Colloidal gold immunochromatography test strip for detecting African swine fever virus antibody and application of colloidal gold immunochromatography test strip

Abstract

The invention provides an African swine fever virus antibody detection colloidal gold immunochromatography test strip and application thereof. The method can be used for detecting various scenes, is beneficial to improving the detection sensitivity, can recognize the African swine fever earlier, faster and more accurately, and solves the problems of retrophication and recessive infection.

Description

Technical Field

[0001] This disclosure relates to the field of immunoassay, and more particularly to a colloidal gold immunochromatographic test strip for detecting African swine fever virus antibodies and its application. Background Technology

[0002] African swine fever is a viral disease caused by infection with the African swine fever virus (ASFV). The disease is highly contagious and deadly, with a morbidity and mortality rate that can reach 100%.

[0003] Due to ASFV's large genomic structure and complex immune escape mechanisms, developing an effective vaccine is extremely difficult, and to date, there is no safe and effective vaccine for epidemic prevention and control. Currently, there are no effective vaccines or drugs to control African swine fever. Large-scale pig farms mainly rely on building high-level biosecurity systems for prevention and control; early diagnosis, identification, and precise culling are key technologies for the success or failure of African swine fever control.

[0004] However, commercially available test cards cannot meet the testing requirements in terms of sensitivity and other detection performance, so there is an urgent need for detection technologies with higher sensitivity. Summary of the Invention

[0005] To address at least one of the above-mentioned problems, this disclosure provides a colloidal gold immunochromatographic test strip for detecting African swine fever virus antibodies and its application. Using the immunochromatographic test strip provided by this disclosure can improve detection performance, such as sensitivity.

[0006] According to one aspect of this disclosure, an immunochromatographic test strip for detecting African swine fever antibodies is provided, comprising a chromatographic membrane disposed on a base plate, wherein an absorbent pad is disposed at one end of the chromatographic membrane, and a blood filtration membrane and a conjugation pad are disposed sequentially at the other end. The chromatography membrane is provided with a control line (C line) and a detection line (T line), with the T line located near the conjugate pad and the C line located near the absorbent pad. The T-line is coated with African swine fever virus P30 protein or a functional fragment thereof, and the C-line is coated with an antibody against the tag protein or an antigen-binding fragment thereof; the binding pad is coated with African swine fever virus P30 protein or a functional fragment thereof bearing the marker and tag protein.

[0007] In some embodiments, the African swine fever virus P30 protein comprises an amino acid sequence as shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0008] In some embodiments, the label includes at least one of colloidal gold, nanoparticles, quantum dot fluorescent microspheres, latex microspheres, protein peptides, nucleic acid sequences, or isotopes.

[0009] In some embodiments, the particle size of the colloidal gold includes 10 nm to 50 nm.

[0010] In some embodiments, the particle size of the colloidal gold includes 20 nm to 40 nm.

[0011] In some embodiments, the particle size of the colloidal gold includes, but is not limited to, 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm, 25nm, 26nm, 27nm, 28nm, 29nm, 30nm, 31nm, 32nm, 33nm, 34nm, 35nm, 36nm, 37nm, 38nm, 39nm, 40nm, 41nm, 42nm, 43nm, 44nm, 45nm, 46nm, 47nm, 48nm, 49nm, 50nm, or 50nm.

[0012] In some embodiments, the tag protein includes one or more of the following: glutathione thiotransferase, staphylococcal protein A, green fluorescent protein, maltose-binding protein, GFP, CFP, YFP, mCherry, SNAP-Tag, Halo Tag, SUMO, His, HA, c-myc, Avi, FLAG, Strep tag, Strep tag II, Twin Strep tag II, or derivatives or fragments thereof.

[0013] In some embodiments, the tag protein is located at the N-terminus or C-terminus of the African swine fever virus P30 protein or a functional fragment thereof.

[0014] In some embodiments, the chromatography membrane includes at least one of nitrocellulose membrane, PVDF membrane, nylon membrane, glass fiber membrane or DEAE cellulose membrane.

[0015] In some embodiments, the substrate includes a weakly fluorescent substrate or a non-fluorescent substrate.

[0016] According to a second aspect of this disclosure, a method for preparing the immunochromatographic test strip described in the first aspect is provided, the method comprising the following steps: (1) Add K2CO3 solution to colloidal gold solution and mix well; dilute African swine fever virus P30 protein with tag protein to 100 μL with deionized water, then add it to colloidal gold solution and mix quickly. After reacting for 20 minutes, add 20 μL of 20% w / w BSA solution and block at room temperature; after centrifugation and discarding the liquid, add a solution composed of BSA, sucrose, sodium chloride and phosphate buffer to the precipitate and mix well to obtain colloidal gold-labeled P30 protein. The ratio of colloidal gold, K2CO3 solution and African swine fever virus P30 protein with tag protein is 1 mL: 1.5 μmol: 50 μg. (2) The above-mentioned colloidal gold-labeled P30 protein is sprayed onto the conjugate pad, preferably and then dried overnight at 37°C, wherein the spraying amount is 6~8 μL / cm, preferably 6 μL / cm; (3) Spray 0.83 mg / mL of P30 protein onto the detection line (T line) of the chromatography membrane, and spray 0.2 mg / mL of His tag antibody onto the control line (C line) of the chromatography membrane. The distance between the detection line (T line) and the control line (C line) is 3~5 mm, and the edge distance of the T line is 6~8 mm. After completion, dry the chromatography membrane. (4) Preparation steps of blood filtration membrane: Soak the blood filtration membrane in the treatment solution until saturated (about 3 to 5 seconds), then take it out and place it horizontally on a metal mesh rack. Dry it at 45°C for about 12 hours. Cut it into 10 mm × 300 mm pieces and store it at room temperature in a drying oven. (5) The blood filtration membrane, conjugate pad, NC membrane and absorbent pad are sequentially pasted onto the PVC base plate, with each component overlapping each other by 1-2 mm to obtain the colloidal gold test strip.

[0017] In some embodiments, step (1) includes: adding 15 μL of 0.1 mol / L K2CO3 solution to 1 mL of colloidal gold solution and mixing well; diluting 2 μL of 2.5 mg / mL African swine fever virus P30 protein with the tagged protein to 100 μL using deionized water, then adding it to the colloidal gold solution and mixing quickly; after reacting for 20 minutes, adding 20 μL of 20% w / w BSA solution and blocking at room temperature; after centrifugation and discarding the liquid, adding a solution composed of BSA, sucrose, sodium chloride and phosphate buffer to the precipitate and mixing well to obtain colloidal gold-labeled P30 protein.

[0018] In some embodiments, the 20-minute reaction in step (1) includes a 20-minute reaction at room temperature (20±5°C) on a rotary mixer.

[0019] In some embodiments, the solution composed of BSA, sucrose, sodium chloride, and phosphate buffer in step (1) contains 0.01 mol / L PB, 1% w / w BSA, 2% w / w sucrose, and 0.15 mol / L sodium chloride.

[0020] In some embodiments, the drying conditions in step (3) are 45°C for 4 hours.

[0021] In some embodiments, the treatment solution used in step (4) consists of: 20 mmol / L PB (pH 7.4), containing 150 mmol / L NaCl, 1% Triton x-100, and 0.5% BSA.

[0022] According to a third aspect of this disclosure, a method for detecting African swine fever virus antibodies is provided, the method comprising using the immunochromatographic test strip described in the first aspect to detect African swine fever virus antibodies in a sample to be tested.

[0023] In some embodiments, the detection method includes the following steps: B1) Load the sample to be tested onto the blood filtration membrane and perform an immunochromatographic reaction on the immunochromatographic test strip; B2) After the reaction is completed, the results of the C-line and T-line tests are used to determine whether the sample contains African swine fever virus antibodies.

[0024] In some embodiments, the sample to be tested in step B1) is diluted and then loaded onto the blood filtration membrane.

[0025] In some implementations, the dilution factor of the sample to be tested includes 3 to 10 times.

[0026] In some implementations, the dilution factor of the sample to be tested includes 3, 4, 5, 6, 7, 8, 9, and 10 times.

[0027] In some implementations, a sample diluent is used to dilute the sample to be tested.

[0028] In some embodiments, the sample diluent includes one or more of 100 mM NaCl, 16 mM Na2HPO4, 4 mM KH2PO4, 0.2% casein, 1% Tween-20, and 0.03% Proclin 300.

[0029] In some embodiments, the pH of the sample diluent includes 7 to 8, for example 7.4, 7.5, or 7.6.

[0030] In some implementations, if both the T and C lines are red, the result is positive for African swine fever virus antibodies, meaning the sample contains antibodies against the African swine fever virus P30 protein. If both the C and T lines are red, the result is negative for African swine fever virus antibodies, meaning the sample does not contain antibodies against the African swine fever virus P30 protein. If the C line is not colored, the test result is invalid regardless of whether the T line is colored.

[0031] In some implementations, the sample to be tested includes a body fluid sample and / or a tissue sample.

[0032] In some embodiments, the sample to be tested includes blood, serum, plasma, dried blood paper, saliva, urine, feces, spleen, lymph nodes, tonsils, bone marrow, etc.

[0033] In some implementations, the sample to be tested comes from a suidae or a rodent.

[0034] In some embodiments, the test samples are from domestic pigs, wild boars, warthogs, bush pigs, rabbits, rats, mice, etc.

[0035] According to the fourth aspect of this disclosure, the use of the immunochromatographic test strip described in the first aspect or the preparation method described in the second aspect in the preparation of products for detecting African swine fever virus antibodies in a sample to be tested is provided.

[0036] In some embodiments, the product can be used to diagnose African swine fever virus infection, evaluate the immunization effect of African swine fever virus vaccines, or distinguish between African swine fever virus infection and immunization by African swine fever virus vaccines.

[0037] In some implementations, the sample to be tested includes a body fluid sample and / or a tissue sample.

[0038] In some embodiments, the sample to be tested includes blood, serum, plasma, dried blood paper, saliva, urine, feces, spleen, lymph nodes, tonsils, bone marrow, etc.

[0039] In some implementations, the sample to be tested comes from a suidae or a rodent.

[0040] In some embodiments, the test samples are from domestic pigs, wild boars, warthogs, bush pigs, rabbits, rats, mice, etc.

[0041] Beneficial effects: This disclosure describes the development of ASFV antibody detection technology and the fabrication of test strips based on positive African swine fever virus samples. These technologies can be used in various scenarios, such as clinical diagnosis, on-site testing, rapid testing, large-scale screening, epidemiological investigation, and inspection and quarantine. This will help improve detection sensitivity, identify African swine fever earlier, faster, and more accurately, and solve the problems of repopulation and latent infection. Attached Figure Description

[0042] Figure 1 The results of PCR identification of the P30 recombinant plasmid are shown.

[0043] Figure 2 The SDS-PAGE identification results of P30 protein are shown. Where M: Marker, 1: Supernatant, 2: Precipitate.

[0044] Figure 3 The SDS-PAGE results of affinity chromatography-purified p30 protein are shown. M: Marker, 1: 30 mM imidazole, 2: 80 mM imidazole, 3: 120 mM imidazole, 4: 500 mM imidazole.

[0045] Figure 4 The comparison results of P30 colloidal gold labeling conditions are shown. Figure 4 A shows different amounts of K2CO3. Figure 4 B shows the comparison results of different amounts of K2CO3 used for electrostatic labeling of P30 protein.

[0046] Figure 5 A structural diagram of the detection card is shown.

[0047] Figure 6 A schematic diagram of serum sample addition and a method for interpreting test results are shown. Figure 6 A is a schematic diagram of sample addition. Figure 6 B is the result determination chart.

[0048] Figure 7 The results show a comparison between the T-line scribing fold and the amount of gold-labeled P30 protein sprayed onto the conjugate pad. From left to right: ...

[0049] Figure 8 The comparison results of P30 labeling amounts are shown.

[0050] Figure 9 The comparison results of gold-labeled antigen dosage are shown. 4, 5, and 6 represent different gold-labeled antigen dosages: 4 μL / cm, 5 μL / cm, and 6 μL / cm, respectively. Figure 9 A shows the test results of African swine fever negative serum; Figure 9 B shows the test results for African swine fever-positive serum.

[0051] Figure 10 The results of the sensitivity experiment are shown.

[0052] Figure 11The results of the specific tests are shown. Among them, 1-4 are positive for CSFV antibody, 5-8 are positive for PRRSV antibody, 9-11 are positive for PCV antibody, 12-14 are positive for PRV antibody, 15-17 are positive for FMDV / O antibody, and 18-20 are positive for PEDV antibody.

[0053] Figure 12 The results of the rapid antibody test for African swine fever virus are shown. Detailed Implementation

[0054] This disclosure uses a prokaryotic expression system to clone and express AFSV-specific proteins, and prepares colloidal gold immunochromatographic test strips for African swine fever antibody screening in large-scale pig farms.

[0055] definition Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0056] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0057] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

[0058] In this disclosure, the term "African swine fever (ASF)" refers to a hemorrhagic, highly fatal viral infectious disease of pigs caused by African swine fever virus (ASFV). Pigs of all ages are susceptible to infection. The incubation period for natural ASF infection is long. Clinical manifestations include high fever, loss of appetite, and bleeding in the skin and internal organs, and its clinical symptoms are similar to those of classical swine fever and swine danscin virus.

[0059] In this disclosure, the term "African swine fever virus (ASFV)" refers to a virus belonging to the genus *Asfarviridae*, the only species within that family. It is a double-stranded DNA virus. ASFV is highly infectious and extremely pathogenic. It has an icosahedral morphology, approximately 200 nanometers in diameter, and is composed of multiple layers: a central protein nucleoid shell, followed by a lipid envelope and a protein capsid. The capsid consists of 8280 major capsid proteins (p72) and 60 p72 proteins. In addition, at least three other proteins maintain the capsid structure's stability by adhering to adjacent proteins. Acute cases are characterized by high fever, short disease course, high mortality, widespread internal organ hemorrhage, and respiratory and nervous system dysfunction. The ASFV p30 protein is an early membrane protein expressed by the African swine fever virus, encoded by the ORFCP204L gene. It is typically produced 2-4 hours after infection and is continuously expressed throughout the infection period. It is involved in viral invasion of host cells and is an important structural protein. The ASFV p54 protein is also an early membrane protein expressed by the African swine fever virus, encoded by the ORFE183L gene. It contains a transmembrane domain and is located in the endoplasmic reticulum-derived inner membrane precursor. It plays a crucial role in viral adsorption to susceptible cells and invasion, and is also an important structural protein. The ASFV p72 protein is produced late in viral infection, encoded by the ORF B646L gene. It is an important antigenic protein of the African swine fever virus, a major component of the viral icosahedron, and is essential for viral capsid formation.

[0060] In this disclosure, the term "immunochromatography" is used primarily based on the principle of the specific binding and color development of antigens and antibodies. Labels can be used to enable easy visual detection or the use of sensors to detect antigen-antibody reactions.

[0061] In this disclosure, the term "marker" as used refers to a substance capable of generating a signal that can be visually perceived or sensed using a sensor. Examples of markers used in this invention include, but are not limited to, latex particles, gold particles, colored polystyrene microparticles, enzymes, fluorescent dyes, conductive polymers, or magnetic particles. Furthermore, signals can be generated by the inherent properties of the marker (e.g., cold light) or by external stimuli (e.g., fluorescence).

[0062] In this disclosure, the term "antibody or antigen-binding fragment thereof" as used encompasses immunoglobulins (whether naturally occurring or partially or completely synthetically produced) and fragments thereof. The term also covers any protein having a binding domain homologous to an immunoglobulin binding domain. The use of the term "antibody" is intended to include complete antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and also includes single-chain antibodies, humanized antibodies, mouse antibodies, chimeric monoclonal antibodies, mouse-human monoclonal antibodies, mouse-primate monoclonal antibodies, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments (e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(ab1)2, Fv, dAb, and Fd fragments), bifunctional antibodies, and antibody-associated peptides. Antibodies include both bispecific and multispecific antibodies, provided they exhibit the desired biological activity or function.

[0063] In this disclosure, the term "antigen" as used means any substance that can be specifically bound by an antibody or an antigen-binding fragment. This includes whole antigens or polypeptide fragments containing antigenic epitopes, and said antigens can be antigens isolated from organisms or bioengineered antigens.

[0064] In this disclosure, the term "tag protein" refers to a class of protein molecules that can bind to a specific ligand. The tag protein is selected from tag proteins known to those skilled in the art or tag proteins designed by computer programs. For example, the tag protein is selected from, but is not limited to, the following proteins: glutathione thiotransferase, staphylococcal protein A, green fluorescent protein, maltose-binding protein, GFP, CFP, YFP, mCherry, SNAP-Tag, Halo Tag, SUMO, His, HA, c-myc, Avi, FLAG, Strep tag, Strep tag II and / or Twin Strep tag II, and / or derivatives thereof, and / or fragments thereof. In one specific embodiment, the tag protein is located at the N-terminus or C-terminus of the polypeptide.

[0065] In this disclosure, the term "sequence identity" as used refers to the "sequence identity percentage" or "identity percentage" between two polynucleotides, that is, the number of identical matching positions common to sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide is present in both the target and reference sequences. Since vacancies are not nucleotides, vacancies present in the target sequence are not counted. Similarly, vacancies present in the reference sequence are not counted because nucleotides from the target sequence are counted but nucleotides from the reference sequence are not. At least 85% sequence identity includes at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the full length of the sequence having sequence identity. Methods for aligning the sequences are well known in the art. Various procedures and alignment algorithms are described in Smith and Waterman, Adv. Appl. Math. 2” 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48” 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85” 2444, 1988; Higgins and Sharp, Gene, 73” 237-44, 1988; Higgins and Sharp, CABIOS 5” 151-3, 1989; Corpet et al., Nuc. Acids Res. 16” 10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. Mol. Bio. .24”307-31, 1994; Altschul et al., J .Mol .Biol .215”403-10, 1990, present detailed considerations on sequence alignment methods and homology calculations.

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. The actual scope of protection of this invention is set forth in the claims. In the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications. Unless otherwise specified, the equipment, instruments, reagents, and / or kits used in the following embodiments are commercially available or obtained through conventional methods known to those skilled in the art.

[0067] Example Example 1: Preparation and Identification of African Swine Fever Diagnostic Antigens Based on the published African swine fever P30 gene sequence (GenBank: NC-044959), after codon optimization in E. coli, Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the DNA sequence. The synthesized DNA sequence was then cloned into the pET-28a(+) vector to form the pET-28a(+)-P30 recombinant plasmid (His tag).

[0068] The amino acid sequence of the recombinant P30 (SEQ ID NO:1): DFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKKHHHHHH Figure 1 The results of PCR identification of the pET-28a(+)-P30 recombinant plasmid are shown in the figure. A specific target band appeared at 2900bp, indicating that the recombinant plasmid was successfully prepared.

[0069] The recombinant plasmid pET-28a(+)-P30 was transformed into *E. coli* BL21(DE3) using a chemical transformation method. The specific steps are as follows: 50 μL of thawed BL21(DE3) competent cells were transferred to a sterile EP tube. 2 μL of the recombinant plasmid pET-28a(+)-P30 was added, and the mixture was gently mixed. The tube was incubated on ice for 30 min, then heat-shocked in a 42℃ water bath for 45 s, followed immediately by an ice bath for 2 min. The centrifuge tube was not shaken during this process. 200 μL of antibiotic-free LB liquid medium was added, and the tube was incubated at 37℃ for 1 h on a shaker at 180 rpm to allow the bacteria to recover. The suspension was then evenly spread onto LB solid medium containing 50 μg / mL kanamycin and incubated upside down at 37℃ for 12 h before bacterial culture was selected for preservation. 1 mM IPTG was added, and the tube was incubated at 15℃ for 5 h to induce soluble expression. The bacterial culture was then collected. Dissolve the protein thoroughly in 8M urea, place it in an ice box, and lyse it using an ultrasonic cell disruptor for 10 minutes, observing the lysis progress every 2 minutes at a pressure of 1300 Bar. Centrifuge the lysate at 8000 rpm for 20 minutes, collect the liquid, and use Ni-NTA agarose to nonspecifically bind to the P30 protein tagged with HIS (histidine). Then, competitive protein elution was performed using gradient concentrations of imidazole (30mM, 80mM, 120mM, 500mM) to purify the recombinant P30 protein.

[0070] The collected supernatant and precipitate were identified by Western blotting, and the results are as follows: Figure 2 As shown, p30 can be successfully expressed, and clear reaction bands appear in both the supernatant and the precipitate, indicating that the protein is expressed in a soluble manner.

[0071] The SDS-PAGE results after purification of the recombinant protein using a Ni-NTA column are as follows: Figure 3 As shown, elution with 500 mM imidazole yielded the highest efficiency, and the SDS-PAGE results showed the most distinct bands. Protein concentration was determined using a Nanodrop 2000 micro-volume spectrophotometer, and protein purity was analyzed using Imagej software. With a p30 concentration of 2.5 mg / mL, the protein purity was 96%.

[0072] Example 2. Immunochromatographic detection method for African swine fever antibodies and manufacturing process of the detection card product 1. Preparation of 30nm colloidal gold: Gold nanoparticles were prepared by reduction with trisodium citrate. The specific steps are as follows: (1) Add 100mL of pure water to a round-bottom flask and bring it to a boil; (2) Add 2.0mL of chloroauric acid (2%w / v) and 1.0mL of trisodium citrate solution (1%w / w) to the round-bottom flask and stir continuously until boiling during heating; (3) Stop heating after boiling for 5min and let it cool naturally until room temperature; (4) Make up to 100mL with distilled water and store at 4℃ for later use.

[0073] 2. Electrostatic labeling of P30 protein: Take 1 mL of the colloidal gold solution prepared above into a 1.5 mL centrifuge tube, add an appropriate volume of 0.1 mol / L K2CO3 to adjust the pH of the colloidal gold solution; dilute 2 μL of the P30 protein (2.5 mg / mL) obtained in Example 1 to 100 μL with deionized water, then add it to the colloidal gold solution and mix quickly. React at room temperature (20±5℃) for 20 min on a rotary mixer; add 20 μL of 20% w / w BSA and mix thoroughly, react at room temperature for 30 min to block the remaining sites on the surface of the colloidal gold particles. Centrifuge at 1,2000g for 10 min (4℃), discard the liquid (containing free antigen), add 200 μL of colloidal gold reconstitution solution (0.01 mol / L PB, containing 1% w / w BSA, 2% w / w sucrose, 0.15 mol / L sodium chloride), mix by sonication, and store at 2-8℃ for later use. The above describes the P30 labeling process for small-volume colloidal gold solutions during method optimization. For large-scale preparation, the reagents can be scaled up proportionally for labeling.

[0074] Optimization of K2CO3 dosage: In the above steps, gold-labeled P30 was prepared using 10, 15, 20, 25, 30, 35, 40, and 50 μL of K2CO3, respectively. Then, the gold-labeled P30 was immobilized on an NC membrane (Sartorius UniSart® CN 95, 25 mm) using a BioDot xyz 3060 membrane scribing apparatus. The results are as follows... Figure 4 The results showed that the test strip exhibited the strongest color development at 15 μL of K₂CO₃. Therefore, 15 μL of K₂CO₃ was used for subsequent experiments.

[0075] 3. Preparation of conjugate pads: First, the glass fiber pads (SB08, Shanghai Jinbiao Biotechnology Co., Ltd.) were thoroughly soaked in the conjugate pad treatment solution (10 mmol / L Tris-HCl, containing 0.5% BSA, 2% sucrose, 0.5% w / w Tween 20, pH 8.0) and dried overnight at 45℃. The protein-labeled colloidal gold was sprayed onto the treated glass fiber conjugate pads at concentrations of 6 μL / cm, 8 μL / cm, and 10 μL / cm using a three-dimensional gold spraying apparatus (HM3030, Shanghai Jinbiao Biotechnology Co., Ltd.). The pads were then dried overnight in a 37℃ forced-air drying oven, cut into 10 mm × 300 mm strips, dried at room temperature, and stored away from light.

[0076] 4. NC membrane coating: The P30 (2.5 mg / mL) obtained in Example 1 was diluted 3 times (scratch ratio 3 times, concentration after dilution 0.83 mg / mL) and 5 times (scratch ratio 5 times, concentration after dilution 0.5 mg / mL) with 0.02 mol / L PB (pH 7.4) as the coating solution for the detection line (T line, 0.8 μL / cm). 0.2 mg / mL His-tagged antibody (GenScript, A00186) was used as the coating solution for the control line (C line, 0.8 μL / cm). The two coating solutions were then fixed onto the NC membrane (Sartorius UniSart® CN 95, 25 mm) using a BioDot xyz 3060 membrane scrubber. The distance between the detection line (T line) and the control line (C line) was 4 mm, and the edge distance of the T line was 7 mm. After completion, the NC membrane was dried at 45°C for 4 h.

[0077] 5. Sample pad preparation: Thoroughly soak glass fiber (RB65, Shanghai Jinbiao Biotechnology Co., Ltd.) in sample pad treatment solution (20 mmol / L PB, containing 150 mmol / L sodium chloride, 1% Triton X-100, 0.5% Tween-20, pH 7.4), dry overnight at 45℃, cut into 12 mm × 300 mm strips, and store at room temperature, dry, and protected from light.

[0078] 6. Preparation steps of blood filtration membrane: Immerse the blood filtration membrane in the treatment solution until saturated (about 3-5 s), then take it out and place it horizontally on a metal mesh frame. Dry it at 45℃ for about 12 h. Cut it into 10 mm × 300 mm pieces using a strip cutter (Shanghai Jinbiao, model CTD300). Store it at room temperature in a drying oven for later use.

[0079] 7. Assembly of the test card: according to... Figure 5As shown, the blood filtration membrane, conjugate pad, NC membrane and absorbent pad are respectively pasted on the PVC base plate, with each component overlapping by 1-2 mm, and then cut into test strips with a width of 4.0 mm. Then, the test strips are inserted into the card holder to assemble into a test card, and stored in a light-proof and dry environment at room temperature (20±5℃).

[0080] 8. Sample diluent The sample diluents included PBS (150 mM NaCl, 16 mM Na2HPO4, 4 mM KH2PO4), 0.2% w / w casein, 1% w / w Tween-20, and 0.03% w / w Proclin 300, pH 7.4. The casein was from Sigma, and the Proclin 300 was from Aladdin.

[0081] 9. Sample processing and testing Serum preparation: After allowing pig whole blood to coagulate at room temperature, centrifuge at 3000 rpm for 5 min at room temperature for later testing. Take the required number of test cards, add 20 μL of serum sample to each well, then add 100 μL of sample diluent to each well, and incubate at room temperature (25±5℃) for 10 min. Figure 6 A); Observe the test results ( Figure 6 B): If the test line does not show color but the control line does, the result is negative for African swine fever virus antibodies, meaning the sample does not contain antibodies against the African swine fever virus P30 protein. If both the test line and the control line are red, the result is positive for African swine fever antibodies, meaning the sample contains antibodies against the African swine fever virus P30 protein. If the control line does not show color, the test result is invalid regardless of whether the test line shows color.

[0082] 10. Parameter Testing 10.1 Testing was performed using African swine fever-negative swine serum. Results were as follows: Figure 7 As shown, at a T-line stretch magnification of 3x, significant nonspecific color development was observed; at a T-line stretch magnification of 5x, the nonspecific color development disappeared. At a T-line stretch magnification of 5x, if the gold-labeled P30 protein spray volume was 10 μL / cm, some negative tests showed nonspecific color development; when the gold-labeled P30 protein spray volume on the conjugate pad was 6 μL / cm and 8 μL / cm, the negative results were clear. Therefore, the maximum spray volume of gold-labeled P30 protein on the conjugate pad should not exceed 8 μL / cm. Subsequent experiments were conducted using gold-labeled P30 protein spray volumes on the conjugate pads of… and a T-line stretch magnification of 3x.

[0083] 10.2 Determination of P30 Labeling Amount: Detection cards were prepared according to the above steps, with labeling amounts of 0.75 μL, 1.25 μL, and 2 μL, and a spray volume of 6 μL / cm for all cases. The results are as follows: Figure 8As shown, the 2 μL group showed the deepest color development, while the negative control showed no nonspecific color development. Therefore, a labeling amount of 2 μL was used for subsequent experiments.

[0084] 10.3 T line: p30 obtained in Example 1, 20mM PBS, 5-fold dilution; C line: His tag antibody, 25mM BB, 6-fold dilution. Other steps were performed to prepare the test card as described above. Different gold-labeled antigen concentrations were tested using swine serum samples that were positive for known African swine fever virus antibodies and swine serum that were negative for African swine fever: 4 μL / cm, 5 μL / cm, and 6 μL / cm. Results are as follows... Figure 9 The display shows that, Figure 9 A represents a negative serum test result. Figure 9 B represents the results of positive serum tests. Negative serum tests were all clean, with the strongest color development observed in positive serum after a spraying volume of 6 μL / cm. Furthermore, compared to drying for less than 2 hours after gold spraying, drying overnight resulted in a cleaner background (NC membrane). Therefore, a gold-labeled antigen volume of 6 μL / cm was used, followed by overnight drying for subsequent experiments.

[0085] Example 3. Sensitivity Experiment A sample of swine serum known to be positive for African swine fever virus antibodies was taken and serially diluted to 1:2048 using African swine fever-negative swine serum. Simultaneously, the test was performed using the detection card prepared in Example 2 of this invention and a commercially available ID-vet African swine fever virus antibody ELISA kit. The detection results of the detection card of this invention are as follows: Figure 10 As shown, the sensitivity is as high as 1:256, and the sensitivity of the ID-vet kit is also 1:256 (the test OD value of 1:512 is slightly lower than the critical value). The detection sensitivity of the two methods is basically the same.

[0086] Example 4. Specificity test Twenty serum samples from African swine fever-negative pigs infected with other pathogens (antibody positive) were selected. These other pathogen antibody-positive samples included: 4 samples of classical swine fever virus (CSFV), 4 samples of porcine reproductive and respiratory syndrome virus (PRRSV), 3 samples of porcine circovirus type 2 (PCV), 3 samples of pseudorabies virus (PRV), 3 samples of foot-and-mouth disease virus type O (FMDV / O), and 3 samples of porcine epidemic diarrhea virus (PEDV). The test cards prepared in Example 2 of this invention were used for detection. The test results are as follows: Figure 11 As shown, all 20 samples tested negative, indicating that the antigen prepared in this invention has high specificity for detecting African swine fever virus antibodies.

[0087] Example 5. Epidemiological survey of African swine fever antibodies Following the sampling and judgment methods described in Example 1, the African swine fever virus antibody rapid detection card prepared in Example 2 was used to detect African swine fever antibodies in 621 serum samples from pig farms belonging to Zhongyu Pig Breeding Company. The results were compared with those from a commercially available kit (African swine fever virus indirect ELISA antibody detection kit, Aidiwei (Qingdao) Biotechnology Co., Ltd., L2615. ELISA antibody detection kit result judgment: S / P% ≤ 30 is negative, S / P% ≥ 40 is positive, and 30 < S / P% < 40 is suspicious). Each sample was tested using both the detection card prepared in Example 2 of this disclosure and the commercially available kit.

[0088] The results of the test card prepared in this disclosure are partially shown as follows: Figure 12 As shown, six samples tested positive for African swine fever virus antibodies. Using the African swine fever virus antibody detection kit from Aidiwei Company (results shown in Table 1), five positive results and one suspected result (which was positive after retesting) were obtained, corresponding one-to-one with the six samples mentioned above. Therefore, the African swine fever virus antibody rapid detection card developed in this disclosure has a 99.84% concordance rate with the test results of the Aidiwei Company's African swine fever virus antibody detection kit; for the single suspected result sample, the detection card's sensitivity is even better.

[0089] The blocking rate described in Table 1 is calculated using the signal value identified by the bar reader according to the following formula: Blocking rate = (1 - T-line signal value of positive sample / T-line signal value of negative control) × 100%; A blocking rate of 0 indicates a negative result. The higher the blocking rate, the higher the content of P30 antibody in the sample. The blocking rate can be used to quantitatively detect P30 antibody in the sample.

[0090] Table 1

[0091] Table 1 (continued)

[0092] Table 1 (continued)

[0093] Table 1 (continued)

[0094] Table 1 (continued)

[0095] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An immunochromatographic test strip for detecting African swine fever virus P30 antibodies, comprising a base plate and a chromatographic membrane disposed on the base plate, wherein an absorbent pad is disposed at one end of the chromatographic membrane and a blood filtration membrane and a conjugate pad are disposed sequentially at the other end; The chromatography membrane is provided with a C-line near the absorbent pad and a T-line near the binding pad; The T-line is coated with African swine fever virus P30 protein or a functional fragment thereof. The C-line is coated with an antibody or antigen-binding fragment against the tag protein; The binding pad is coated with African swine fever virus P30 protein or a functional fragment thereof with markers and tagged proteins.

2. The immunochromatographic test strip according to claim 1, characterized in that, The label includes at least one of colloidal gold, nanoparticles, quantum dot fluorescent microspheres, latex microspheres, protein peptides, nucleic acid sequences or isotopes, more preferably colloidal gold.

3. The immunochromatographic test strip according to claim 1, characterized in that, The colloidal gold particles have a diameter of approximately 10 nm to 50 nm, more preferably approximately 20 nm to 40 nm, and most preferably approximately 30 nm.

4. The immunochromatographic test strip according to claim 1, characterized in that, The tag protein includes one or more of the following: glutathione thiotransferase, staphylococcal protein A, green fluorescent protein, maltose-binding protein, GFP, CFP, YFP, mCherry, SNAP-Tag, Halo Tag, SUMO, His, HA, c-myc, Avi, FLAG, Strep tag, Strep tag II, Twin Strep tag II, or their derivatives or fragments, preferably His tag; Preferably, the tag protein is located at the N-terminus or C-terminus of the African swine fever virus P30 protein or a functional fragment thereof.

5. The immunochromatographic test strip according to claim 1, characterized in that, The African swine fever virus P30 protein with the tagged protein includes the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it.

6. The immunochromatographic test strip according to claim 1, characterized in that, The chromatography membrane includes at least one of nitrocellulose membrane, PVDF membrane, nylon membrane, glass fiber membrane or DEAE cellulose membrane; and / or, the substrate includes a weakly fluorescent substrate or a non-fluorescent substrate.

7. A method for preparing the immunochromatographic test strip according to any one of claims 1 to 6, the method comprising the following steps: (1) Add K2CO3 solution to colloidal gold solution and mix well; dilute African swine fever virus P30 protein with tag protein to 100 μL with deionized water, then add it to colloidal gold solution and mix quickly. After reacting for 20 minutes, add 20 μL of 20% w / w BSA solution and block at room temperature; after centrifugation and discarding the liquid, add a solution composed of BSA, sucrose, sodium chloride and phosphate buffer to the precipitate and mix well to obtain colloidal gold-labeled P30 protein. The ratio of colloidal gold, K2CO3 solution and African swine fever virus P30 protein with tag protein is 1 mL: 1.5 μmol: 50 μg. (2) The above-mentioned colloidal gold-labeled P30 protein is sprayed onto the conjugate pad, preferably and then dried overnight at 37°C, wherein the spraying amount is 6~8 μL / cm, preferably 6 μL / cm; (3) Spray 0.83 mg / mL of P30 protein onto the detection line (T line) of the chromatography membrane, and spray 0.2 mg / mL of His tag antibody onto the control line (C line) of the chromatography membrane. The distance between the detection line (T line) and the control line (C line) is 3~5 mm, and the edge distance of the T line is 6~8 mm. After completion, dry the chromatography membrane. (4) Preparation steps of blood filtration membrane: Soak the blood filtration membrane in the treatment solution until saturated (about 3 to 5 seconds), then take it out and place it horizontally on a metal mesh rack. Dry it at 45°C for about 12 hours. Cut it into 10 mm × 300 mm pieces and store it at room temperature in a drying oven. (5) The blood filtration membrane, conjugate pad, NC membrane and absorbent pad are sequentially pasted onto the PVC base plate, with each component overlapping each other by 1-2 mm to obtain the colloidal gold test strip.

8. The use of the immunochromatographic test strip according to any one of claims 1 to 6 or the preparation method according to claim 7 in the preparation of a product for detecting African swine fever virus antibodies in a sample to be tested.

9. The application according to claim 8, characterized in that, The product can be used to diagnose African swine fever virus infection, evaluate the immunization effect of African swine fever virus vaccine, or distinguish between African swine fever virus infection and African swine fever virus vaccine immunization.

10. The application according to claim 8, characterized in that, The samples to be tested include body fluid samples and / or tissue samples; Preferably, the sample to be tested includes one or more of the following: blood, serum, plasma, dried blood paper, saliva, urine, feces, spleen, lymph nodes, tonsils, and bone marrow; Preferably, the sample to be tested comes from a suidae or a rodent; Preferably, the sample to be tested comes from one or more of the following: domestic pig, wild boar, warthog, bush pig, rabbit, rat, and mouse.

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