicates for each dilution.

[0170] The test results show that the Prussian blue nano strip can accurately detect AHSV-positive serum, and there is no cross-reactivity with other virus-positive serum, indicating that this test strip has high specificity. The test results are shown in Table 5.

[0171] Table 5. Specificity test results of Prussian blue nano test strips

[0172]

[0173] 5. Prussian Blue Nano Test Strips for Clinical Sample Testing and Health Screening of Horse Herds in Key Areas

[0174] The prepared Prussian blue nano-test strips were used to test 743 serum samples from common horses in Inner Mongolia and Yunnan and 149 serum samples from racehorses in Shanghai and Qingdao to assess the risk of African horse sickness virus infection in horses.

[0175] Clinical serum samples were diluted 1:100 with diluent for testing, and simultaneously tested using an African horse sickness virus blocking ELISA antibody detection kit. A total of 892 samples were tested, of which 0 samples were positive and 892 samples were negative using Prussian blue nano strips. ELISA testing also revealed 0 positive and 892 negative samples.

[0176] Under the condition of using ELISA as the detection standard, a true positive (TP) is when both ELISA and Prussian blue nano-test strips detect the virus simultaneously; a true negative (TN) is when neither ELISA nor Prussian blue nano-test strips detect the virus simultaneously; a false negative (FN) is when ELISA detects the virus but Prussian blue nano-test strips do not; and a false positive (FP) is when ELISA does not detect the virus but Prussian blue nano-test strips detect the virus.

[0177] According to the sensitivity formula: sensitivity = TP / (TP+FN), the sensitivity of the Prussian blue nano test strip is 100%.

[0178] According to the specificity formula: Specificity = TN / (FP+TN), the specificity of the Prussian blue nano test strip is 100.0%.

[0179] Clinical results have demonstrated that Prussian blue nano-immunochromatographic test strips can be fully applied to clinical testing.

[0180] Comparative Example 1:

[0181] AHSV VP7 recombinant protein for immunochromatographic test strips

[0182] 1. Recombinant Protein Preparation This invention designed and expressed three recombinant AHSV VP7 proteins:

[0183] VP7 FL: Full-length protein, VP7 ΔN 1-129 The preferred truncated protein of this invention, VP7 ΔC 264-345 : Compare truncated proteins.

[0184] All proteins were expressed and purified according to Example 1 of this invention, with a purity >90%.

[0185] 2. Assembly and testing of immunochromatographic test strips

[0186] Equal amounts of the three proteins were coated onto the conjugation pad as antigens, and the detection line was coated with the same concentration of SPA protein, assembling them into an immunochromatographic test strip. The same conjugation pad and sample pad were used to eliminate systematic errors. Since this experiment focuses on screening for superior recombinant proteins with high sensitivity, strong specificity, and stable coupling effects, there are no specific requirements for the quality control line.

[0187] 2.1 Sensitivity Comparison

[0188] The test was performed using AHSV-positive alpaca serum (prepared in Example 1) calibrated using standard methods. The serum was serially diluted and then applied to three different test strips.

[0189] Result: Contains VP7 ΔN 1-129 The protein test strip can detect serum at a maximum dilution of 1:25600, and the test line is clearly visible.

[0190] In comparison, those containing VP7 FL and VP7 ΔC 264-345 The detection sensitivities of the protein test strips were only 1:6400 and 1:800, respectively.

[0191] 2.2 Specificity comparison

[0192] To assess cross-reactivity, positive sera for equine arteritis virus (EAV), equine rhinopulmonary virus (EHV), and equine influenza virus (EIV) were used as samples for testing.

[0193] Result: Contains VP7 ΔN 1-129 VP7 ΔC 264-345 The test strip for the recombinant protein showed no cross-reaction with any of the three heterologous viral positive sera mentioned above, the test line did not develop color, and the results were clearly interpretable.

[0194] In contrast, test strips containing VP7 FL protein showed a weak cross-reactivity with EIV-positive serum, leading to a risk of false positives.

[0195] 2.3 Observation of Coupling Effect

[0196] In effective detection, VP7 ΔN 1-129 The protein used as the antigen in the detection line displays a band with sharp edges, uniform color, and a clean background, making it easy to interpret. In contrast, the detection line displayed by the VP7 FL protein has slightly blurred edges.

[0197] Based on the above comparison results, VP7 ΔN1 -129 When used as the detection line antigen in immunochromatographic test strips, recombinant proteins exhibit significantly superior sensitivity, specificity, and linear clarity compared to full-length proteins and other truncated variants: sensitivity (1:25600) is four times that of VP7 FL protein (1:6400). It demonstrates high specificity, showing no cross-reactivity with positive sera from common equine disease viruses, effectively avoiding false positives. Excellent conjugation results in clear chromatographic bands, facilitating accurate result interpretation.

[0198] Therefore, VP7 ΔN 1-129 This is the preferred antigen of the present invention, and is particularly suitable for preparing highly sensitive and highly specific immunochromatographic test strips for rapid detection of AHSV antibodies.

[0199] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A Prussian blue nano-test strip for detecting African horse sickness virus antibodies, characterized in that: A sample pad, a conjugate pad, a chromatography detection membrane, and an absorbent pad are sequentially fixed on a supporting base plate. The chromatography detection membrane has a detection line and a control line. The detection line is coated with Staphylococcus aureus protein A, and the control line is coated with a monoclonal antibody containing recombinant African horse sickness virus VP7 protein. The conjugate pad is coated with Prussian blue nanoparticles conjugated with African horse sickness virus VP7 ΔN. 1-129 Recombinant protein fragments; Among them, the African horse sickness virus VP7 ΔN 1-129 The amino acid sequence of the recombinant protein fragment is: GAVEVQQSGRYYVPQGRTRGGYINSNIAEVCMDAGAAGQVNALLAPRRGDAVMIYFVWRPLRIFCDPQGASLESAPGTFVTVDGVNVAAGDVVAWNTIAPVNVGNPGARRSILQFEVLWYTSLDRSLDTVPELAPTLTRCYAYVSPTWHALRAVIFQQMNMQPINPPIFPPTERNEIVAYLLVASLADVYAALRPDFRMNGVVAPVGQINRALVL.

2. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to claim 1, characterized in that: The monoclonal antibody against the recombinant VP7 protein of African horse sickness virus has the antigenic epitope PPIFPP.

3. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to claim 2, characterized in that: The monoclonal antibody against the recombinant VP7 protein of African horse sickness virus was secreted by hybridoma cells with accession number GDMCC No:67154.

4. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to any one of claims 1-3, characterized in that: The Prussian blue nanoparticles coupled with African horse sickness virus VP7 ΔN 1-129 Recombinant protein fragments, including African horse sickness virus VP7 ΔN 1-129 The amount of recombinant protein fragment on the binding pad is 1-3 μg / cm 2 The amount of Prussian blue nanoparticles on the conjugate pad was 3-9 μg / cm. 2 .

5. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to any one of claims 1-3, characterized in that: The amount of Staphylococcus A protein coated on the detection line is 1.5-2.5 μg / cm.

6. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to any one of claims 1-3, characterized in that: The amount of monoclonal antibody coated on the quality control line with the recombinant African horse sickness virus VP7 protein is 1-1.2 μg / cm.

7. The Prussian blue nano test strip for detecting African horse sickness virus antibodies according to any one of claims 1-3, characterized in that: The chromatographic detection membrane is made of nitrocellulose; the conjugate pad is made of glass cellulose.

8. The method for preparing the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to claim 1, characterized in that: This includes fixing the absorbent pad to one side of the chromatographic detection membrane with overlapping edges, fixing the conjugate pad to the other side of the chromatographic detection membrane with overlapping edges, and fixing the sample pad to the conjugate pad on the side away from the absorbent pad with overlapping edges. Among them, the Prussian blue nanoparticles are coupled with African horse sickness virus VP7 ΔN 1-129 The preparation method of the recombinant protein fragment includes: adding a suspension of Prussian blue nanoparticles to a 2-(N-morpholino)ethanesulfonic acid solution, centrifuging, resuspending the precipitate in a 2-(N-morpholino)ethanesulfonic acid solution, dispersing evenly, adding carbodiimide and N-hydroxysuccinimide, incubating, centrifuging, resuspending in a borate-borax buffer solution at pH 8.0, and adding African horse sickness virus VP7 ΔN. 1-129 The recombinant protein fragment was reacted at 4 °C, blocked with BSA, centrifuged, and resuspended in resuspending buffer to obtain Prussian blue nanoparticle-conjugated African horse sickness virus VP7 ΔN. 1-129 Recombinant protein fragment solution; The process of adding a resuspension solution and resuspending the virus yields Prussian blue nanoparticles coupled with African horse sickness virus VP7 ΔN. 1-129 Recombinant protein fragment solution, containing African horse sickness virus VP7 ΔN 1-129 The concentration of the recombinant protein fragment was 0.025-0.075 μg / μL, and the concentration of the Prussian blue nanoparticles was 0.08-0.26 μg / μL.

9. The method for preparing the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to claim 8, characterized in that: The method for preparing the Prussian blue nanoparticles includes: dissolving citric acid or hyaluronic acid in water to obtain solution A, which is then divided into two portions; adding a soluble ferrous salt to one portion of solution A to obtain solution B; adding potassium ferricyanide to the other portion of solution A to obtain solution C; heating solution B and adding solution C dropwise under high-speed stirring to obtain Prussian blue nanoparticles; the average particle size of the Prussian blue nanoparticles is 70-80 nm.

10. The application of the Prussian blue nano-test strip for detecting African horse sickness virus antibodies according to claim 1 in the preparation of a kit for detecting African horse sickness virus antibodies, characterized in that: It also includes a sample diluent, which is formulated as follows: a 20-30 mM HEPES solution containing 0.5-1% BSA (bovine serum albumin), 0.05-0.1% sodium ascorbate, 2-4% trehalose, 2-4% sucrose, 5-8% glycerol, and 0.5-1 M betaine, by mass percentage.

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