Anti-ASFV P30 protein monoclonal antibody and its application

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to an anti-ASFV P30 protein monoclonal antibody and its application. The present invention provides an anti-ASFV P30 protein monoclonal antibody, wherein the anti-ASFV P30 protein monoclonal antibody has a heavy chain sequence as shown in SEQ ID NO: 1 and a light chain sequence as shown in SEQ ID NO: 2. The anti-ASFV P30 protein monoclonal antibody of the present invention not only has high specificity and sensitivity, but also has a simple preparation process and low cost, and provides a new and powerful tool for the rapid diagnosis of ASFV.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to an anti-ASFV P30 protein monoclonal antibody and an application thereof. Background Art

[0002] African swine fever (ASF) is an infectious disease of pigs caused by African swine fever virus (ASFV). ASFV is an enveloped, linear, double-stranded DNA virus, the only member of the order DsDNAvirales, the family Aspreviridae, and the genus Asprevirus, and is the only known insect-borne DNA virus.

[0003] ASFV is highly contagious and can be transmitted through soft ticks or direct contact with infected pigs and their products, and persists in the environment. The ASFV genome is approximately 170-190 kb in length, containing 150-167 open reading frames (ORFs), encoding approximately 150-200 proteins, of which approximately 50 are structural proteins. Among these structural proteins, proteins such as P72, P54, and P30 have attracted much attention due to their important biological functions and immunogenicity.

[0004] P30 protein is a membrane protein of ASFV, encoded by the CP204L gene, expressed in the early stage of virus infection, and can induce immune response in the body, so it has important antigenicity. Studies have shown that antibodies against P30 protein can inhibit virus internalization, indicating that P30 protein is involved in the process of virus entry into cells.

[0005] Although ASFV has caused huge economic losses to the pig farming industry, no effective vaccines and drugs have been developed to prevent or treat ASF. Therefore, the development of rapid, accurate and efficient ASFV detection technology is of great significance for timely detection of epidemics and control of disease spread. Monoclonal antibodies have broad application prospects in the field of virus detection due to their strong specificity, high efficiency, sensitivity, convenience and low cost. In this context, the present invention is committed to the development of monoclonal antibodies against ASFV, in order to achieve breakthroughs in the rapid diagnosis, pathogenic mechanism and immune mechanism of ASFV. Summary of the invention

[0006] Based on the above background, the present invention has successfully developed a monoclonal antibody that can effectively detect ASFV by in-depth research on the P30 protein of ASFV. The antibody not only has high specificity and sensitivity, but also has a simple preparation process and low cost, providing a new and powerful tool for the rapid diagnosis of ASFV. The publication of this research result not only enriches the theoretical basis of ASFV detection technology, but also provides new ideas and methods for the prevention and control of ASFV and vaccine development.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides an anti-ASFV P30 protein monoclonal antibody, wherein the anti-ASFV P30 protein monoclonal antibody has a heavy chain sequence as shown in SEQ ID NO: 1 and a light chain sequence as shown in SEQ ID NO: 2.

[0009] Preferably,

[0010] The heavy chain sequence has a heavy chain constant region as shown in SEQ ID NO: 3;

[0011] The light chain sequence has a light chain constant region as shown in SEQ ID NO:4;

[0012] A second aspect provides a polynucleotide, wherein:

[0013] a) encoding the anti-ASFV P30 protein monoclonal antibody of the present invention;

[0014] b) having a first nucleotide sequence as shown in SEQ ID NO: 5; and / or

[0015] c) having a second nucleotide sequence as shown in SEQ ID NO: 6; and / or

[0016] d) is complementary to a) or b) or c).

[0017] Preferably,

[0018] The first nucleotide sequence has a first constant region as shown in SEQ ID NO:7;

[0019] The second nucleotide sequence has a second constant region as shown in SEQ ID NO:8.

[0020] It should be noted that the terms "first" and "second" are only used for descriptive purposes in the context of nucleotide sequences or constant regions to distinguish the specified substances; they do not limit the order or priority in any way, nor do they limit the specific types of substances.

[0021] In a third aspect, a hybridoma cell line 1E6 is provided, wherein the hybridoma cell line 1E6 secretes the anti-ASFV P30 protein monoclonal antibody described in the present invention.

[0022] In a fourth aspect, a kit is provided, comprising the anti-ASFV P30 protein monoclonal antibody described in the present invention or the hybridoma cell line 1E6 described in the present invention.

[0023] In a fifth aspect, a colloidal gold test strip is provided, wherein the colloidal gold test strip comprises a base plate, a coating film, a sample pad, a gold label pad and a water absorbent pad, wherein:

[0024] A detection line and a quality control line are sequentially arranged on the coating membrane along the flow direction of the sample;

[0025] The detection line is coated with Protein A protein;

[0026] The quality control line is coated with the anti-ASFV P30 protein monoclonal antibody described in the present invention.

[0027] In a sixth aspect, a packaging box is provided, comprising the colloidal gold test strip and a sample diluent as described in the present invention.

[0028] Preferably, the sample diluent is prepared as follows: weigh 0.2543 g of disodium hydrogen phosphate dodecahydrate, 0.0452 g of sodium dihydrogen phosphate dihydrate and 0.85 g of sodium chloride, measure 80 mL of pure water and pour them into a beaker, stir evenly, measure the pH value using a pH meter, add 0.1 mol / L hydrochloric acid titration solution to adjust the pH value to 7.4, make up to 100 mL with pure water, and filter and sterilize with a 0.22 μm filter.

[0029] The packaging box of the present invention may include a container, a chip, instructions for use, a buffer, an immunoadjuvant and / or other materials, structures and / or reagents required for diagnosis / detection as required.

[0030] Other materials required for diagnosis / detection in the packaging box of the present invention include, but are not limited to, other anti-ASFV P30 protein antibodies other than the antibodies of the present invention, antigens bound to ASFV P30 protein and / or ASFV P30 protein. The above-mentioned other materials may be present in a conventional manner in the art, for example, in a dissolved or dried form in a container, coated on a solid phase carrier (e.g., a membrane, a plate, a bead, a particle (such as a magnetic particle), etc.), in a dissolved or dried form in a chamber of a chip, but the present invention is not limited thereto.

[0031] Other structures required for diagnosis / detection in the packaging box of the present invention include, but are not limited to, structures for sampling, structures for control, and / or structures for observing the detection process or results.

[0032] Other reagents required for diagnosis / detection in the package of the present invention include, but are not limited to, detergents, color developing agents and / or terminators.

[0033] In a seventh aspect, provided is the use of the anti-ASFV P30 protein monoclonal antibody of the present invention in the preparation of a kit for the diagnosis of African swine fever virus.

[0034] In an eighth aspect, there is provided the use of the anti-ASFV P30 protein monoclonal antibody described in the present invention in the preparation of a colloidal gold test strip for the diagnosis of African swine fever virus.

[0035] In the present invention, the term "antibody" refers to an immunoglobulin molecule and also relates to recombinantly or synthetically produced / synthesized antibodies. The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific for a single antigenic site. Monoclonal antibodies are advantageous because they can be obtained by hybridoma cell line culture and are substantially free from contamination by other immunoglobulins.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] First, the antibody titer is very high and has a good immune effect; second, the antibody of the present invention has no cross reaction with CSFV, PRRSV, PCV2, and PRV, and has a good specific binding ability; third, clinical experiments show that the monoclonal antibody of the present invention can be used for colloidal gold test strips for the diagnosis of African swine fever virus, with a detection sensitivity of up to 96.7% and a detection specificity of up to 100%.

[0038] That is to say, the monoclonal antibody of the present invention has shown excellent performance in many aspects such as antibody titer, cross-reactivity and detection effect. Therefore, the present invention provides an anti-ASFV P30 protein monoclonal antibody that has been systematically evaluated and can be used for in vitro diagnosis and has outstanding comprehensive capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solution of the present invention, the drawings involved in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the description are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 This is the SDS-PAGE electrophoresis diagram of the anti-ASFV P30 protein monoclonal antibody of the embodiment of the present invention; wherein, lane M represents maker, lane 1 represents the monoclonal antibody in the reduced state, and lane 2 represents the monoclonal antibody in the non-reduced state.

[0041] Figure 2 The figure is a schematic diagram of assembling the colloidal gold test strip according to the embodiment of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0043] Example 1 Bacterial strain inspection

[0044] The strain used in the present invention is recombinant Escherichia coli E. coli -ASFV-P30 strain, purchased from Nanjing Mingyan Biotechnology Co., Ltd. This bacterium is a Gram-negative bacillus.

[0045] 1) Culture characteristics: Pick a small amount of bacterial liquid with a loop, streak inoculate on LB solid medium, and culture at 37℃ for 12 hours. A round colony with a size of 1~2mm, neat edges, smooth surface, and translucent will be formed.

[0046] Among them, the preparation method of LB solid culture medium is as follows: weigh 5g yeast extract powder, 10g peptone, 10g sodium chloride, and 15g agar powder and dissolve them in 950mL purified water, adjust the pH to 7.0 with 5moL / L NaOH, make the volume to 1L, sterilize at 121.0℃ for 20 minutes, wait for the culture medium to cool to about 55℃, add kanamycin with a final concentration of 50μg / mL, shake gently, and immediately pour the culture medium into the culture dish (about 25mL / plate), be careful not to generate bubbles when pouring, and store it at 2~8℃ for later use after cooling and solidification.

[0047] 2) Specificity:

[0048] ① The strain was tested by PCR. A 580bp target band was amplified.

[0049] The total reaction system for bacterial PCR detection is 13 μL: 2×Taq PCR Mix 6.5 μL, upstream and downstream primers 0.5 μL each, template 1 μL, ddH2O 4.5 μL.

[0050] PCR reaction parameters for bacterial species: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 10 min, 30 cycles; extension at 72°C for 7 min.

[0051] Primer sequences:

[0052] F1: 5'-ACATTAAATGTAGGTGAGATAAAAGC-3' (SEQ ID NO: 9);

[0053] R1: 5'-ATATTAGAGGGCATCTTCTTATTA-3' (SEQ ID NO: 10).

[0054] Positive and negative controls should be set up for each PCR amplification. The positive control uses pET-21b-ASFV-P30 containing the African swine fever virus P30 target gene as a template, and the negative control uses the empty vector plasmid pET-21b as a template.

[0055] Test establishment conditions: If the positive control amplifies a specific band of 580bp and the negative control does not amplify a specific band of 580bp, the test result is valid, otherwise the test should be repeated.

[0056] Result determination: If the sample under test amplifies a specific band of 558 bp, it is judged as positive; if the sample under test does not amplify a specific band of 558 bp, it is judged as negative.

[0057] ② The concentration of the target protein (P30 protein) was determined by the BCA method, and the purity of the target protein was determined by the density analysis method. The concentration of the target protein per milliliter was ≥ 0.8 mg, and the purity of the target protein was ≥ 94%.

[0058] ③ Perform Western-blot test and the target band of about 35kDa can be seen.

[0059] The Western-blot test process is as follows:

[0060] Sample preparation: Take 60 μL of the sample to be tested and mix it evenly with 20 μL of 4× loading buffer, boil it in a water bath for 10 minutes, and place it on ice for later use.

[0061] Gel preparation and electrophoresis: prepare 12% separation gel as the lower gel, prepare 5% concentration gel as the upper gel after the lower gel solidifies, prepare protein gel with a thickness of 1.5 mm, load protein marker, recombinant protein (P30 protein) sample and control sample into the gel wells in sequence, 35 μL per well, perform SDS-PAGE gel electrophoresis at 80V for 40min for the upper gel and 120V for 90min for the separation gel.

[0062] Transfer: After electrophoresis, transfer the proteins on the separation gel to the PVDF membrane (PVDF membrane needs to be activated by immersing in pure methanol solution for 30 seconds and equilibrated in PBS for 3 minutes before use), and transfer the membrane at 350mA for 30 minutes.

[0063] Blocking: Place the protein-loaded PVDF membrane in a plastic washing box, add 25 ml of 5% skimmed milk powder, and block at room temperature for 1 hour.

[0064] Primary antibody incubation: Pour off the skim milk, wash three times with PBST, add 30 mL of primary antibody (use the serum sample to be tested or monoclonal antibody as the primary antibody) (1% BSA dilution, ratio 1:500), and react with slow shaking at room temperature for 1 hour.

[0065] Secondary antibody incubation: Pour away the primary antibody, wash three times with PBST, add 30 mL of HRP-labeled rabbit anti-pig IgG (secondary antibody) (1% BSA dilution, ratio 1:2000), and react at room temperature with slow shaking for 1 hour.

[0066] Color development: Pour away the secondary antibody, wash the membrane 3 times with PBST, and then wash the membrane 2 times with PBS. Prepare the color development solution (color development solution A: color development solution B = 1:1), place the membrane on the color development plate, absorb the PBS on the membrane with filter paper, and then add 100μL of color development solution, and expose with the ECL imaging system.

[0067] Judgment: After color development, a specific band should appear at about 35 kDa.

[0068] Among them, color solution A is: weigh 5.76g of citric acid, 0.5g of urea peroxide, and 6.21g of sodium acetate, add 800mL of purified water and stir to dissolve, then weigh 0.5mL of Proclin-300 and add, add pure water to make up to 1000mL, aseptically quantitatively package, 12mL / bottle, and store at 2~8℃. Color solution B is: weigh 5.76g of citric acid and 0.2g of TMB, add 100mL of methanol to dissolve, then add 0.5mL of Proclin-300, finally add pure water to make up to 1000mL, aseptically quantitatively package, 12mL / bottle, and store at 2~8℃.

[0069] 3) Stability of expressing African swine fever virus P30 protein:

[0070] Will E. coli -ASFV-P30 was propagated on LB solid culture medium for 10 generations, and the F1, F3, F5, F7, and F10 generations of bacteria were induced and cultured in a constant temperature incubator at 37°C for 6-8 hours. The induced expressed P30 protein was purified and the amount of P30 protein expressed per liter of bacterial liquid was determined by the BCA method to be ≥6 mg, and the purity of P30 protein was determined by density analysis to be ≥95%.

[0071] The purification process of P30 protein is as follows:

[0072] Take the supernatant and perform nickel column purification. The steps are as follows:

[0073] (1) Take Ni-Agarose Resin filler and mix well, then add it to the chromatography column. Let it stand at room temperature for 5 minutes. After the gel and solution are separated, open the liquid outlet at the bottom to allow the ethanol to slowly flow out by gravity.

[0074] (2) Add deionized water (the amount used is 5 times the amount of filler) to the packed column to rinse out the ethanol, and then equilibrate the column with Binding Buffer (the amount used is 5 times the amount of filler). After equilibration, the sample can be loaded.

[0075] (3) Use Binding Buffer (20mmol / L Tris-HCl pH7.9, 10mmol / L Imidazole, 0.5mol / L NaCl) (the amount used is 15 times the amount of filler) and discard the waste liquid.

[0076] (4) Rinse the column with Soluble Binding Buffer (15 times the amount of filler) to remove impurities. Invert the column for 5 minutes each time, open the bottom outlet, and discard the waste liquid.

[0077] (5) Add Soluble Elution Buffer (20mmol / L Tris-HCl pH7.9, 500mmol / L Imidazole, 0.5mol / L NaCl) (the amount used is 2.5 times the amount of filler) to elute the target protein, invert the tube upside down for 15 minutes, open the bottom outlet, and collect the eluate.

[0078] (6) After elution, wash the column with deionized water (the amount used is 5 times the amount of filler used), then balance it with 20% ethanol (the amount used is 5 times the amount of filler used), seal the column and store it at 2~8℃.

[0079] 4) Reactivity and specificity of P30 protein:

[0080] The extracted ASFV-P30 was coated on ELISA plates, and the indirect ELISA antibody detection method of African swine fever virus P30 protein was used to determine the P30 protein and the positive serum of classical swine fever virus (CSFV) antibody, porcine reproductive and respiratory syndrome virus (PRRSV) antibody, porcine circovirus type 2 (PCV2) antibody, pseudorabies virus (PRV) antibody and empty vector pET-21b Escherichia coli. E. coli Specific binding of BL21 (DE3) antibody-positive serum and African swine fever virus (ASFV) antibody-positive serum. The results showed that the S / N value of P30 protein with African swine fever virus (ASFV) antibody-positive serum was ≥2.1, and the S / N value with other antibody-positive serum was <2.1.

[0081] Among them, the indirect ELISA antibody detection method for African swine fever virus P30 protein is as follows:

[0082] The recombinantly expressed P30 protein is coated, the serum sample to be tested or the monoclonal antibody is used as the primary antibody, and the horseradish peroxidase-labeled pig secondary antibody or mouse secondary antibody is used as the enzyme-labeled secondary antibody.

[0083] Sample addition: Add 100 μL of African swine fever negative serum as a negative control, add two wells for each test; add 100 μL of African swine fever positive serum as a positive control, add two wells for each test; add 100 μL of diluted sample to the corresponding well.

[0084] Incubation: Cover the plate with sealing film and incubate at 37°C for 60 minutes (± 1 minute).

[0085] Wash the plate: discard the liquid in each well into the waste liquid cylinder, wash the plate wells with 280µL (±20µL) washing solution, and wash 5 times in total. Discard the liquid in the well after each wash. After the last wash solution is discarded, pat the remaining washing solution in the well dry on absorbent paper.

[0086] Incubation: Add 100 µL of enzyme binding solution to each well. Cover with sealing film and incubate at 37°C for 30 minutes (± 1 minute).

[0087] Wash plate: Repeat the above steps.

[0088] Color development: Add 50µL of color development solution A and 50µL of color development solution B to each well (you can also mix color development solution A and B in equal proportions and then add 100µL).

[0089] Incubation: Cover the plate with sealing film and incubate at 37°C for 15 minutes (±1 minute).

[0090] Stop: Add 50 µL of stop solution to each well to terminate the reaction.

[0091] Assay: Measure and record the OD value (450 nm) of the sample and control.

[0092] determination:

[0093] The mean value of negative control (NC ): NC =(NC1+NC2) / 2

[0094] The average value of positive control (PC ): PC =(PC1+PC2) / 2

[0095] Test establishment criteria: PC ≥0.80; NC <0.30.

[0096] If the test is invalid, the test should be repeated according to the operating instructions.

[0097] Result determination: S / N=sample / NC

[0098] If the S / N value is <2.1, the sample should be judged as antibody negative.

[0099] If the S / N value is ≥2.1, the sample should be judged as antibody positive.

[0100] Among them, the reagents used in the indirect ELISA antibody detection method for African swine fever virus P30 protein are specifically described as follows:

[0101] Washing solution (PBST): Measure 1 mL of Tween-20, mix thoroughly with 10 mM PBS buffer, and make up to 1000 mL. Store at room temperature for later use.

[0102] Color developing solution A: Weigh 5.76 g of citric acid, 0.5 g of urea peroxide, and 6.21 g of sodium acetate, add 800 mL of purified water and stir to dissolve, then add 0.5 mL of Proclin-300, add purified water to make up to 1000 mL, aseptically divide into 12 mL / bottle, and store at 2-8°C.

[0103] Color developing solution B: Weigh 5.76 g of citric acid and 0.2 g of TMB, add 100 mL of methanol to dissolve, then add 0.5 mL of Proclin-3000, and finally add purified water to make up to 1000 mL. Aseptically divide the solution into 12 mL / bottle and store at 2-8°C.

[0104] Stop solution: Measure 876 mL of purified water, slowly add 124 mL of concentrated sulfuric acid (2 mol / L), stir evenly, aseptically divide into quantitative portions, 12 mL / bottle, and store at 2-8°C.

[0105] 5) Purity test: Take F1, F3, F5, F7, and F10 bacterial solutions and conduct purity test according to the three appendices of the current "Pharmacopoeia of Veterinary Medicine of the People's Republic of China". They should be pure.

[0106] 6) Bacterial strain generation: Recombinant Escherichia coli E. coli -The original seed generations of ASFV-P30 strain are 1st to 2nd generations, the basic seed generations are 3rd to 5th generations, and the production seed generations are 6th to 10th generations.

[0107] 7) Storage conditions of bacteria: Recombinant Escherichia coli E. coli -Seeds of each generation of ASFV-P30 strain were mixed with sterile glycerol at a ratio of 1:1 (V / V) and stored below -70°C.

[0108] Example 2 Hybridoma cell line testing

[0109] The cells secreting anti-ASFV P30 protein monoclonal antibodies are hybridoma cell line 1E6, which were prepared, identified, stored and supplied by Northwest Agriculture and Forestry University.

[0110] 1) Cell morphology and culture characteristics: Hybridoma cell line 1E6 is spherical with smooth edges under an optical microscope and has a good refractive index. It can be cultured in RPMI 1640 culture medium containing 10% FBS in an incubator at 37°C and 5% CO2, proliferating once every 18-24 hours and growing into a good monolayer.

[0111] 2) The ability of hybridoma cell line 1E6 to secrete anti-ASFV P30 protein monoclonal antibodies: The hybridoma cell line 1E61 / 4 was subcultured, and the cell culture supernatant was collected every 2 generations for determination of the anti-ASFV P30 protein monoclonal antibody titer using the indirect ELISA method. The titer of the culture supernatant should not be less than 1:1024.

[0112] 3) Reactivity and specificity of secreted monoclonal antibodies against ASFV P30 protein: The cell culture supernatant was serially diluted with PBST and added to ELISA plates coated with 100ng / 100μL / well P30 protein. The titer of monoclonal antibodies against ASFV P30 protein was determined by indirect ELISA method. The maximum dilution of monoclonal antibodies when the S / N ratio was ≥2.1 was used as the antibody titer, which should be no less than 1:1024. The S / N value should be <2.1 when the bacterial protein cultured in BL21(DE3) with 100ng / 100μL / well pET-21b empty vector was coated on the ELISA plate.

[0113] 4) The ability of cells to produce anti-ASFV P30 protein monoclonal antibodies after intraperitoneal inoculation of mice: 1×10 6 Ascites was induced by hybridoma cell line 1E6, and the collected ascites was purified by protein G affinity chromatography, and no less than 1 mg of purified antibody was obtained per milliliter of ascites. The purified antibody was subjected to SDS-PAGE electrophoresis, and two clear bands were observed by naked eye, one heavy chain 50kDa (having the heavy chain sequence shown in SEQ ID NO:1, and the heavy chain sequence had the heavy chain constant region shown in SEQ ID NO:3) and one light chain 25kDa (having the light chain sequence shown in SEQ ID NO:2, and the light chain sequence had the light chain constant region shown in SEQ ID NO:4). After the amino acid sequence of the anti-ASFV P30 protein monoclonal antibody was converted into a nucleotide sequence, a first nucleotide sequence as shown in SEQ ID NO:5 (the first nucleotide sequence had the first constant region shown in SEQ ID NO:7) was obtained, and the first nucleotide sequence shown in SEQ ID NO:5 was the heavy chain base sequence; and a second nucleotide sequence as shown in SEQ ID NO:6 (the second nucleotide sequence had the second constant region shown in SEQ ID NO:8), and the second nucleotide sequence shown in SEQ ID NO:6 was the light chain base sequence.

[0114] 5) Reactivity and specificity of anti-ASFV P30 protein monoclonal antibodies produced after cell inoculation into the peritoneal cavity of mice: The collected ascites and purified anti-ASFV P30 protein monoclonal antibodies were serially diluted with PBST and added to 100 ng / 100 μL / well P30 protein-coated ELISA plates. The antibody titer was determined by indirect ELISA method. The maximum dilution of anti-ASFV P30 protein monoclonal antibodies when the S / N ratio was ≥2.1 was used as the antibody titer, which should be no less than 1:10. 6 ELISA plates were coated with bacterial proteins cultured in BL21 (DE3) with 100 ng / 100 μL / well of pET-21b empty vector, and the S / N value was <2.1.

[0115] 6) Cell passage stability: After the hybridoma cell line 1E6 was revived, it was cultured in RPMI 1640 containing 10% FBS in an incubator at 37°C and 5% CO2 for 5 generations. The antibody titer of the cell culture supernatant was determined by indirect ELISA. The antibody titer of each generation should not be less than 1:1024.

[0116] 7) Cell storage conditions: The hybridoma cell line 1E6 was placed in cryopreservation protection solution (10% dimethyl sulfoxide, 90% fetal bovine serum) and stored in liquid nitrogen.

[0117] 8) Cell generations: 1~15.

[0118] Example 3 Preparation and testing of the main components of colloidal gold test strips

[0119] 1. Preparation and testing of African swine fever virus P30 protein:

[0120] 1.1 Preparation of P30 protein

[0121] 1.1.1 Preparation of primary seeds: Use a loop to pick a small amount of bacterial liquid for production, streak inoculate on LB solid culture medium plate, incubate at 37℃ for 12~16 hours, pick a single colony and inoculate it into LB liquid culture medium, incubate at 37℃ and 200r / min for 12~16 hours.

[0122] 1.1.2 Preparation of secondary seeds: Inoculate 5% of the primary seeds into LB liquid culture medium and culture at 37°C and 200 r / min for 12 to 16 hours.

[0123] 1.1.3 Fermentation and expression: Inoculate 5% of the secondary seeds into LB liquid medium. Ferment at 37℃ with aeration for 5-7 hours and dissolved oxygen at 30%-40% until the OD of the bacterial solution reaches 600nmWhen the value was 1.1-1.3, lactose was added to a final concentration of 0.02 mol / L, the feed rate was 70 mL / h, the induction culture was carried out at 25 °C for 11 hours, and the fermentation was stopped.

[0124] 1.1.4 Bacterial disruption: The fermentation product was centrifuged at 12000r / min at room temperature for 10min in a tubular centrifuge, and the bacteria were collected and washed twice with PBS (0.015moL / L, pH7.2). The collected bacteria were resuspended at a ratio of 1g wet bacteria to 9mL PBS (0.015moL / L, pH7.2), and the bacteria were disrupted by a high-pressure homogenizer at 2~8℃ until more than 98% of the bacteria were disrupted or the bacteria passed through the homogenizer twice in succession. The disrupted bacterial solution was smeared and stained with 0.1% crystal violet solution for 0.5min. 3~5 fields of view were observed under a microscope. When the disruption was complete, the field of view was full of cell fragments and no complete bacteria could be seen. The bacterial solution was disrupted until the bacterial disruption rate no longer changed. The tubular centrifuge was centrifuged at 12000r / min at room temperature for 10min, and the supernatant was collected. Then, a high-speed centrifuge was used to centrifuge for a second time at 17000r / min at 4℃ for 10min, and the supernatant was collected.

[0125] 1.1.5 Protein purification: Purify the protein according to the above P30 protein purification process until the purity of the target protein is not less than 95% as determined by grayscale scanning after SDS-PAGE electrophoresis.

[0126] Among them, LB solid culture medium is: weigh 5g yeast extract powder, 10g peptone, 10g sodium chloride, and 15g agar powder, dissolve in 950mL purified water, adjust the pH to 7.0 with 5moL / L NaOH, make up to 1L, sterilize at 121.0℃ for 20 minutes, wait for the culture medium to cool to about 55℃, add kanamycin with a final concentration of 50μg / mL, shake gently, and immediately pour the culture medium into the culture dish (about 25mL / plate), be careful not to generate bubbles when pouring, and store at 2~8℃ for later use after cooling and solidification. LB liquid culture medium is as follows: weigh 5 g yeast extract powder, 10 g peptone, and 10 g sodium chloride, dissolve in 950 mL sterile nuclease-free water, adjust the pH to 7.0 with 5 mol / L NaOH, make up to 1 L, sterilize at 121.0°C for 20 min, wait for the culture medium to cool to about 55°C, add kanamycin at a final concentration of 50 μg / mL, shake gently, and store at 2-8°C for later use.

[0127] 1.2 P30 protein test

[0128] 1.2.1 Properties: It should be a colorless, clear liquid without precipitation.

[0129] 1.2.2 Determination of target protein concentration and purity: The target protein concentration was determined by BCA method, and the target protein purity was determined by density analysis. Protein concentration per ml ≥ 0.8 mg, protein purity ≥ 94%.

[0130] 1.2.3 Reactivity and specificity of P30 protein: The extracted ASFV-P30 protein was coated on ELISA plates, and the indirect ELISA antibody detection method of African swine fever virus P30 protein was used to determine the recombinant protein and serum positive for antibodies against classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV) and empty vector pET-21b Escherichia coli. E. coli Specific binding of BL21 (DE3) antibody positive serum and African swine fever virus (ASFV) antibody positive serum. The S / N value with African swine fever virus (ASFV) antibody positive serum is ≥2.1, and the S / N value with other antibody positive serum is <2.1.

[0131] 1.3 Storage and shelf life: Store below -20℃ for no more than 6 months.

[0132] 2. Preparation and testing of gold-labeled African swine fever virus P30 protein:

[0133] 2.1 Preparation of gold-labeled African swine fever virus P30 protein:

[0134] 2.1.1 Preparation of gold-labeled African swine fever virus P30 protein: Taking the production of 2500 African swine fever virus antibody colloidal gold test strips as an example, take 90mL of colloidal gold solution, add 720μL 0.1moL / L K2CO3 (1.38g K2CO3, 100mL pure water) solution to adjust the pH value of the colloidal gold solution to 7.4, take 2700μL of purified P30 protein (concentration 0.2mg / mL) and add it to the colloidal gold solution with adjusted pH value, and react at room temperature for 1h; add 18mL of blocking solution FB002 (2.5g BSA, 50mL pure water) to the system, block at room temperature for 30 minutes; 11000rpm, 15 minutes, use a pipette to discard the supernatant, and use 1500μL of gold-labeled diluent JX002 (100mL 0.01moL / L Tris pH8.5, 5g sucrose, 1g trehalose, 1.5g BSA) was reconstituted (i.e., concentrated 60 times, 60×).

[0135] Among them, the preparation of 0.01% colloidal gold solution is as follows: measure 99.5mL of purified water into a beaker, take 0.5mL of 2% chloroauric acid into the beaker and mix well, adjust the power of the electric furnace to 500W, heat to boiling, quickly add 0.9mL of 2% trisodium citrate (the amount can be adjusted according to the final result), observe the color change of the solution (colorless-black-burgundy), continue heating for 5 minutes after the color stabilizes, then stop heating, cool the solution to room temperature, and then make up to 100mL.

[0136] 2.2 Testing of Gold Label African Swine Fever Virus P30 Protein:

[0137] 2.2.1 Properties: Observe the appearance and color of the colloidal gold solution, which should be a dark red or purple-red, transparent solution without particles.

[0138] 2.2.2 Test of the labeling effect of the gold-labeled African swine fever virus P30 protein: Use a UV spectrophotometer to scan the gold-labeled African swine fever virus P30 protein at full wavelength in the visible light range (400~600nm). The absorption wavelength should be between 528.20~529.20nm, and the OD value should be between 0.840~0.866.

[0139] 2.2.3 Storage and shelf life: Store at 2~8℃ for no more than 1 month.

[0140] 3. Preparation and testing of monoclonal antibodies against ASFV P30 protein:

[0141] 3.1 Preparation of anti-ASFV P30 protein monoclonal antibodies:

[0142] ①Resuscitation of hybridoma cells

[0143] Take out the cryovial of hybridoma cells from liquid nitrogen and immediately put it into a 38℃~42℃ water bath. After the cell suspension is quickly thawed, centrifuge at 300g for 10 minutes, discard the cryopreservation solution after centrifugation, wash the cells twice with RPMI-1640 culture medium, and then make a cell suspension with complete RPMI-1640 culture medium and drop it into the wells of a 24-well culture plate or a 25ml culture bottle. Culture in a 37℃ 5% CO2 incubator. When the revived cells grow well, subculture for 2~3 days.

[0144] ② Preparation of monoclonal antibodies:

[0145] (1) 0.5 mL of paraffin oil was injected intraperitoneally into mice, and cells were implanted within 1 to 9 weeks after injection.

[0146] (2) The hybridoma cells in the logarithmic growth phase were collected, washed once with 5% FCS-1640 solution, and centrifuged at 1000 rpm for 10 min.

[0147] (3) Take a sample, stain it with trypan blue, count the number of live cells, and reconstitute it with 5% FCS-1640 solution to 1.0×10 7 cells / mL suspension.

[0148] (4) Hybridoma cells were inoculated into mice injected with paraffin oil. Each mouse was intraperitoneally injected with 1 mL (containing 1.0 × 10 7 cells / mL).

[0149] (5) The tumor volume is at its maximum about 10 days after inoculation. At this time, ascites can be extracted from the abdominal cavity, once every 1 to 3 days, for a total of 10 times. Serum can be separated after blood is collected from the axillary artery or the heart.

[0150] ③Purification of monoclonal antibodies

[0151] i) Disposal of dialysis bags

[0152] (1) Select a dialysis bag with appropriate pore size according to your needs;

[0153] (2) Cut the dialysis bag into small pieces of the required length (about 10 cm);

[0154] (3) Place the dialysis bag in 400 mL of 1 M EDTA solution (pH 8.0) containing 2% (m / v) NaHCO3 and boil for 10 min;

[0155] (4) Rinse the dialysis bag thoroughly with distilled water;

[0156] (5) Place the dialysis bag in 1 M EDTA solution (pH 8.0) and boil for 10 min;

[0157] (6) After the dialysis bag has cooled, store it in distilled water at 4°C, making sure that the dialysis bag is always submerged in the liquid.

[0158] ii) IgG crude extraction (saturated ammonium sulfate treatment)

[0159] (1) Pipette 1 mL of ascites into a 25 mL beaker, add an equal amount of PBS to dilute, place the beaker on ice or at 4°C, slowly add 2 mL of saturated ammonium sulfate solution dropwise, stirring while adding until the ammonium sulfate concentration reaches 50%, and stir at 4°C for 2 h;

[0160] (2) Centrifuge at 10,000 rpm for 10 min, discard the supernatant, resuspend the precipitate in 1 mL of PBS, place it in a dialysis bag and seal both ends of the dialysis bag;

[0161] (3) Dialyze the IgG sample with PBS at 4°C overnight (change PBS 1-2 times in between) to remove residual ammonium sulfate in the sample;

[0162] (4) If the ascites fluid after dialysis cannot be purified in time, it should be stored at -20℃.

[0163] iii) Further purification of crude IgG

[0164] (1) The dialyzed crude IgG sample was centrifuged at 10,000 r / min for 5 min, and the supernatant was collected to obtain the sample to be further purified.

[0165] (2) Mix the Protein G Resin and add an appropriate amount to the purification column as needed, then wash the column with PBS until the OD value of the effluent reaches 0. 280 Close to 0 (<0.001).

[0166] (3) Add the sample from (1) to the column at a slow flow rate (1 mL / min). After the effluent is recovered, repeat the loading five times.

[0167] (4) Elute the impurities with PBS at a slow flow rate (2 mL / min) until the OD of the effluent reaches 280 <0.001.

[0168] (5) Elute the target antibody with a glycine solution 3-4 times the original volume of ascites, and collect the sample in a sterile centrifuge tube until the OD value of the effluent is 280 <0.2-0.3, stop collecting samples and immediately neutralize the collected samples with Tris-HCl to pH 7-8.

[0169] (6) Dialyze the sample in (5) with PBS at 4°C overnight (change PBS 1-2 times in between). Finally, collect the purified target antibody, clearly mark the name, concentration, date, etc., and store at -20°C.

[0170] (7) Column post-treatment: Continue to add glycine solution to the column until the OD of the effluent is 280 <0.001, after the glycine solution has run out, wash the column with PBS until the OD of the effluent is 280 =0.

[0171] (8) Column storage: After PBS has drained off, if the column will be used again within one week, add tetra-distilled water and store at 4°C. If the column will not be used for a long time, add 20% ethanol [containing sodium azide (antibacterial)] and store it, or store it according to the specific method in the product manual.

[0172] ④Monoclonal antibody testing

[0173] (1) The purity of the purified antibody was detected by SDS-PAGE.

[0174] (2) The antibody titer was detected by ELISA method.

[0175] (3) IFA and Western blot methods were used to detect whether the purified antibodies obtained had normal biological functions.

[0176] 3.1.1 Preparation of ascites

[0177] (1) Select five 6-8 week old female BALB / c mice. 7 days in advance, inject 200 µL of autoclaved paraffin oil into the peritoneum of each mouse.

[0178] (2) The hybridoma cell line 1E6 was cloned and expanded. When the number of cells was sufficient for injection into five mice (10 6 When the number of cells / mouse is 4, discard the waste liquid, use a 1mL pipette to draw 4mL of RPMI-1640 culture medium and add it to the cell bottle, then gently blow up the hybridoma cell line 1E6. After counting, collect the hybridoma cell line 1E6 into a 50mL centrifuge tube and centrifuge at 100×g for 10min. Resuspend the cells with 1mL of incomplete RPMI-1640 culture medium, draw the cell suspension with a 1mL syringe, and immunize mice intraperitoneally, 200µL per mouse;

[0179] (3) Observe the abdominal swelling of the mouse every day from 5 days after immunization. Usually, the abdomen of the mouse will swell after 7-10 days, and then the ascites can be collected. The tool for collecting ascites is a large syringe needle, which is inserted into the mouse abdomen with the needle facing downward for drainage. After the ascites is collected, centrifuge it at 8000×g for 10 minutes at 4℃. The supernatant is the slightly yellow clear ascites, which is stored at -80℃.

[0180] 3.1.2 Purification of ascites

[0181] (1) Prepare an ice box in advance and add an appropriate amount of water to make it an ice-water mixture. Place a 50 mL beaker in the ice box for an ice bath. Place the ice box on a magnetic stirrer. Use a pipette to draw an appropriate amount of ascites into the 50 mL beaker. Then add an equal amount of 1× PBS and mix well with the ascites.

[0182] (2) Slowly add ascites and 1×PBS to the saturated ammonium sulfate precipitate drop by drop and stir on ice for about 6 hours;

[0183] (3) Collect the liquid from the previous step into a 1.5 mL centrifuge tube, and then centrifuge at 4°C, 12,000 × g, 15 min. Discard the supernatant. The precipitate is the crude antibody, and then add 1 × PBS to resuspend and dissolve. Add the resuspended liquid to a 14 kDa dialysis bag and place it in 500 mL 1 × PBS. Place it overnight once, or every 2 hours, and change it at least twice. The whole process should be dialysis at 4°C or in an ice bath.

[0184] (4) Filter the crude antibody after dialyzing to remove saturated ammonium sulfate through a 0.22 μm filter, take an appropriate amount of Protein G and load it onto the column, and then balance the filler: balance with 5 times the column volume of 1× PBS, then cycle 5-8 times for sample loading, adjust the throttle to control the flow rate to about 1 mL / min; retain the effluent sample.

[0185] (5) Use PBS to elute the impurities through the column, controlling the flow rate to about 2 mL / min; retain the washing solution.

[0186] (6) Elute the specific antibody with glycine (pH 2.4). Neutralize with Tris-HCl every time 1 mL of elution is added, and adjust the pH value to between 7.2 and 7.4. Control the flow rate to about 1 mL / min; pay attention to find out the neutralization ratio of glycine and Tris-HCl in advance.

[0187] (7) Add the anti-ASFV P30 protein monoclonal antibody purified by Protein G affinity chromatography to a 10kD dialysis bag and place it in 500mL 1×PBS solution for dialyzing at 4°C. Change the dialysate every 2 hours for 2-3 times. After dialysis, concentrate it at 4°C and 1800×g to a concentration of 1-1.5mg / mL, divide it into 1mL / tube, and store it at -20°C.

[0188] 4. Testing for monoclonal antibodies against ASFV P30 protein:

[0189] (1) Properties: It should be a colorless, clear liquid without precipitation.

[0190] (2) Concentration determination of anti-ASFV P30 protein monoclonal antibody: the concentration should not be less than 0.5 mg / mL.

[0191] (3) Purity determination of anti-ASFV P30 protein monoclonal antibodies:

[0192] Perform SDS-PAGE, scan the electrophoresis pattern, and analyze the protein purity using Bandscan software. Calculate the purity according to the following formula: Purity (%) = (optical density of the target protein band / sum of the optical density of all protein bands in the lane where the target protein is located) × 100%, and the purity should not be less than 95.0%. The SDS-PAGE electrophoresis pattern of the anti-ASFV P30 protein monoclonal antibody is shown in Figure 2. Figure 1 As shown, lane 1 represents the monoclonal antibody in a reduced state (β-mercaptoethanol was added during sample preparation); lane 2 represents the monoclonal antibody in a non-reduced state (β-mercaptoethanol was not added during sample preparation).

[0193] (4) Determination of the titer of monoclonal antibodies against ASFV P30 protein:

[0194] Coating (anti-ASFV P30 protein monoclonal antibody): first mark the plate, then dilute the antigen with ELISA coating buffer to a coating concentration of 1μg / mL, add 100μL to each well of the ELISA plate, incubate at 4℃ for 12-16h, discard the liquid in the plate after incubation, wash the ELISA plate with ELISA washing buffer, add 200uL washing buffer to each well, discard the liquid in the plate and repeat the above operation, wash twice in total, and pat dry the residual liquid in the well.

[0195] Blocking: Add 150 μL of blocking solution (5% skim milk) to each well and incubate at 37°C for 3 hours. After the incubation, discard the liquid in the plate and wash the enzyme-linked plate with ELISA washing solution. Add 200 μL of washing solution to each well, discard the liquid in the plate and repeat the above operation. After washing 3 times, pat dry the residual liquid in the well.

[0196] Add primary antibody (goat anti-mouse IgG): dilute the antibody to be tested to 1 μg / μL with 5% skim milk, and then dilute it to 10 2 , 10 3 , 10 4 , 10 5 , 10 6 After that, add the concentrations from high to low to the ELISA plate, add the negative control to the remaining wells, 100 μL per well, and incubate at 37°C for 1 hour. After the incubation, discard the liquid in the plate, wash the ELISA plate with ELISA washing solution, add 200 μL washing solution to each well, discard the liquid in the plate, and repeat the above operation. After washing 5 times in total, pat dry the residual liquid in the well.

[0197] Add secondary antibody (goat anti-mouse-HRP): Determine the type of secondary antibody-HRP to be used based on the source of the test sample. Use goat anti-mouse-HRP, dilute with blocking solution, the dilution multiple is 1:5000, add 100μL to each well, incubate at 37℃ for 30min, discard the liquid in the plate after incubation, wash the enzyme-linked plate with ELISA washing solution, add 200μL washing solution to each well, discard the liquid in the plate and repeat the above operation, wash 5 times in total, and pat dry the residual liquid in the well.

[0198] Color development: Mix color development solution A and color development solution B (1:1), add 100 μL to each well, and react at 37°C for 15 min.

[0199] Stop and read: 50 μL of stop solution was added to each well, and the reading was immediately after the stop (wavelength 450 nm). The data was archived and analyzed, and it was concluded that the OD value of the first well was >2.5, and the OD value of the negative control was in the range of 0.04~0.1.

[0200] in,

[0201] ELISA coating buffer (0.05 mol / L, pH 9.6) is as follows: weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, add 800 mL of purified water, stir to dissolve, add purified water to make up to 1000 mL, and adjust the pH to 9.6.

[0202] The ELISA washing solution is: measure out 800mL of 10mmoL / L PBS buffer (pH7.4), add 1mL of Tween-20, and then add 10mmoL / L PBS buffer (pH7.4) to make up to 1000mL.

[0203] The blocking solution is: weigh 0.58g of disodium hydrogen phosphate (containing 12 crystal waters), 0.059g of potassium dihydrogen phosphate, and 0.9g of sodium chloride, add 800mL of purified water and stir to dissolve, then add 0.4mL of Proclin-300, 1mL of Tween-20, 50g of sucrose, and 10g of BSA, stir evenly, and then add purified water to make up to 1000mL.

[0204] Color developing solution A is: weigh 5.76g of citric acid, 0.5g of urea peroxide, and 6.21g of sodium acetate, add 800mL of purified water and stir to dissolve, then add 0.5mL of Proclin-300, add purified water to make up to 1000mL, aseptically quantitatively package, 12ml / bottle, and store at 2~8℃.

[0205] Color developing solution B is: weigh 5.76g of citric acid and 0.2g of TMB, add 100mL of methanol to dissolve, then add 0.5mL of Proclin-300, and finally add pure water to make up to 1000mL, aseptically quantitatively package, 12mL / bottle, and store at 2~8℃.

[0206] The stop solution is: measure 876 mL of purified water, slowly add 124 mL of concentrated sulfuric acid (2 mol / L), stir evenly, aseptically divide into quantitative portions, 12 mL / bottle, and store at 2~8°C.

[0207] The results showed that the titer of anti-ASFV P30 protein monoclonal antibody was 1:1024.

[0208] (5) Specificity determination of monoclonal antibodies against ASFV P30 protein:

[0209] When the indirect ELISA method was used for detection, the ELISA plate was coated with P30 protein, and the S / N value was ≥2.1; when the ELISA plate was coated with pET-21b empty vector protein, the S / N value was <2.1.

[0210] (6) Storage and shelf life: Store below -20°C for no more than 3 months.

[0211] 5. Protein A preparation and testing:

[0212] (1) Protein A: Protein A, His (Shanghai Puxin Biotechnology Co., Ltd., Catalog No.: 1001-01).

[0213] (2) Protein A test:

[0214] ① Properties: Observe the appearance and color of Protein A, which should be a colorless and transparent liquid.

[0215] ② Protein content determination: Use a microphotometer to determine the protein content, which should not be less than 5 mg / ml.

[0216] ③Purity: Perform SDS-PAGE and scan the electrophoresis pattern. There should be no impurity bands.

[0217] ④Storage and shelf life: Store below -20℃ for no more than 24 months.

[0218] 6. Preparation and testing of sample diluent:

[0219] (1) Preparation of sample diluent: Weigh 0.2543 g of disodium hydrogen phosphate dodecahydrate, 0.0452 g of sodium dihydrogen phosphate dihydrate, and 0.85 g of sodium chloride, and pour 80 mL of pure water into a beaker. Stir evenly, measure the pH value using a pH meter, add an appropriate amount of 0.1 mol / L hydrochloric acid titration solution to adjust the pH value to 7.4, make up to 100 mL with pure water, and filter through a 0.22 μm filter for sterilization.

[0220] (2) Inspection of sample diluent

[0221] ①Properties: Colorless clear liquid, no dirt, no precipitation.

[0222] ② Sterility test: Test according to the Appendix III of the current "Pharmacopoeia of Veterinary Medicine of the People's Republic of China". The sample dilution solution should show sterile growth.

[0223] (3) Specification: 1mL / tube.

[0224] (4) Storage and shelf life: Store at 4-30℃ away from light. The shelf life is 18 months.

[0225] 7. Pre-treatment of sample pad: Spread the entire sheet of sample pad glass fiber 8964 flat on the acrylic plate, pour 50mL of sample pad treatment solution (100mL0.1moL / L Tris pH8.5, 0.1g S9 surfactant (Tetronic1307), 0.2mL Tween-20, 0.05g casein sodium) on the glass fiber 8964, roll it flat with a roller, and dry it at 37℃ for more than 3h (you can touch it with your hands and it is dry).

[0226] 8. Pre-treat the binding pad: Soak the polyester film 6613 in the binding pad treatment solution (100mL of 10mmol / L PB buffer pH7.4, 0.5g PVPK30, 0.2mL Tween-20). The membrane material will float on the surface at first, and then slowly soak through for 20 minutes. After laying the paper towel flat on the table, use tweezers to pick up the polyester film and lay it flat on the paper towel to absorb the excess water. Place it flat at 37℃ and dry it for 1h~3h. Put it in a sealed bag with desiccant, seal it and store it away from light.

[0227] 9. Preparation of gold-labeled pad: prepare a pre-treated binding pad of 0.8 cm × 30 cm, spray 60× gold-labeled African swine fever virus P30 protein on the pre-treated binding pad at a spraying volume of 2 μL / cm, and dry at 37°C for 2 h for use.

[0228] 10. Preparation of coating membrane: Place the frosted side of the NC membrane upwards, and stick the smooth (mirror) side to the bottom plate for drawing. Use coating solution BB001 (100mL of 10mmol / L PBS pH7.4, 1g sucrose) to dilute the anti-ASFV P30 protein monoclonal antibody to 1.0mg / mL, and the drawing volume is 0.8μL / cm; use coating solution BB001 to dilute Protein A to 0.4mg / mL, and the drawing volume is 0.6μL / cm. At the same time, draw lines on the appropriate position of the NC membrane, and dry it at 37℃ overnight after drawing.

[0229] 11. Test strip packaging:

[0230] Assembly: Stick the absorbent pad, gold label pad and sample pad to the corresponding positions of the coated rubber plate. The absorbent pad covers the coating film by 1~2mm, the gold label pad covers the coating film by 1~2mm, and the sample pad is flush with the bottom of the base plate.

[0231] Cutting: Cut the assembled large board into test strips with a specification of 4.0mm×60mm;

[0232] Shell pressing: Put the cut test strips into a special plastic shell;

[0233] Bag packing: Take 1 test strip and 1 pack of desiccant and seal them in aluminum foil bag.

[0234] The test strip assembly results are as follows Figure 2 shown.

[0235] Example 4 Usage and determination of finished colloidal gold test strips

[0236] 1. Usage

[0237] (1) Sample collection: Aseptically collect plasma separated from anticoagulated blood or serum separated from non-anticoagulated blood.

[0238] (2) Sample storage: The collected samples should be stored at 2-8°C for no more than 24 hours and at -70°C to avoid repeated freezing and thawing.

[0239] (3) Sample transportation: Styrofoam boxes with ice packs should be sealed for transportation. Packaging and transportation should comply with the "Specifications for the Transportation and Packaging of Highly Pathogenic Animal Pathogenic Microorganisms, Bacteria (Viruses) or Samples" of the Ministry of Agriculture and Rural Affairs and relevant regulations of the transportation department on the management of dangerous goods transportation.

[0240] 2. Sample Processing

[0241] Blood sample processing: Anticoagulated blood samples were placed in centrifuge tubes, centrifuged at 8000r / min for 2 minutes, and 200μL of upper plasma was collected and numbered for testing. Non-anticoagulated blood samples were placed in centrifuge tubes, and after coagulation, they were centrifuged at 8000r / min for 2 minutes, and 200μL of upper serum was collected and numbered for testing.

[0242] 3. Sample requirements

[0243] (1) Applicable to serum and plasma samples. Fresh samples are recommended.

[0244] (2) After sample collection, serum / plasma should be separated as soon as possible to avoid hemolysis. Severely hemolyzed samples cannot be used for testing and need to be recollected;

[0245] (3) Before testing, refrigerated samples must be returned to room temperature, and frozen samples must be completely thawed, mixed, and returned to room temperature before testing.

[0246] 4. Inspection methods

[0247] (1) Sample processing: Take 20 μL of the separated serum or plasma sample, add 80 μL of sample diluent, and mix well;

[0248] (2) Sample addition: Pipette 80 μL of the treated sample and add it to the sample well of the test card. Be careful not to produce obvious bubbles when adding the sample. Set the timer to count the time and read the results after 15 minutes.

[0249] 5. Result determination

[0250] (1) Negative result: Only one red band appears in the control area (C), and no band appears in the test area (T).

[0251] (2) Positive result: A red stripe appears in the control area (C) and the test area (T).

[0252] (3) Invalid: When no red strip appears in the quality control area (C), the test strip is considered invalid regardless of whether a red strip appears in the test area (T).

[0253] Example 5: Finished colloidal gold test strip inspection

[0254] 1. Sensitivity test: The sensitivity control samples S1~S7 (the African swine fever positive serum purchased from the China Veterinary Drug Administration was diluted 1:10, and the African swine fever positive serum diluted 1:10 was diluted in multiples of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128, respectively marked as S1, S2, S3, S4, S5, S6, S7, sterile quantitative packaging, 200μL / tube, as sensitivity quality control samples) were tested according to the "Usage and Judgment", and 2 test strips were tested for each dilution. The results showed that the sensitivity met the following standards: S1~S6 were all positive and S7 was negative.

[0255] 2. Specificity test: According to the "Usage and Determination", one sample each of the positive serum for classical swine fever virus (CSFV) antibody, one sample each of the positive serum for porcine reproductive and respiratory syndrome virus (PRRSV) antibody, one sample each of the positive serum for porcine circovirus type 2 (PCV2) antibody, one sample each of the positive serum for pseudorabies virus (PRV) antibody and the empty vector pET-21b Escherichia coli BL21 (DE3) antibody (purchased from Beijing Animal Disease Prevention and Control Center) were tested, and the results showed that all were negative.

[0256] Example 6 Clinical diagnosis of pathogens

[0257] The colloidal gold test strips of the present invention were used to test 30 positive samples and 30 negative samples respectively. Table 1 lists the comparative experiments of the test strips prepared by the anti-ASFV P30 protein monoclonal antibody of the present invention and the commercial test strip African swine fever virus antibody rapid detection card (Zhengzhou Zhongnong Quick Detection Technology Co., Ltd.). The test results show that the test strips prepared by the anti-ASFV P30 protein monoclonal antibody of the present invention have the same sensitivity and specificity as the commercial test strips. The present invention has successfully developed a monoclonal antibody that can effectively detect ASFV, and the results are shown in Table 1.

[0258] Table 1 Test results of positive and negative samples

[0259]

[0260] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0261] Those skilled in the art will also recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also included in the appended claims. It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and the same and similar parts between the various embodiments can be referred to each other.

[0262] Although the optional embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including optional embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

Claims

1. An anti-ASFV P30 protein monoclonal antibody, characterized in that The anti-ASFV P30 protein monoclonal antibody has a heavy chain sequence as shown in SEQ ID NO: 1 and a light chain sequence as shown in SEQ ID NO:

2.

2. The anti-ASFV P30 protein monoclonal antibody according to claim 1, characterized in that The heavy chain sequence has a heavy chain constant region as shown in SEQ ID NO: 3; The light chain sequence has a light chain constant region as shown in SEQ ID NO:

4.

3. A polynucleotide, characterized in that The polynucleotide encodes the anti-ASFV P30 protein monoclonal antibody according to claim 1.

4. The polynucleotide according to claim 3, characterized in that The polynucleotide has a first nucleotide sequence as shown in SEQ ID NO:5 and a second nucleotide sequence as shown in SEQ ID NO:

6.

5. The polynucleotide according to claim 4, characterized in that The first nucleotide sequence has a first constant region as shown in SEQ ID NO:7; The second nucleotide sequence has a second constant region as shown in SEQ ID NO:

8.

6. A kit, characterized in that: The kit comprises the anti-ASFV P30 protein monoclonal antibody according to claim 1.

7. A colloidal gold test strip, characterized in that: The colloidal gold test strip comprises a base plate, a coating film, a sample pad, a gold label pad and a water absorbent pad, wherein: A detection line and a quality control line are sequentially arranged on the coating membrane along the flow direction of the sample; The detection line is coated with Protein A protein; The quality control line is coated with the anti-ASFV P30 protein monoclonal antibody according to claim 1 or 2.

8. A packaging box, characterized in that: The packaging box comprises the colloidal gold test strip according to claim 7 and a sample diluent.

9. Use of the anti-ASFV P30 protein monoclonal antibody according to claim 1 in the preparation of a kit for diagnosing African swine fever virus.

10. Use of the anti-ASFV P30 protein monoclonal antibody according to claim 1 in the preparation of a colloidal gold test strip for the diagnosis of African swine fever virus.

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