Colloidal gold immunochromatography test strip for detecting total antibodies of african swine fever virus

Abstract

The application discloses an African swine fever virus total antibody detection colloidal gold immunochromatography test paper card. The test paper card comprises a backing and a sample pad, a marker pad, a chromatography membrane and a water absorption pad on the backing. The detection line on the chromatography membrane is sprayed with an African swine fever virus antigen epitope polypeptide composition. The antigen epitope polypeptide composition is one or more than two arbitrary combinations of a polypeptide shown in sequence 1 in a sequence table, a polypeptide shown in sequence 2 in the sequence table and a polypeptide shown in sequence 3 in the sequence table. The quality control line is sprayed with goat anti-pig IgG. The obtained test paper card has very high sensitivity, meanwhile, the test paper card has very high specificity and accuracy, the result can be judged by naked eyes, is more intuitive, can rapidly and specifically detect specific antibodies of the African swine fever virus in serum, and has good practical value and market prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and more particularly, the present application relates to a colloidal gold immunochromatography test strip card for detecting total antibodies of African swine fever virus. BACKGROUND

[0002] The pathogen of African swine fever (ASF) is African swine fever virus (ASFV), which belongs to the Asfarviridae Asfivirus. ASFV is a capsid virus with a regular icosahedral structure composed of 5 concentric layers, with an average particle diameter of about 200 nm. The virus genome is a double-stranded DNA molecule, 170-193 kb in length, encoding more than 150 polypeptides, at least 50 of which form different domains of the virus particle. The 80 nm virus core contains the virus genome and nucleoproteins p10 and pA104R, which is wrapped by a virus capsid composed of p35, p15, p150, p37, p34 and p14 proteins; surrounding the virus capsid is a two-layer lipid molecule, the inner capsid membrane is composed of p54, p17, pE248R and p12, and the capsid structure contains p72, pE120R and pB438L; the virus is released from the host cell by budding through the plasma membrane, and the outer capsid membrane obtained in this process contains proteins p24, CD2v, p30 and p12.

[0003] African swine fever (ASF) is an acute, febrile, highly contagious zoonosis of pigs caused by the African swine fever virus (ASFV). The disease is characterized by high fever, reticuloendothelial system hemorrhage and high mortality rate in susceptible pigs. The disease is listed as a notifiable disease by the World Organization for Animal Health (OIE) and is classified as a category I animal epidemic disease in China. Currently, there is no available vaccine for ASF, and in fact, no effective commercial vaccine for ASF has ever been developed. The control of the disease relies strictly on animal quarantine, biosafety measures and culling. The technical challenges for the development of a vaccine include the following aspects: as one of the largest known DNA viruses, the genes of ASFV have not been identified and their functions have not been determined; there is no virus propagation cell line available for vaccine production; there are multiple genotypes of ASFV with different phenotypic characteristics, and there is no cross-protection in the development of candidate vaccines; the developed vaccine should be applicable for injection immunization in domestic pigs and also for oral immunization in wild boars and potentially other wild animals in the jungle transmission cycle. After the infection of animals with African swine fever virus, the immune system of the body produces specific antibodies for immune defense against the virus. Within 2 weeks after infection, IgM and IgG antibodies are produced in sequence. IgM is the first antibody to appear in the immune system, and the detection of IgM indicates that the infection has recently occurred, which is generally used for early diagnosis of infection. IgG is an indicator of past infection. The detection of ASFV-specific IgM and IgG total antibodies can improve the sensitivity of ASFV infection detection, and in addition, it can be used for the detection of IgA antibodies in milk and saliva, thus having important clinical significance for the effective control of ASFV.

[0004] There are many methods for detecting antibodies against African swine fever virus. The diagnostic techniques recommended by the World Organization for Animal Health (WOAH) are divided into etiological detection and serological test. ELISA is the international trade inspection method specified by WOAH. Virus isolation and identification and nucleic acid detection can be used as detection means for the incubation period, early stage of disease and symptomatic period, mainly including virus isolation test, red blood cell adsorption test, PCR detection, real-time fluorescent quantitative PCR test, etc. Serological test is used for detecting antigens and antibodies of African swine fever virus, mainly including fluorescence antibody technology, ELISA and immunochromatography test, etc. At present, due to the use of live virus for detection, it is time-consuming and laborious, and the etiological detection, fluorescence antibody technology and immunological blotting test require higher technical and equipment requirements and are not suitable for clinical promotion. ELISA needs to be operated in a special laboratory, which also limits its promotion and use in the grassroots.

[0005] Colloidal gold immunochromatography is a rapid detection technique that can be performed with the naked eye. It is easy to operate, has a short reaction time, requires no instruments, and produces clear, easily interpretable results, making it a suitable rapid detection technique for point-of-care testing (POCT). On the other hand, since there is currently no commercially available vaccine for African swine fever (ASF), the detection of ASF virus antibodies indicates the presence of ASF virus infection in pigs. Given the current trend of covert transmission of ASF, the detection of ASF virus-specific antibodies requires high sensitivity and specificity, as well as ease of operation and simple result interpretation, to facilitate its widespread use at the grassroots level. Summary of the Invention

[0006] The purpose of this invention is to provide a colloidal gold immunochromatographic test strip for detecting African swine fever virus antibodies. This test strip utilizes the antigenic epitope polypeptide on the African swine fever virus P54 protein (sequence 4 GenBank No. MH717102) as the specific antigen for detecting antibodies, thus establishing an antibody detection test strip with good specificity, sensitivity, and repeatability, for detecting whether the sample to be tested contains African swine fever virus antibodies.

[0007] To achieve the above objectives, this invention first screened and obtained a high-performance African swine fever virus antigenic epitope polypeptide composition. The African swine fever virus antigenic epitope polypeptide composition provided by this invention is one or more of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3 of the sequence listing. When the polypeptide composition consists of two of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3, the mass ratio of the two polypeptides is (0.5-1.5):(0.5-1.5); preferably, their mass ratio is 1:1. When the polypeptide composition consists of three of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3, the mass ratio of any three polypeptides is (0.5-1.5):(0.5-1.5):(0.5-1.5); preferably, their mass ratio is 1:1:1.

[0008] This invention also claims protection for African swine fever virus antigenic epitope peptides, which are peptides shown in Sequence 1 of the sequence listing, peptides shown in Sequence 2 of the sequence listing, or peptides shown in Sequence 3 of the sequence listing.

[0009] The immunochromatographic test strip for detecting African swine fever virus antibodies of the present invention includes a backing and a sample pad, a marker pad, a chromatography membrane, and an absorbent pad on the backing. The marker pad is labeled with a marker for Staphylococcus aureus protein A (SPA), and the chromatography membrane is provided with a detection line and a control line. The detection line (T line) is coated with the above-mentioned African swine fever virus antigen polypeptide composition.

[0010] The quality control line (C line) on the chromatography membrane is coated with goat anti-swine IgG; the African swine fever virus antigenic epitope polypeptide is obtained through chemical artificial synthesis.

[0011] The chromatography membrane is preferably a nitrocellulose membrane (NC membrane). The absorbent pad is made of absorbent filter paper; the marker pad is a glass cellulose membrane.

[0012] The backing of the test strip is made of polyethylene.

[0013] The colloidal gold immunochromatographic test strip can be used to specifically detect African swine fever virus antibodies. The test samples include, but are not limited to, whole blood, serum, plasma, saliva swabs, and milk.

[0014] The antibody categories detected by the test strip include, but are not limited to, IgG, IgA, and IgM, thus the test strip has higher sensitivity.

[0015] Qualitative detection method and result determination of the test strip card of this invention:

[0016] The method for detecting African swine fever virus antibodies using the test strip card and sample diluent of the present invention includes the following steps:

[0017] (1) Sample preparation: The types of samples to be tested include whole blood, serum, blood plasma, saliva, milk, etc. Take 2 drops of the sample to be tested (about 60 μL) and add 6 drops of sample diluent to dilute (i.e., perform a 1:4 dilution) to obtain the test sample.

[0018] (2) Tear open the aluminum foil bag of the test strip, take out the test strip, and place it on a clean work surface.

[0019] (3) Use a Pasteur tube to draw up the test sample and add 3 drops on the sample pad, adding it slowly and vertically.

[0020] (4) After the addition is complete, wait 10-15 minutes and determine the result.

[0021] (5) Result interpretation: If both the test line and the control line are purple-red, the result is positive for African swine fever virus antibody; if the test line is not colored but the control line is purple-red, the result is negative for African swine fever virus antibody; if the control line is not colored, the test strip is invalid.

[0022] The test strip of the present invention can be used to detect African swine fever virus antibodies to determine whether the tested animal has African swine fever virus antibodies produced after infection.

[0023] The advantages of this invention are as follows: This invention employs bioinformatics methods to accurately analyze the antigenic epitopes of African swine fever virus, screening peptides suitable for colloidal gold immunochromatographic detection from the major antigenic epitopes on the P54 protein (sequence 4). These peptides concentrate the antigenic epitopes of the P54 protein, exhibiting advantages of high sensitivity and strong specificity.

[0024] Meanwhile, advanced solid-phase peptide synthesis technology is used to synthesize polypeptide antigens for the preparation of antigens for the test strips of this invention.

[0025] In addition, since the polypeptide antigen used in the test strip is a chemically synthesized polypeptide, it is free of impurities and has high purity, which further improves the efficiency of detecting African swine fever virus antibodies, so as to determine whether the tested animal is infected with African swine fever virus.

[0026] In summary, this test strip uses an antigenic peptide from the major antigenic site of the chemically synthesized structural protein P54 as the detection antigen. It requires a small amount of antigen, has high sensitivity and specificity, and can effectively detect antibodies produced after African swine fever virus infection to determine whether the tested animal is infected with the virus. Experimental results show that the test strip of this invention has good repeatability, high specificity, and high sensitivity. It can meet the needs of grassroots personnel and has broad market prospects and good economic and social benefits.

[0027] The colloidal gold immunochromatographic test strip for African swine fever virus antibodies of the present invention can simultaneously detect IgM, IgG and total IgA antibodies, with higher sensitivity and specificity, and can greatly reduce the false negative rate.

[0028] The African swine fever virus antibody test strip involved in this invention is used to detect whether animals are infected with African swine fever virus, which is beneficial to the establishment of my country's African swine fever virus prevention and control system. Attached Figure Description

[0029] Figure 1 This is a colloidal gold immunochromatographic test strip for the specific detection of antibodies against African swine fever virus. Components include: 1, sample pad; 2, label pad (SPA gold-labeled pad coated with colloidal gold); 3, test line; 4, control line; 5, chromatographic membrane (nitrocellulose membrane); 6, absorbent pad; and 7, backing. Detailed Implementation

[0030] Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0031] Example 1: Preparation of colloidal gold immunochromatographic test strip for detecting total antibodies against African swine fever virus.

[0032] This experiment employed bioinformatics methods to precisely analyze the major antigenic epitopes of the P54 protein (Sequence 4) of the Pig / HLJ / 2018 strain sequence (MK333180.1) published in GenBank. Suitable peptides were screened, and polypeptide antigens were synthesized using a fully automated polypeptide synthesizer. The sequences are shown in Sequence 1, Sequence 2, and Sequence 3 of the sequence listing, respectively. The resulting polypeptide antigens, with a purity of approximately 80%, can cover the major neutralizing antigenic epitopes of the African swine fever virus P54 protein, thereby improving the detection rate of antibody positivity. Conventional methods can be used for polypeptide synthesis. This invention uses the following method to synthesize the three polypeptides of this invention, which serve as the detection antigens for the test strip of this invention.

[0033] The detection antigen for African swine fever, a polypeptide antigen of this invention, can be prepared using an Applied Biosystems fully automated polypeptide synthesizer (model 433A). The Merrifield solid-phase synthesis method is employed, using Fmoc (9-fluorenylmethyloxycarbonyl) modified amino acids and Rink Amide MBHA resin as the solid-phase support. The production process includes five parts: polypeptide antigen solid-phase synthesis, polypeptide cleavage and identification, antigen purification, freeze-drying, and preservation. These are described below:

[0034] I. Solid-phase synthesis of polypeptide antigens

[0035] 1. Preparation of Synthetic Reagents

[0036] The amino acid sequences of the antigens are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0037] SEQ ID NO:1 is shown below:

[0038] FFQPVYPRHY GECLSPVTTP 20SEQ ID NO:2 is shown below:

[0039] SRKKKAAAIE EEDIQFINPY QDQQWVEVTP QPGTSKPAGA TTASVGKPVT GRPATNRPAT60NKPVTDNPVT DRL 73SEQ ID NO:3 is shown below:

[0040] GPAAAPAAAS APAHPAEPYT TVTTQNTASQ TMSAIENLRQ RNTYTHKDL 49

[0041] Based on the peptide antigen sequence and the desired synthesis scale, prepare suitable Fmoc-modified amino acids (purchased from NOVA) and add them to the corresponding cartridges. Similarly, weigh 5g of resin according to the required synthesis scale, place it in the reaction chamber, tighten the top and bottom caps, label it, and record the name of the synthesized peptide, batch number, TARE of the reaction chamber, and the weight of the resin. Install the reaction chamber into the synthesizer. Prepare appropriate amounts of synthesis reagents, including 100% NMP, 3% AIM (hexamethylenetetramine), 35% PIP (piperidine), and 100% MeOH (methanol), and place them in the corresponding reagent bottles.

[0042] 2. Synthesizer status detection

[0043] Check if the 433A peptide synthesis instrument is operating normally. After powering on, run the Run Self Test program to check if all instrument self-test indicators are normal. Also check if nitrogen is sufficient and if the system gauge pressure is normal (normal gauge pressure for the 433A is 10.2 psi). Before synthesis, understand the instrument's performance; therefore, measure the flow rate of each synthetic reagent. For the 433A synthesizer: Send Flow Rate1-18 to the synthesizer, select Main Menu—Module Test—press Prer or next to find Module A, Module D, Module I, Module A—press Start—press more to measure or observe. If the flow rate is unsuitable, adjust the valve pressure until the requirements are met (see Table 1 below for specific testing requirements).

[0044] Table 1 Standards for Flow Rate Detection in Peptide Synthesizers

[0045] Reagents Vial No. Module Standard Range 35% Piperidine 1 A 1.0～1.2ml 3% AIM 4 D 1.0～1.2ml 100% MeOH 9 I 3.5～4.0ml DIC 8 I 0.45～0.55g 100% NMP 10 A 2.6～2.8ml

[0046] 3. Polypeptide antigen synthesis begins

[0047] In the 433A synthesizer program, send the amino acid sequence to be synthesized to the synthesizer via Std Fmoc 1.0Sol DIC90. Go to File-New-Sequence to edit the peptide sequence and save. Go to File-New-Run, check if the Chemistry is Std Fmoc 1.0Sol DIC 90 and if the Sequence matches the saved name; set the Cycles; and save. Finally, send the sequence to the synthesizer.

[0048] Main Menu—Cycle Monitor—begin, start running.

[0049] 4. Polypeptide antigen synthesis proceeds

[0050] The removal of the Fmoc group, due to the electron-withdrawing effect of the fluorene ring system, makes the 9-H acidic and easily removed by a weak base. During the reaction, piperidine (PIP) attacks the 9-H, leading to β-elimination and the formation of diphenylfluorene, which is readily attacked by a secondary cyclic amine to form a stable adduct. The removal of the Fmov group exposes the "-NH2" group for the synthetic reaction. Then, an activated amino acid protected by the next Fmoc group and 1-hydroxybenzotriazole (HOBT) are added to the reactor.

[0051] For the polypeptide sequence described above, the synthesis process begins from the C-terminus to the N-terminus, following a specific sequence of steps that are repeated continuously (the synthesizer operates automatically according to a program; the specific cycle steps are shown in Table 2 below). The amount of reagents used and the operating status are observed and recorded during the process.

[0052] Table 2. Cyclic Steps for Peptide Antigen Synthesis

[0053]

[0054] 5. Polypeptide antigen synthesis is complete.

[0055] After the peptide antigen synthesis is complete, the synthesizer will automatically stop, and the peptide resin (the peptide is still attached to the resin) will be basically clean. Then, remove the reactor from the peptide synthesizer, wash the peptide resin three times with 100% methanol, dry it in a fume hood, and then transfer all the peptide resin to a brown polyethylene bottle, place it in a -20°C freezer, and seal it with sealing film for later use.

[0056] II. Cleavage and Identification of Polypeptide Antigens

[0057] 1. Cleavage of polypeptide antigens

[0058] The polypeptide obtained through the above reaction is chemically bonded to the solid support and must be separated from the solid support by acid hydrolysis with a specific strong organic acid. Acid hydrolysis also removes the protecting groups on the functional groups of each amino acid. The steps are as follows:

[0059] Remove the synthesized polypeptide resin (meaning the peptide is still attached to the resin) from the refrigerator and place it in a 2L round-bottom flask. In a fume hood, add 90ml of trifluoroacetic acid (TFA), 10ml of tripropylsilane (TIS), and a magnetic stir bar to the flask. Then, stably place the flask on the magnetic stirrer and stir continuously at room temperature for 1 hour until the reaction is complete. After the reaction, use a rotary evaporator with a cold trap to continuously evaporate for 30–120 minutes to remove TFA from the crude product. Then, wash the crude polypeptide antigen several times with dimethylformamide (DMF). Finally, filter the mixed resin through a sintered glass funnel to obtain the polypeptide antigen.

[0060] 2. Identification of polypeptide antigens

[0061] After the peptide antigen was synthesized, qualitative and quantitative analysis was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MODAL-TOF) and reversed-phase high-performance liquid chromatography (RP-HPLC). Common amino acid analysis was used to identify the synthesized peptide.

[0062] 3. Polypeptide antigen purification

[0063] The cyclized peptide antigen was ultrafiltered using a circulating tangential filtration membrane (PALL's Tangential Flow Device circulating tangential filtration membrane and its matching peristaltic pump). As a large molecule, the peptide antigen could not pass through the membrane with its specific pore size, while small molecule impurities formed or introduced during the initial synthesis and subsequent cyclization reactions could pass through. The solution was then sterilized using a 0.2 μm filter. The final solution was aliquoted into sterile plastic bottles and labeled. The labels indicated the peptide name, serial number, batch number, concentration, production date, shelf life, and storage conditions. After aliquoting, the solutions were stored at -20°C or -40°C for later use.

[0064] 4. Freeze-drying of polypeptide antigens

[0065] To facilitate long-term preservation and transportation, the peptide antigen needs to be freeze-dried to obtain a solid peptide. The pre-frozen peptide antigen is placed in a Labconco freeze dryer for drying, resulting in a solid peptide antigen. After packaging, it is labeled with the peptide's name, serial number, batch number, concentration, production date, shelf life, and storage conditions.

[0066] Example 2: Preparation of colloidal gold immunochromatographic test strip for detecting total antibodies against African swine fever virus

[0067] 1. Preparation of sample pads: The sample pads are made of glass fiber membranes. Each glass fiber membrane is soaked in 5 mL of treatment solution (containing 3% sucrose, 0.5% BSA, 0.1% Triton X100, and 0.5% trehalose) and dried overnight at 37°C for later use.

[0068] 2. Preparation of colloidal gold-labeled Staphylococcus aureus protein A (SPA)

[0069] (1) Take 1 mL of 50 nm colloidal gold particles, add 5 μL of 1 M potassium carbonate solution (K2CO3), mix quickly for 3 minutes using a micro vortex mixer, add 25 μg of Staphylococcus A protein (SPA, concentration 5 mg / mL), mix quickly, and then incubate at room temperature for 1 hour on a 3D rotator.

[0070] (2) Add 200 μL of PBS buffer containing 10 mg / mL bovine serum albumin (BSA) at pH 7.4 and incubate at room temperature for 1 hour on a D-rotator for blocking.

[0071] (3) After sealing in step (2), the colloidal gold-labeled Staphylococcus A suspension was centrifuged at 12000 r / min for 20 minutes at 4℃. The supernatant was carefully discarded, and the precipitate was the prepared colloidal gold-labeled Staphylococcus A protein (SPA) particles. After resuspending in gold-labeled resuspension solution (containing 3% sucrose, 0.5% casein, 0.5% BSA, 0.1% Triton X100, and 0.5% trehalose), it was stored at 4℃ for later use.

[0072] 3. Preparation of gold-labeled pads (marker pads)

[0073] (1) Pretreatment: The material of the gold standard pad is a glass fiber membrane. Each glass fiber membrane is soaked in 5 mL of treatment solution (containing 3% sucrose, 0.5% BSA, 0.1% Triton X100, and 0.5% trehalose) and dried overnight at 37°C for later use.

[0074] (2) Spraying with gold: Take 180 μL of colloidal gold-labeled SPA solution (concentration of 0.14 mg / mL), spray the film at a speed of 6 μL / cm, and dry it at 37℃ for 2 hours for later use.

[0075] 4. Nitrocellulose membrane marking

[0076] (1) Spraying the detection line (T line): Spray the African swine fever virus polypeptide antigen with 0.01 mol / M PBS was diluted to a total antigen concentration of 1.5 mg / mL. Seven treatment groups were set up: ZM202201 (stretched peptide is the peptide shown in Sequence 1), ZM202202 (stretched peptide is the peptide shown in Sequence 2), ZM202203 (stretched peptide is the peptide shown in Sequence 3), ZM202204 (stretched peptide is the peptide shown in Sequence 1 and the peptide shown in Sequence 2 in a 1:1 mass ratio), ZM202205 (stretched peptide is the peptide shown in Sequence 2 and the peptide shown in Sequence 3 in a 1:1 mass ratio), ZM202206 (stretched peptide is the peptide shown in Sequence 1 and the peptide shown in Sequence 3 in a 1:1 mass ratio), and ZM202207 (stretched peptide is the peptide shown in Sequence 1, the peptide shown in Sequence 2, and the peptide shown in Sequence 3 in the sequence listing in a 1:1 mass ratio). The peptides were uniformly drawn on the T-line of the nitrocellulose membrane at a speed of 1 μL / cm using a streak scrubber.

[0077] (2) Quality control line (C line) spraying: Goat anti-pig IgG (Sigma SAB 4600047, 2mg / mL) was diluted with 0.01mol / MPBS to 1mg / mL. The solution was then evenly applied to the C line position of the nitrocellulose membrane at a speed of 1μl / cm using a membrane scribing instrument. The membrane was then dried overnight at 37℃ for later use.

[0078] 5. Assembly of the test strip: Assemble the test strip as follows: On a clean operating table under normal humidity and temperature, attach the prepared sample pad (1), the gold-labeled pad (2) coated with the marker pad - colloidal gold-labeled SPA, the chromatography membrane - nitrocellulose membrane (5) coated with the detection line (3) and control line (4), and the absorbent pad (6) (made of absorbent filter paper) in sequence with a 2-4mm overlap to the backing (7), see Figure 1 .

[0079] 6. Cutting and Assembling the Test Strips: Cut the assembled test strips into 4mm colloidal gold test strips using a strip cutter. Select intact and neat test strips, place them into the card holder, and after capping, seal them together with the desiccant in an aluminum foil bag for storage.

[0080] 7. Preparation of sample diluent: The sample diluent is 0.01M PBS buffer with pH 7.4 containing 0.5% Tween 20.

[0081] Example 3, Sensitivity Test

[0082] I. Instructions for using the colloidal gold immunochromatographic test strip for total antibody detection of African swine fever virus

[0083] (1) Sample preparation: The types of samples to be tested include whole blood, serum, saliva, milk and other samples. Take 2 drops of the sample to be tested (about 60 μL) and add 6 drops of sample diluent to dilute (i.e., dilute 1:4) as the test sample.

[0084] (2) Tear open the aluminum foil bag of the test strip, take out the test strip, and place it on a clean work surface.

[0085] (3) Use a Pasteur tube to draw up the test sample and add 3 drops on the sample pad, adding it slowly and vertically.

[0086] (4) After the addition is complete, wait 10-15 minutes and determine the result.

[0087] (5) Result interpretation: If both the test line and the control line are purple-red, the result is positive for African swine fever virus antibody; if the test line is not colored but the control line is purple-red, the result is negative for African swine fever virus antibody; if the control line is not colored, the test strip is invalid.

[0088] II. Sensitivity Test

[0089] 1. Sensitivity test

[0090] Three batches of colloidal gold immunochromatographic test strips for detecting total African swine fever virus antibodies (batch numbers ZM202201 to ZM202207) prepared according to the method in Example 2 were used to test African swine fever standard positive serum (purchased from the China Institute of Veterinary Drug Control, with an indirect immunofluorescence assay titer of 1:4000 and CVCC number Z286, detected using the gold standard method for detecting African swine fever virus antibodies). First, the African swine fever standard positive serum was diluted 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600 times using sample dilution buffer (0.01M PBS buffer containing 0.5% Tween 20, pH 7.4), and then tested using the test strips of this invention. The results showed that the three batches of test strips of the present invention were still positive for African swine fever standard positive serum at a dilution of 1:12800. That is, the lowest detection limit of the test strips of the present invention for African swine fever can reach 1:12800, which is higher than the sensitivity of indirect immunofluorescence assay.

[0091] 2. Sensitivity test

[0092] Three batches of colloidal gold immunochromatographic test strips for detecting total African swine fever virus antibodies (batches ZM202201 to ZM202207) prepared according to the method in Example 2 were used to test 55 swine serum samples infected with African swine fever (which tested positive for African swine fever virus antibodies using the Korean Jinno African swine fever virus ELISA antibody detection kit). The results (Table 3) show that the test strips of the present invention detected a total of 55 samples, indicating that the sensitivity of the test strips to 55 known positive sera is 100.0%.

[0093] Table 3. Results of Sensitivity Test

[0094]

[0095] 3. Specificity test

[0096] Using three batches of colloidal gold immunochromatographic test strips (batches ZM202201 to ZM202207) prepared according to the method in Example 2, 20 healthy pig serum samples, 2 positive sera for foot-and-mouth disease virus type O (FMD-O), 2 positive sera for foot-and-mouth disease virus type A (FMD-A), 2 positive sera for porcine circovirus (PCV2), and 2 positive sera for porcine reproductive and respiratory syndrome (PRRS) were tested according to the above-described method for using the colloidal gold immunochromatographic test strips for total African swine fever virus detection.

[0097] The specificity test results of the test strips are shown in Table 4 below. The results of testing 20 healthy pig serum samples showed that the specificity of all 7 batches of test strips was 100.0%. The test results for 2 positive sera for porcine foot-and-mouth disease virus type O (FMD-O), 2 positive sera for porcine foot-and-mouth disease virus type A (FMD-A), 2 positive sera for porcine circovirus (PCV2), and 2 positive sera for porcine reproductive and respiratory syndrome (PRRS) were all negative. Therefore, the specificity of all 7 batches of test strips for detecting these 8 related pathogen positive sera was 100%.

[0098] Table 4. Results of Specificity Tests

[0099]

[0100]

[0101] Example 4: Compliance test with imported reagent kits

[0102] The ELISA method is a diagnostic technique for African swine fever virus antibodies recommended by the World Organisation for Animal Health (WOAH). In this experiment, seven batches of colloidal gold immunochromatographic test strips for detecting total African swine fever virus antibodies, prepared according to the method in Example 2, were used, along with the Korean Jinno African swine fever antibody detection kit, to test 35 swine serum samples.

[0103] The results of the concordance rate test (Table 5) show that the sensitivity of the colloidal gold immunochromatographic test strip for detecting total antibodies against African swine fever virus (batch number ZM202201) was 48.6% for 35 samples of swine serum, while the sensitivity of the imported kit was 77.1%. The results were consistent for 21 samples. Therefore, the concordance rate between the kit of this invention and the imported kit is 60.0%.

[0104] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202202) showed a sensitivity of 42.9% for 35 swine serum samples, while the sensitivity of the imported kit was 77.1%. Results were consistent for 19 samples detected by both methods. Therefore, the concordance rate between the present invention's kit and the imported kit is 54.2%.

[0105] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202203) showed a sensitivity of 42.9% for 35 swine serum samples, while the sensitivity of the imported kit was 77.1%. Results were consistent for 21 samples detected by both methods. Therefore, the concordance rate between the present invention's kit and the imported kit is 60.0%.

[0106] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202204) showed a sensitivity of 68.6% for 35 swine serum samples, while the sensitivity of the imported kit was 77.1%. Results were consistent for 26 samples detected by both methods. Therefore, the concordance rate between the present invention's kit and the imported kit is 74.3%.

[0107] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202205) showed a sensitivity of 60.0% for 35 swine serum samples, while the imported kit showed a sensitivity of 77.1%. Results from both methods were consistent for 27 samples. Therefore, the concordance rate between this invention's kit and the imported kit is 77.1%.

[0108] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202206) showed a sensitivity of 62.9% for 35 swine serum samples, while the sensitivity of the imported kit was 77.1%. Results from both methods were consistent for 26 samples. Therefore, the concordance rate between the present invention's kit and the imported kit is 74.3%.

[0109] The colloidal gold immunochromatographic assay strip for detecting total antibodies against African swine fever virus (batch number ZM202207) showed a sensitivity of 82.9% for 35 swine serum samples, compared to 77.1% for an imported kit. Results were consistent for 33 samples. Therefore, the kit from this invention has a concordance rate of 94.3% with the imported kit, demonstrating high accuracy and suitability for African swine fever antibody detection.

[0110] Table 5. Results of the Compliance Rate Test

[0111]

[0112]

[0113]

[0114]

[0115]

Claims

1. A colloidal gold immunochromatographic test strip for detecting total antibodies against African swine fever virus, characterized in that, The invention includes a backing and a sample pad, a marker pad, a chromatography membrane, and an absorbent pad on the backing; characterized in that the marker pad is embedded with colloidal gold-labeled Staphylococcus A protein, the chromatography membrane is provided with a detection line and a control line, the detection line is coated with an African swine fever virus antigenic epitope polypeptide composition, wherein the African swine fever virus antigenic epitope polypeptide composition is the polypeptide shown in Sequence 1 of the sequence listing, the polypeptide shown in Sequence 2 of the sequence listing, and the polypeptide shown in Sequence 3 of the sequence listing.

2. The colloidal gold immunochromatographic test strip card according to claim 1, characterized in that: The polypeptide composition comprises the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3 in a mass ratio of (0.5~1.5):(0.5~1.5):(0.5~1.5).

3. The colloidal gold immunochromatographic test strip card according to claim 2, characterized in that: The polypeptide composition comprises the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, and the polypeptide shown in sequence 3 in a mass ratio of 1:1:

1.

4. The colloidal gold immunochromatographic test strip card according to claim 1, characterized in that: The quality control lines on the chromatography membrane are coated with goat anti-swine IgG; the African swine fever virus antigenic epitope polypeptide is obtained through chemical artificial synthesis.

5. The colloidal gold immunochromatographic test strip card according to claim 1, characterized in that: The backing is a polyethylene backing.

6. The colloidal gold immunochromatographic test strip according to any one of claims 1-5, characterized in that, It also includes loading the casing.

7. The colloidal gold immunochromatographic test strip according to any one of claims 1-5, characterized in that, The backing is made of polyethylene material; The absorbent pad is made of absorbent filter paper; the marker pad is made of glass fiber membrane. The chromatography membrane is a nitrocellulose membrane.

8. The use of the colloidal gold immunochromatographic test strip according to any one of claims 1-7 in the preparation of a kit for the specific detection of African swine fever virus antibodies.

9. The application according to claim 8, characterized in that, The test samples for the kits that specifically detect antibodies against African swine fever virus include whole blood, serum, plasma, saliva swabs, and milk from pigs.

10. The application according to claim 8, characterized in that, The kit detects African swine fever virus antibody categories including IgG, IgA, and IgM.

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