Truncated fragments of the african swine fever virus pB602L protein and antibodies or antigen-binding fragments thereof and uses

Abstract

This application relates to the field of antibody technology, specifically providing a truncated fragment of the African swine fever virus pB602L protein, an antibody thereof, or an antigen-binding fragment thereof, and their applications. The truncated fragment, encoded by SEQ ID NO.1, eliminates mismatched disulfide bonds and repetitive sequences by deleting the first 200 amino acids from the N-terminus, thereby improving protein solubility and immunogenicity. The antibody thereof or its antigen-binding fragment contains the heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.4, exhibiting a blocking rate of greater than 90% against the pB602L protein. This truncated fragment and antibody demonstrate high specificity and sensitivity, and can be used for the detection, diagnosis, and evaluation of vaccine immunization efficacy for African swine fever virus, providing an important tool for the prevention and control of African swine fever.

Description

Technical Field

[0001] This application relates to the field of antibody technology, specifically providing a truncated fragment of the African swine fever virus pB602L protein, an antibody or its antigen-binding fragment, and their applications. Background Technology

[0002] African swine fever virus (ASFV) is a pathogen that causes highly contagious diseases in pigs, resulting in significant economic losses to the global pig industry. ASFV is an enveloped double-stranded DNA virus with a multilayered viral particle structure. Its genome is approximately 170–193 kb in length and encodes 150–200 proteins.

[0003] Among them, the pB602L protein is an important non-structural protein of ASFV, involved in the formation of the viral icosahedral capsid, and can be expressed in the later stages of infection, inducing a high level of antibody response in the body. Studies have shown that this protein can be neutralized by ASFV hyperimmune serum, making it an important target for viral structural analysis and serological detection.

[0004] However, specific diagnostic tools targeting the pB602L protein, especially monoclonal antibodies with high blocking activity and their derived detection reagents, are currently lacking. The absence of monoclonal antibodies capable of efficiently blocking the binding of the pB602L protein to antibodies limits the application of this protein in accurate serological detection and immunological research of ASFV. Therefore, developing monoclonal antibodies that specifically bind to the pB602L protein and possess strong blocking activity, along with related detection kits, is of great significance for the prevention and control of ASFV and basic research. Summary of the Invention

[0005] One of the purposes of this application is to provide a truncated fragment of the African swine fever virus pB602L protein and an antibody or its antigen-binding fragment and application, so as to provide an antibody and its derivative tools that can bind to the ASFV pB602L protein with high specificity and high affinity, especially suitable for constructing competitive inhibition detection systems (such as blocking ELISA).

[0006] To achieve the above objectives, this application adopts the following technical solution:

[0007] A truncated fragment of the pB602L protein of African swine fever virus, named The truncated fragment has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0008] An antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus pB602L protein, said antibody or antigen-binding fragment comprising: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.4.

[0009] Furthermore, the antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment;

[0010] Optionally, the antibody is a monoclonal antibody 3A3, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

[0011] The biological material related to the antibody or its antigen-binding fragment described in this application is any one of the following:

[0012] (a) A nucleic acid molecule containing a sequence encoding a heavy chain variable region and / or a light chain variable region encoding the antibody or an antigen-binding fragment thereof;

[0013] (b) Expression cassette containing the nucleic acid molecules in (a);

[0014] (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b);

[0015] (d) Recombinant host cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0016] Further, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3; and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.5.

[0017] This application relates to the use of the aforementioned truncated fragments or antibodies, or their antigen-binding fragments or biological materials, in the preparation of African swine fever virus ELISA antibody detection products;

[0018] Optionally, the product is an African swine fever virus indirect ELISA antibody detection kit or an African swine fever virus blocking ELISA antibody detection kit.

[0019] An indirect ELISA antibody detection kit for African swine fever virus, the kit comprising: a support medium coated with the truncated fragment described above, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled anti-pig IgG antibody.

[0020] Furthermore, the aforementioned The coating concentration of the (truncated fragment) is 0.6-1.0 μg / mL, preferably 0.8 μg / mL;

[0021] Optionally, the support medium is a microtiter plate;

[0022] Optionally, the enzyme labeled in the enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0023] Optionally, the detection reagent includes a colorimetric solution and a stop solution;

[0024] Optionally, the kit may further include at least one of a positive control, a negative control, a washing solution, and a sample diluent.

[0025] An African swine fever virus blocking ELISA antibody detection kit, the kit comprising: a support medium coated with the truncated fragment of the present application, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody of the present application or its antigen-binding fragment.

[0026] Furthermore, the enzyme-labeled reagent is an enzyme-labeled monoclonal antibody 3A3;

[0027] Optionally, the The coating concentration of the (truncated fragment) is 1.2-1.8 μg / mL, preferably 1.6 μg / mL;

[0028] Optionally, the support medium is a microtiter plate;

[0029] Optionally, the enzyme labeled in the enzyme-labeled reagent is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0030] Furthermore, the detection reagent includes a colorimetric solution and a stop solution;

[0031] Optionally, the kit may further include at least one of a positive control, a negative control, a washing solution, and a sample diluent.

[0032] The technical effects of this application are as follows:

[0033] By rationally truncating the African swine fever virus pB602L protein, a protein fragment encoded by SEQ ID NO.1 was obtained. This design effectively overcomes the technical challenge of easy aggregation of full-length proteins in prokaryotic expression systems by removing the N-terminal region that is prone to mismatched disulfide bonds. It significantly improves the soluble expression level and yield of recombinant proteins, while also significantly enhancing their immunogenicity, providing a stable and efficient core antigen raw material for the development of subsequent detection reagents.

[0034] The antibody or antigen-binding fragment provided in this application, by including the heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.4, exhibits extremely high affinity and specificity for the target antigen, and demonstrates excellent blocking effect. This antibody can be flexibly prepared into various forms of antigen-binding fragments, such as monoclonal antibody 3A3 (IgG1 / kappa type) and single-chain antibodies; its corresponding encoding nucleic acids, expression vectors, and recombinant cells also provide a reliable basis for large-scale production and modification.

[0035] The indirect ELISA and blocking ELISA detection methods established based on this truncated fragment and antibody, through systematic optimization of key parameters such as coating concentration, reaction system and reagent formulation, have enabled the developed kits to exhibit excellent performance in terms of detection specificity, sensitivity and repeatability. They can accurately distinguish ASFV-infected serum from other common swine disease positive serum, effectively meeting the needs of large-scale screening, early monitoring and accurate diagnosis of clinical samples. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions described in this application will be further described in detail below with reference to specific embodiments.

[0037] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0038] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0039] In this application, terms such as "multiple", "various", "multiple times", and "multi-source" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "at least one" means one or more than or equal to two.

[0040] "Truncation fragment" refers to a protein fragment obtained by truncating the N-terminus of the full-length African swine fever virus pB602L protein. Specifically, it is the protein sequence remaining after removing the first 200 amino acids from the N-terminus, having the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.1. This truncation design aims to eliminate mismatched disulfide bond formation and protein aggregation problems caused by the cysteine-rich N-terminal region, while also removing repetitive sequences in the central variable region to improve protein solubility, stability, and immunogenicity.

[0041] "Antibody" refers to an immunoglobulin molecule that specifically binds to the African swine fever virus pB602L protein. An example is monoclonal antibody 3A3, whose heavy chain subclass is IgG1 and light chain subclass is kappa. "Antigen-binding fragment" refers to a fragment form that retains the antigen-binding activity of a complete antibody, including but not limited to Fab fragments (antigen-binding fragments composed of a heavy chain variable region, a light chain variable region, and a first constant region), Fab' fragments (containing a partial hinge region on top of the Fab fragment), F(ab')2 fragments (two Fab' fragments linked by disulfide bonds), scFv fragments (single-chain antibodies formed by heavy chain variable regions and light chain variable regions linked by linker peptides), and Fv fragments (the smallest antigen-binding unit containing both heavy chain and light chain variable regions). These fragments can all be obtained from intact antibodies through enzymatic digestion or genetic engineering methods and retain the same antigen-binding specificity as the intact antibody.

[0042] "Heavy chain variable region" and "light chain variable region" refer to the variable regions at the N-terminus of the heavy chain and light chain of the antibody molecule, respectively. They contain three hypervariable regions (complementarity-determining regions, CDRs) and four frame regions (FRs), responsible for recognizing and binding to specific antigenic epitopes. In this application, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0043] "Biomaterials" include nucleic acid molecules (DNA or RNA) encoding the antibodies or their antigen-binding fragments, expression cassettes containing such nucleic acid molecules (containing regulatory elements such as promoters and signal peptide sequences), recombinant vectors (plasmids, phages, or viral vectors), and recombinant host cells containing the genetic material described above (including eukaryotic cells such as mammalian cells and yeast cells, or prokaryotic cells such as Escherichia coli).

[0044] The "African Swine Fever Virus Indirect ELISA Antibody Detection Kit" refers to an antibody detection kit constructed based on the principle of indirect ELISA. This kit uses a truncated fragment of the pB602L protein described in this application as the coating antigen and an enzyme-labeled anti-pig immunoglobulin antibody (such as HRP-labeled goat anti-pig IgG or rabbit anti-pig IgG) as the enzyme-labeled reagent. The detection principle is as follows: the sample to be tested is added to an ELISA plate coated with the truncated pB602L protein fragment and incubated. If the sample contains African swine fever virus-specific antibodies, it binds to the coating antigen to form an antigen-antibody complex. After washing, enzyme-labeled anti-pig IgG antibody is added, which reacts with the bound sample antibody to form an antigen-antibody-enzyme-labeled antibody complex. The absorbance is measured by a colorimetric reaction, and the result is determined based on the ratio of the sample absorbance to the positive control absorbance (S / P value). The result was positive. This kit is primarily used to detect the presence of specific antibodies against the pB602L protein in porcine serum.

[0045] The "African Swine Fever Virus Blocking ELISA Antibody Detection Kit" refers to an antibody detection kit constructed based on the principle of competitive blocking ELISA. This kit uses a truncated fragment of the pB602L protein described in this application as the coating antigen, but its enzyme-labeled reagent is an enzyme-labeled specific monoclonal antibody (such as HRP-labeled monoclonal antibody 3A3). Its detection principle is as follows: the sample to be tested is pre-incubated or simultaneously incubated with the coating antigen. If the sample contains African swine fever virus-specific antibodies, these antibodies compete with the enzyme-labeled monoclonal antibody for binding to the same or similar epitopes on the coating antigen, thereby blocking the binding of the enzyme-labeled monoclonal antibody to the antigen. The result is determined by measuring the absorbance of the colorimetric reaction and the ratio (S / N value) between the sample absorbance and the mean absorbance of the negative control. This kit significantly improves detection sensitivity through competitive inhibition, enabling the detection of antibodies at lower titers, and is suitable for early diagnosis and immune monitoring of African swine fever.

[0046] This application provides a truncated fragment of the African swine fever virus pB602L protein, having an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.1.

[0047] The truncated fragment refers to a protein fragment obtained by artificially modifying the African swine fever virus pB602L protein. Specifically, its amino acid sequence is encoded by the nucleotide sequence shown in SEQ ID NO.1 as described in this specification. Based on the correspondence of genetic codons, this nucleotide sequence uniquely determines the primary structure (i.e., the amino acid sequence) of the truncated fragment. Those skilled in the art will understand that, due to the degeneracy of the genetic code, the nucleotide sequence can be equivalently substituted or optimized without changing the encoded amino acid sequence. For example, it can be adapted to the codon preferences of different expression hosts (such as Escherichia coli, yeast, or mammalian cells). Such optimized nucleotide variants encoding the same amino acid sequence all fall within the scope of the meaning of "encoded by the nucleotide sequence shown in SEQ ID NO.1" in this application.

[0048] This truncated fragment was obtained by cloning a DNA fragment encoding the specific amino acid sequence (e.g., SEQ ID NO.1 or its optimized variant) into a suitable expression vector using molecular biology techniques, followed by expression and purification in host cells. This design significantly improved the soluble expression level and stability of the full-length pB602L protein in prokaryotic systems such as E. coli by removing the N-terminal domain that is prone to misfolding and aggregation, while retaining good immunogenicity and antigenicity, laying the material foundation for its subsequent application as a diagnostic antigen.

[0049] Therefore, the core of the statement "having an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.1" lies in defining the specific amino acid sequence composition of the fragment and the technical source of its preparation by means of the nucleotide sequence shown in SEQ ID NO.1 or its functionally equivalent nucleotide sequence.

[0050] This application provides the use of the above-mentioned truncated fragment in the preparation of an indirect ELISA antibody detection kit for African swine fever virus.

[0051] "Indirect ELISA Antibody Detection Kit" refers to a reagent combination based on the principle of indirect enzyme-linked immunosorbent assay (ELISA) used to detect whether a sample contains specific antibodies against African swine fever virus. In this application, a truncated fragment (i.e., encoded by SEQ ID NO.1) is used. Protein fragments are used as coating antigens, which are fixed on the surface of a solid support (such as a polystyrene microtiter plate) by physical adsorption or chemical cross-linking to form an antigen-coated plate.

[0052] During the detection process, specific antibodies in the sample to be tested (such as serum) (if present) will bind to the coating antigen. Subsequently, the bound antibody will react with an enzyme-labeled secondary antibody (such as horseradish peroxidase-labeled goat anti-pig IgG). Finally, the substrate will be catalyzed by enzyme to achieve qualitative and semi-quantitative detection of the target antibody.

[0053] This application provides an antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus pB602L protein, comprising: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.4. These two variable regions together constitute the antigen-binding site of the antibody, determining its high specificity and high affinity binding ability to the unique epitope of the pB602L protein.

[0054] Based on the aforementioned variable region structure, the antibodies of this application can exist in the form of intact immunoglobulins or as functional antigen-binding fragments. These fragments include, but are not limited to: Fab fragments, which consist of a variable region of an intact light chain and heavy chain, and a first constant region; Fab' fragments, derivatives of Fab, containing additional cysteine ​​residues in the heavy chain hinge region; F(ab')2 fragments, formed by two Fab' fragments linked by disulfide bonds, possessing bivalent binding capability; scFv (single-chain antibody), a single polypeptide chain formed by linking VH and VL through a flexible peptide chain; and the smallest Fv fragment, containing only non-covalently bound variable regions. All of these fragments retain the specific binding activity derived from the sequences of SEQ ID NO.2 and SEQ ID NO.4.

[0055] In a preferred embodiment, this application obtained a monoclonal antibody named 3A3 using hybridoma technology. This antibody contains the specific heavy and light chain variable region sequences described above. Identification revealed that the heavy chain constant region of monoclonal antibody 3A3 belongs to the IgG1 subclass, and the light chain is kappa type. This antibody exhibited excellent performance in blocking assays against the pB602L protein, with a blocking rate exceeding 90%, reaching 94%, confirming its efficient and competitive blocking of the binding of the target antigen to the antibody in serum. This antibody or its fragments can be prepared using conventional hybridoma technology, or through molecular biology methods, by cloning the nucleic acid sequence encoding its variable region (such as SEQ ID NO.3 and SEQ ID NO.5) into an expression vector for recombinant expression and production in eukaryotic or prokaryotic host systems. Therefore, this application not only protects complete antibodies (such as 3A3) containing the specific variable region sequence, but also covers all antibody derivatives and functional fragments containing this core variable region sequence and maintaining specific binding ability to the pB602L protein.

[0056] This application also provides biological materials related to the aforementioned antibodies or their antigen-binding fragments, including any of the following forms:

[0057] (a) Nucleic acid molecule: refers to the nucleotide sequence encoding the heavy chain variable region and / or light chain variable region of the antibody or its antigen-binding fragment of this application. The nucleic acid molecule may be in the form of DNA, such as a gene fragment obtained by chemical synthesis, PCR amplification, or cloning from hybridoma cells; or in the form of RNA, such as in vitro transcribed mRNA. In a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 3, and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO. 5. Those skilled in the art will understand that the above sequences can be optimized based on codon bias without altering the encoded amino acid sequence, and such optimized equivalent nucleotide sequences also fall within the scope of the nucleic acid molecules described in this application.

[0058] (b) Expression cassette: refers to a genetic unit containing the nucleic acid molecules described in (a) above. This unit contains not only the structural gene encoding the antibody variable region, but also regulatory elements necessary to ensure effective transcription and translation in the host cell. These regulatory elements include, but are not limited to, promoters, enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals. Expression cassettes can exist independently or be integrated into a vector.

[0059] (c) Recombinant vector: refers to a DNA molecule carrying the nucleic acid molecule in (a) or the expression cassette in (b) above, used to introduce the target gene into a host cell. The backbone of the vector can be a plasmid, granulosome, bacteriophage, or viral vector (such as adenovirus or lentiviral vector). Recombinant vectors usually also contain selection markers (such as antibiotic resistance genes) and origins of replication to facilitate selection and amplification in host cells.

[0060] (d) Recombinant host cell: refers to a cell that has been genetically engineered to contain the nucleic acid molecule described in (a), the expression cassette described in (b), or the recombinant vector described in (c). The host cell may be a prokaryotic cell, such as E. coli strain BL21(DE3); or a eukaryotic cell, such as mammalian cells (e.g., HEK293, CHO cells), yeast cells, etc. These recombinant host cells are capable of expressing and producing the antibody or its antigen-binding fragment as described in this application under suitable conditions.

[0061] The truncated fragments, antibodies or their antigen-binding fragments provided in this application, as well as the biological materials, can all be used to prepare African swine fever virus blocking ELISA antibody detection kits.

[0062] This application provides an African swine fever virus blocking ELISA antibody detection kit, the core components of which include: a support medium coated with a truncated fragment, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody of this application or its antigen-binding fragment.

[0063] In some embodiments, the support medium is preferably a polystyrene or other suitable microtiter plate (ELISA plate) for protein adsorption. It is coated with the protein provided in this application through conventional physical adsorption or chemical cross-linking methods. A truncated antigen fragment was used. After experimental optimization, the coating concentration of this antigen was found to be 1.2-1.8 μg / mL, preferably 1.6 μg / mL.

[0064] In some embodiments, the enzyme-labeled reagent is enzyme-labeled monoclonal antibody 3A3. The labeled enzyme can be horseradish peroxidase (HRP), alkaline phosphatase (ALP), or β-D-galactosidase, among which HRP is the preferred choice due to its high specific activity, stability, and economy. The enzyme-labeled antibody can be prepared using conventional methods, such as the sodium periodate method (suitable for HRP) or the glutaraldehyde cross-linking method.

[0065] In some embodiments, the detection reagent includes a colorimetric solution for generating a detectable signal and a stop solution for terminating the reaction.

[0066] Colorimetric reagent: Typically a two-component system. For example, when using HRP, the colorimetric reagent may include:

[0067] Developing solution A: Contains oxidizing agents, such as a buffer solution containing 1.47% (w / v) disodium hydrogen phosphate, 0.93% (w / v) citric acid and 0.03% (w / v) urea peroxide, providing a stable acidic reaction environment and peroxides.

[0068] Developing solution B: Contains a chromogenic substrate, such as a solution containing 0.02% (w / v) tetramethylbenzidine (TMB) and 1% (v / v) anhydrous ethanol. TMB is oxidized under HRP catalysis to produce a blue product. The reaction is terminated under acidic conditions and turns yellow, facilitating detection at a wavelength of 450 nm.

[0069] Termination solution: Used to terminate the enzymatic reaction and stabilize the final color, usually a strong acid solution, such as 10% (v / v) hydrochloric acid solution.

[0070] In some implementations, to ensure the kit is fully functional, standardized in operation, and easy to interpret results, the kit may also include one or more of the following components:

[0071] Positive control: A standard known to contain high-titer ASFV pB602L antibody. For example, it could be a standard containing 2.5% (v / v) of ASFV pB602L antibody. Protein-immunized porcine positive serum, 20% (v / v) bovine serum, and PBS buffer containing 0.1% (v / v) Proclin 300 preservative were used to validate the validity of each test and to participate in the calculation of the decision threshold.

[0072] Negative control: A standard to confirm the absence of ASFV pB602L antibody. For example, a PBS buffer containing 2.5% (v / v) healthy swine negative serum, 20% (v / v) bovine serum, and 0.1% (v / v) Proclin 300 can be used to determine the background signal for detection.

[0073] Washing solution: Used to wash unbound substances, typically a buffer salt solution containing surfactants. For example, it can be... The concentrated washing solution is formulated with: 160 g sodium chloride, 58 g disodium hydrogen phosphate, 4.8 g potassium dihydrogen phosphate, 4 g potassium chloride, and 10 ml Tween 20, diluted to 1000 ml with purified water. Before use, dilute 20 times with purified water. Its suitable ionic strength and pH value effectively remove non-specific adsorption and reduce background.

[0074] Sample diluent: Used to dilute the serum sample to be tested, typically a buffer containing protein stabilizers and preservatives. For example, a PBS buffer containing 20% ​​(v / v) newborn calf serum (for blocking non-specific sites) and 0.1% (v / v) Proclin 300 (preservative) can help reduce sample matrix interference and improve the accuracy and stability of the test.

[0075] This application provides an indirect ELISA antibody detection kit for African swine fever virus, the core components of which include: a support medium coated with a truncated fragment, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled anti-pig IgG antibody, used to recognize the antibody to be detected bound to the coated antigen.

[0076] In some embodiments, the support medium is preferably a polystyrene or other suitable microtiter plate (ELISA plate) for protein adsorption. The plate is coated with the protein provided in this application using conventional physical adsorption methods. A truncated antigen fragment was used. After experimental optimization, the optimal coating concentration of this antigen was determined to be 0.6-1.0 μg / mL, preferably 0.8 μg / mL. This concentration achieves the best balance between ensuring sufficient antibody capture and background signal absorption.

[0077] In some implementations, the enzyme-labeled reagent is an enzyme-labeled anti-pig IgG antibody (secondary antibody). The labeled enzyme can be horseradish peroxidase (HRP), alkaline phosphatase (ALP), or β-D-galactosidase, among which HRP is the preferred choice due to its high specific activity, stability, and cost-effectiveness. The enzyme-labeled secondary antibody can be prepared using conventional methods, such as the sodium periodate method (suitable for HRP) or the glutaraldehyde cross-linking method. It must be diluted to the working concentration before use to ensure optimal differentiation between the detection signal and background signal in positive serum.

[0078] In some embodiments, the detection reagent includes a colorimetric solution for generating a detectable signal and a stop solution for terminating the reaction, which together enable the visualization and quantitative detection of the signal.

[0079] Colorimetric reagent: Typically a two-component system. For example, when using HRP, the colorimetric reagent may include:

[0080] Developing solution A: Contains oxidizing agents, such as a buffer solution containing 1.47% (w / v) disodium hydrogen phosphate, 0.93% (w / v) citric acid and 0.03% (w / v) urea peroxide, providing a stable acidic reaction environment and peroxides.

[0081] Developing solution B: Contains a chromogenic substrate, such as a solution containing 0.02% (w / v) tetramethylbenzidine (TMB) and 1% (v / v) anhydrous ethanol. TMB is oxidized under HRP catalysis to produce a blue product. The reaction is terminated under acidic conditions and turns yellow, facilitating detection at a wavelength of 450 nm.

[0082] Termination solution: Used to terminate the enzymatic reaction and stabilize the final color, usually a strong acid solution, such as 10% (v / v) hydrochloric acid solution.

[0083] In some implementations, to ensure the kit is fully functional, standardized in operation, and easy to interpret results, the kit may also include one or more of the following components:

[0084] Positive control: A standard known to contain high-titer ASFV pB602L antibody. For example, it could be a standard containing 0.125% (v / v) of the antibody. Protein-immunized porcine positive serum, 20% (v / v) bovine serum, and PBS buffer with 0.1% (v / v) Proclin 300 preservative were used to validate the validity of each test.

[0085] Negative control: A standard to confirm the absence of ASFV pB602L antibody. For example, a PBS buffer containing 0.125% (v / v) healthy swine negative serum, 20% (v / v) bovine serum, and 0.1% (v / v) Proclin 300 can be used to determine the background signal for detection and participate in result calculation.

[0086] Washing solution: Used to wash unbound substances, typically a buffer salt solution containing surfactants. For example, it can be... The concentrated washing solution is formulated with: 160 g sodium chloride, 58 g disodium hydrogen phosphate, 4.8 g potassium dihydrogen phosphate, 4 g potassium chloride, and 10 ml Tween 20, diluted to 1000 ml with purified water. Before use, dilute 20 times with purified water. Its suitable ionic strength and pH value effectively remove non-specific adsorption and reduce background.

[0087] Sample diluent: Used to dilute the serum sample to be tested, typically a buffer containing protein stabilizers and preservatives. For example, a PBS buffer containing 20% ​​(v / v) newborn calf serum (for blocking non-specific sites) and 0.1% (v / v) Proclin 300 (preservative) can help reduce sample matrix interference and improve the accuracy and stability of the test.

[0088] The present application is further described below with reference to specific embodiments. The advantages and features of the present application will become clear from the description. The embodiments described are merely exemplary and do not constitute any limitation on the scope of the present application. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present application without departing from the spirit and scope of the present application, but such modifications and substitutions all fall within the protection scope of the present application.

[0089] Unless otherwise specified, the experimental methods described in this application are all conventional methods; and the biological materials described are all commercially available unless otherwise specified.

[0090] Example 1: Preparation and content determination of ASFV pB602LΔN protein

[0091] Analysis of the full-length pB602L protein revealed that its N-terminus is rich in cysteine ​​(Cys) residues, with nine Cys residues in the first 200 amino acids. However, the C-terminus only contains Cys residues at positions 465. This dense concentration of Cys residues at the N-terminus makes it highly susceptible to mismatched disulfide bonds during exogenous expression, leading to protein aggregation and affecting immunogenicity. Furthermore, amino acids 160–200 of pB602L constitute the central variable region (CRV), containing small repetitive sequences, which are detrimental to inducing diverse antibody production. To enhance both the immunogenicity and antibody diversity of pB602L, a design was developed to truncate the first 200 amino acids from the N-terminus for expression. The expressed protein was named […]. .

[0092] Synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The sequence SEQ ID NO.1 was used to clone the gene fragment into the pNVC1 expression vector via PCR amplification, enzyme digestion, and ligation, and the result was named the recombinant plasmid. Recombinant plasmid The expression strain was constructed by transforming competent *E. coli* BL21(DE3) into kanamycin-resistant LB broth. After incubation at 37°C and 220 rpm for 12 hours with shaking, the culture was transferred to 0.2 L LB broth and incubated at 37°C and 220 rpm for 2 hours with shaking. The temperature was then lowered to 22°C, and IPTG solution (final concentration 0.5 mmol / L) was added for induction culture for 12 hours. The bacterial cells were collected by centrifugation. The cells were resuspended and homogenized three times at 800 bar. After centrifugation at 13500 rpm for 40 min, the supernatant was collected, and the expression level was detected by 12% SDS-PAGE electrophoresis. Subsequent affinity chromatography and gel filtration chromatography were performed to purify the ASFV. Protein. ASFV was measured using the BCA protein concentration assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) The protein concentration was 1.2 mg / mL.

[0093] SEQ ID NO.1

[0094] GAATACACCGACCTGGCTGACCCGGAACGTATCCCGCTGCACATCATGCAGAAAACCCTGAACGTTCCGAACGAACTGCAGGCTGACATCGACGCTATCACCCAGACCCCGCAGGGTTACCGTGCTGCTGCTCACATCCTGCAGAACATCGAACTGCACCAGTCTATCAAACACATGCTGGAAAACCCGCGTGCTTTCAAACCGATCCTGTTCAACACCAAAATCACCCGTTACCTGTCTCAGCACATCCCGCCGCAGGACACCTTCTACAAATGGAACTACTACATCGAAGACAACTACGAAGAACTGCGTGCTGCTACCGAATCTATCTACCCGGAAAAACCGGACCTGGAATTCGCTTTCATCATCTACGACGTTGTTGACTCTTCTAACCAGCAGAAAGTTGACGAATTCTACTACAAATACAAAGACCAGATCTTCTCTGAAGTTTCTTCTATCCAGCTGGGTAACTGGACCCTGCTGGGTTCTTTCAAAGCTAACCGTGAACGTTACAACTACTTCAACCAGAACAACGAAATCATCAAACGTATCCTGGACCGTCACGAAGAAGACCTGAAAATCGGTAAAGAAATCCTGCGTAACACCATCTACCACAAAAAAGCTAAAAACATCCAGGAAACCGGTCCGGACGCTCCGGGTCTGTCTATCTACAACTCTACCTTCCACACCGACTCTGGTATCAAAGGTCTGCTGTCTTTCAAAGAACTGAAAAACCTGGAAAAAGCTTCTGGTAACATCAAAAAAGCTCGTGAATACGACTTCATCGACGACTGCGAAGAAAAAATCAAACAGCTGCTGTCTAAAGAAAACCTGACCCCGGACGAAGAATCTGAACTGATCAAAACCAAAAAACAGCTGGACAACGCTCTGGAAATGCTGAACGTTCCGGACGACACCATCCGTGTTGACATGTGGGTTAACAACAACAACAAACTGGAAAAAGAAATCCTGTACACCAAAGCTGAACTG。

[0095] Example 2: Preparation and application of ASFV pB602L protein indirect ELISA antibody detection kit

[0096] 2.1 Preparation of the reagent kit

[0097] Antigen-coated plate: The antigen prepared in Example 1 was coated with carbonate buffer (pH 9.6, 0.05 mol / L). Dilute the protein to 0.8 μg / ml for coating, 100 μl / well, and incubate at 2–8℃ for 16–24 hours. After washing with washing buffer, add blocking buffer (weigh 10 g bovine serum albumin, 50 g sucrose, 1 ml Proclin 300, dissolve in PBS buffer (0.01 mol / L, pH 7.4) and bring the volume to 1000 ml) at 2–8℃ for 16–24 hours. Discard the blocking buffer, dry, seal and store at 2–8℃ for later use.

[0098] Enzyme-labeled reagent: Commercially available HRP-labeled goat anti-pig IgG or rabbit anti-pig IgG was diluted 60,000 times with enzyme-labeled dilution buffer (weigh 200 ml newborn calf serum, 1 ml Proclin 300, 0.5 ml Tween 20, 0.04 g AM dye, dissolved in PBS buffer (0.01 mol / L, pH 7.4) and brought to a final volume of 1000 ml) and stored at 2–8 °C.

[0099] Positive control: Take 1.25ml Positive serum from protein-immunized pigs, 200 ml of newborn calf serum, and 1 ml of Proclin 300 were diluted to 1000 ml with PBS buffer (0.01 mol / L, pH 7.4), mixed well, filtered through a 0.22 μm filter, and aseptically aliquoted as positive controls. Store at 2–8 °C.

[0100] Negative control: Take 1.25 ml of negative serum from healthy pigs, 200 ml of newborn calf serum, and 1 ml of Proclin 300, and bring the volume to 1000 ml with PBS buffer (0.01 mol / L, pH 7.4). Mix well and filter through a 0.22 μm filter. Aseptically aliquot the mixture as a negative control and store at 2–8 °C.

[0101] Sample dilution solution: Take 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 200ml of newborn calf serum, and 1ml of Proclin 300. Dissolve them in purified water and bring the volume to 1000ml. Mix well and filter through a 0.22μm filter. Aseptically dispense and store at 2-8℃.

[0102] Concentrated washing solution: Dissolve 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, and 10ml Tween 20 in purified water and bring the volume to 1000ml. Mix well and filter through a 0.22μm filter. Aseptically dispense and store at 2–8℃. Dilute 20 times with purified water before use.

[0103] Colorimetric solution A: Dissolve 14.7g of disodium hydrogen phosphate, 9.3g of citric acid, and 0.3g of urea peroxide in purified water and bring the volume to 1000ml. Mix well and dispense into containers. Dissolve 0.2g of 3,3',5,5'-tetramethylbenzidine (TMB) and 10ml of anhydrous ethanol in purified water and bring the volume to 1000ml. Mix well and dispense into containers.

[0104] Termination solution: 10% hydrochloric acid solution.

[0105] The antigen-coated plate, enzyme-labeled reagent, positive control, negative control, and sample diluent were prepared. Concentrated washing solution, colorimetric solutions A and B, and stop solution were assembled into an indirect ELISA antibody detection kit for African swine fever virus pB602L protein.

[0106] 2.2 Establishment of Detection Method

[0107] The testing steps are as follows:

[0108] (1) Sample addition: Dilute the sample to be tested 100 times with the sample diluent and add it to the corresponding wells, 100 μl / well. At the same time, set up 2 positive control wells, 2 negative control wells, and 1 blank control well (blank control is not required for dual-wavelength detection). After sealing the plate with the sealing film, incubate at 37°C for 30 minutes.

[0109] (2) Washing: Wash with detergent 5 times, and dry it on the last time.

[0110] (3) Add enzyme-labeled reagent: except for the blank control well, add 100 μl of enzyme-labeled reagent to each well, seal the plate with sealing film and incubate at 37°C for 30 minutes.

[0111] (4) Washing: Wash with detergent 5 times, and dry it on the last time.

[0112] (5) Color development: Add 50µl of color development solution A and color development solution B to each well in sequence, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0113] (6) Termination: Add 50µl of stop solution to each well and measure the results with an enzyme-linked immunosorbent assay (ELISA) reader within 10 minutes.

[0114] (7) Measurement:

[0115] When performing dual-wavelength measurements, it is necessary to set the dual wavelengths to 450nm / 600-650nm and measure the A value of each well.

[0116] For single-wavelength assays, the microplate reader was set to 450 nm, and the A value of each well was measured after zeroing the instrument with a blank control well.

[0117] (8) Result determination:

[0118] Calculation method: S / P value = Sample A value / Mean A value of positive control.

[0119] Judgment method:

[0120] S / P value The patient tested positive for ASFV pB602L protein antibody.

[0121] Individuals with an S / P value < 0.5 are negative for ASFV pB602L protein antibody.

[0122] 2.3 Evaluation of the reagent kit

[0123] 2.3.1 Specificity

[0124] The kit prepared in Example 2.1 was used to test 8 positive serum samples of common porcine viruses (including positive serum of classical swine fever virus, positive serum of porcine pseudorabies virus, positive serum of porcine reproductive and respiratory syndrome virus, positive serum of porcine circovirus type 2, positive serum of porcine parvovirus, positive serum of porcine epidemic diarrhea virus, positive serum of porcine transmissible gastroenteritis virus, and positive serum of porcine rotavirus), 20 SPF porcine serum samples, and 50 routinely immunized porcine serum samples that were negative for ASFV antigen. The results showed that the S / P values ​​were all <0.5, indicating that all samples were negative, which shows that the kit has good specificity.

[0125] 2.3.2 Sensitivity

[0126] The ASF standard positive serum serially diluted with the kit prepared in Example 2.1 was tested. The results showed that the 1:8 dilution was positive and the 1:16 dilution was negative.

[0127] The kit prepared in Example 2.1 was used to detect... Twenty positive serum samples from protein-immunized pigs were obtained, and all were positive.

[0128] 2.3.3 Clinical Application

[0129] The kit prepared in Example 2.1 was used in clinical applications to detect 20 ASF-positive swine serum samples and 1000 negative swine serum samples collected before 2018. The results showed that the S / P values ​​of the 20 ASF-positive swine serum samples were all >0.5, indicating that they were all positive; the S / P values ​​of the 1000 negative swine serum samples collected before 2018 were all <0.5, indicating that they were all negative. The kit specificity was 100%.

[0130] Example 3: Preparation and Identification of ASFV pB602L Protein Monoclonal Antibody

[0131] 3.1 Preparation of Monoclonal Antibodies

[0132] Purified Female BALB / c mice aged 4–6 weeks were subcutaneously immunized at multiple sites, 0.1 mg per mouse. The initial immunization used Freund's complete adjuvant, followed by immunizations using Freund's incomplete adjuvant, once every 2 weeks for a total of 3 immunizations. Serum antibody titers after the three immunizations were detected using the indirect ELISA method described in Example 2; all titers were not lower than 1:512,000. Mice with the highest antibody titers were selected and intraperitoneally injected with 100 μg of unadjuvanted protein. Proteins were used to enhance immunization, and cell fusion was performed 3 days after immunization.

[0133] The supernatant of the fused cells was screened using a positive serum blocking assay: the cells prepared in Example 1 were... Dilute the protein to 0.5 μg / ml and add 100 μl to each well of an ELISA plate. Incubate at 2–8 °C for 16–24 hours. Wash the plate and add 200 μl of blocking buffer to each well. Block at 37 °C for 2 hours. Wash the plate, add 100 μl of cell supernatant to each well, and set up wells with PBS buffer (0.01 mol / L, pH 7.4) as negative controls. Incubate at 37°C for 60 minutes, then wash the plate. Add 100 μl of ASF-positive serum diluted 1:800 to each well, incubate at 37°C for 60 minutes, then wash the plate. Add 100 μl of HRP-labeled goat anti-pig IgG diluted 1:20000 to each well, incubate at 37°C for 30 minutes, then wash the plate. Add 50 μl of chromogenic reagent A and B to each well sequentially, vortex to mix, incubate at 37°C in the dark for 15 minutes, then add 50 μl of 10% hydrochloric acid solution as stop solution. Set the microplate reader to 450 nm and measure the OD value of each well. Sample blocking rate = (OD value of negative control wells - OD value of sample wells) / OD value of negative control wells. Multiple subclonings were performed on the cell wells with a screening blocking rate higher than 50%, ultimately yielding one hybridoma cell line (3A3).

[0134] Ascites fluid was prepared from one hybridoma cell line (3A3), and the ascites fluid was diluted 100 times. The blocking rate of the antibody was determined by a positive serum blocking test. The result showed that the blocking rate of the monoclonal antibody 3A3 was 94%.

[0135] 3.2 Identification of Monoclonal Antibody Subclasses

[0136] The subclasses of monoclonal antibody 3A3 were identified using a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclass was IgG1 and the light chain subclass was kappa.

[0137] 3.3 Identification of monoclonal antibodies by Western blot

[0138] First, ASFV pB602L protein was subjected to SDS-PAGE electrophoresis. After transfer to a membrane, Western blot was performed using a diluted monoclonal antibody 3A3 as the primary antibody and a diluted HRP-labeled goat anti-mouse IgG as the secondary antibody. Results: Monoclonal antibody 3A3 reacted with pB602L protein to produce a specific band, indicating that monoclonal antibody 3A3 can recognize pB602L protein.

[0139] 3.4 Identification of the reactivity of monoclonal antibodies with ASFV antigen

[0140] ASFV antigen was diluted 1:1600 and used to coat an antigen plate. Monoclonal antibody 3A3 dilution was added as the primary antibody, and HRP-labeled goat anti-mouse IgG dilution was added as the secondary antibody for indirect ELISA detection. Results: The OD of the reaction between monoclonal antibody 3A3 and ASFV antigen was... 450nm The value is 1.108, indicating that monoclonal antibody 3A3 can react with ASFV antigen.

[0141] 3.5 Monoclonal antibody specificity identification

[0142] Classical swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, and porcine parvovirus were prepared into IFA antigen plates. After fixation with 80% cold acetone, the plates were warmed, washed once with PBS buffer, and then monoclonal antibody 3A3 was added. The plates were then detected using the conventional IFA method. The results showed that no yellow-green fluorescence was observed in the cell wells inoculated with monoclonal antibody 3A3 for different viruses, indicating that monoclonal antibody 3A3 did not react with other porcine viruses and had good specificity.

[0143] Example 4: Establishment of an ASFV pB602L protein blocking ELISA antibody detection kit

[0144] 4.1 Preparation and identification of enzyme-labeled antibodies

[0145] 4.1.1 Preparation

[0146] Horseradish peroxidase (HRP) labeling of monoclonal antibody 3A3 was performed using a modified sodium periodate method. 20 mg of horseradish peroxidase (HRP) was dissolved in 1 ml of ultrapure water, and 1 ml of freshly prepared NaIO4 solution (30 mg NaIO4 dissolved in 1 ml of ultrapure water) was added. The mixture was incubated at 4°C in the dark for 30 minutes. 40 μl of ethylene glycol was added to the above solution, and the mixture was incubated at 4°C in the dark for 30 minutes. Following this, 100 μl of the above mixture was added to 1 mg of purified monoclonal antibody, and the mixture was added to a dialysis bag. After mixing, the mixture was dialyzed against CB buffer for 6 hours. The entire procedure was performed in the dark. The dialyzed mixture was transferred to a 1.5 ml EP tube, and 10 μl of freshly prepared NaBH4 solution (20 mg NaBH4 dissolved in 1 ml of ultrapure water) was added. The mixture was incubated at room temperature for 2 hours, mixing every 30 minutes. An equal volume of saturated ammonium sulfate was added, and the mixture was incubated at 4°C for 15 minutes. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant. Resuspend the precipitate in a mixture of PBS buffer and glycerol (V:V=1:1) equal in volume to the purified antibody.

[0147] 4.1.2 Identification

[0148] Appearance: At room temperature, it is a reddish-brown liquid with no flocculent precipitate observed.

[0149] Quality evaluation: The enzyme-labeled antibody was diluted 10-fold and its absorbance (A) at 403 nm and 280 nm was measured using a UV spectrophotometer. The corresponding enzyme parameters were calculated according to the formula:

[0150] Enzyme amount Dilution factor.

[0151] IgG amount Dilution factor.

[0152] molar ratio quantity.

[0153] .

[0154] The specific results after absorbance detection and calculation are shown in Table 1:

[0155] Table 1 Quality evaluation of enzyme-labeled antibody 3A3

[0156] 4.2 Preparation of ASFV pB602L protein blocking ELISA antibody detection kit

[0157] Antigen-coated plate: The antigen prepared in Example 1 was coated with carbonate buffer (pH 9.6, 0.05 mol / L). Dilute the protein to 1.6 μg / ml for coating, 100 μl / well, and incubate at 2–8 °C for 16–24 hours. After washing with washing buffer, add blocking buffer (weigh 10 g bovine serum albumin, 50 g sucrose, and 1 ml Proclin 300, dissolve in PBS buffer (0.01 mol / L, pH 7.4) and bring the volume to 1000 ml) at 2–8 °C for 16–24 hours. Discard the blocking buffer, dry, seal, and store at 2–8 °C for later use.

[0158] Enzyme-labeled reagent: The enzyme-labeled monoclonal antibody 3A3 prepared in Example 4.1.1 was diluted with enzyme-labeled dilution buffer (weigh 200 ml newborn calf serum, 1 ml Proclin 300, 0.5 ml Tween 20, 0.04 g AM dye, dissolved in PBS buffer (0.01 mol / L, pH 7.4) and brought to a final volume of 1000 ml) and stored at 2–8 °C.

[0159] Positive control: Take 25ml Positive serum from protein-immunized pigs, 200 ml of newborn calf serum, and 1 ml of Proclin 300 were diluted to 1000 ml with PBS buffer (0.01 mol / L, pH 7.4), mixed well, filtered through a 0.22 μm filter, and aseptically aliquoted as positive controls. Store at 2–8 °C.

[0160] Negative control: Take 25 ml of negative serum from healthy pigs, 200 ml of newborn calf serum, and 1 ml of Proclin 300, and bring the volume to 1000 ml with PBS buffer (0.01 mol / L, pH 7.4). Mix well and filter through a 0.22 μm filter. Aseptically aliquot the mixture as a negative control and store at 2–8 °C.

[0161] Sample dilution solution: Take 8g of sodium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 200ml of newborn calf serum, and 1ml of Proclin 300. Dissolve them in purified water and bring the volume to 1000ml. Mix well and filter through a 0.22μm filter. Aseptically dispense and store at 2-8℃.

[0162] Concentrated washing solution: Dissolve 160g sodium chloride, 58g disodium hydrogen phosphate, 4.8g potassium dihydrogen phosphate, 4g potassium chloride, and 10ml Tween 20 in purified water and bring the volume to 1000ml. Mix well and filter through a 0.22μm filter. Aseptically dispense and store at 2–8℃. Dilute 20 times with purified water before use.

[0163] Colorimetric solution A: Dissolve 14.7g of disodium hydrogen phosphate, 9.3g of citric acid, and 0.3g of urea peroxide in purified water and bring the volume to 1000ml. Mix well and dispense into containers. Dissolve 0.2g of 3,3',5,5'-tetramethylbenzidine (TMB) and 10ml of anhydrous ethanol in purified water and bring the volume to 1000ml. Mix well and dispense into containers.

[0164] Termination solution: 10% hydrochloric acid solution.

[0165] The antigen-coated plate, enzyme-labeled reagent, positive control, negative control, and sample diluent were prepared. Concentrated washing solution, colorimetric solutions A and B, and stop solution were assembled into an African swine fever virus pB602L protein blocking ELISA antibody detection kit.

[0166] 4.3 Establishment of Detection Methods

[0167] The testing steps are as follows:

[0168] (1) Sample addition: Dilute the sample to be tested by 2 times with the sample diluent and add it to the corresponding wells, 100 μl / well. At the same time, set up 2 positive control wells, 2 negative control wells, and 1 blank control well (blank control can be omitted for dual-wavelength detection). After sealing with sealing film, incubate at 37°C for 45 minutes.

[0169] (2) Washing: Wash with detergent 3 times, and dry after the last wash.

[0170] (3) Add enzyme-labeled reagent: except for the blank control well, add 100 μl of enzyme-labeled reagent to each well, seal the plate with sealing film and incubate at 37°C for 30 minutes.

[0171] (4) Washing: Wash 3 times with detergent, and dry after the last wash.

[0172] (5) Color development: Add 50µl of color development solution A and color development solution B to each well in sequence, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0173] (6) Termination: Add 50µl of stop solution to each well and measure the results with an enzyme-linked immunosorbent assay (ELISA) reader within 10 minutes.

[0174] (7) Measurement:

[0175] When performing dual-wavelength measurements, it is necessary to set the dual wavelengths to 450nm / 600-650nm and measure the A value of each well.

[0176] For single-wavelength assays, the microplate reader was set to 450 nm, and the A value of each well was measured after zeroing the instrument with a blank control well.

[0177] (8) Result determination:

[0178] Calculation method: S / N value = Sample A value / Mean A value of negative control.

[0179] Judgment method: When When the S / N ratio is greater than 0.5, the sample is positive; when the S / N ratio is greater than 0.5, the sample is negative.

[0180] 4.4 Evaluation of the African swine fever virus pB602L protein blocking ELISA antibody detection kit

[0181] 4.4.1 Specificity

[0182] The kit prepared in Example 4.2 was used to test 8 positive serum samples of common porcine viruses (including positive serum samples of classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine parvovirus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine rotavirus), 20 SPF porcine serum samples, and 50 routinely immunized porcine serum samples that were negative for ASFV antigen. The results showed that the S / N values ​​were all >0.5, indicating that all samples were negative, demonstrating that the kit had good specificity.

[0183] 4.4.2 Sensitivity

[0184] The ASF standard positive serum serially diluted with the kit prepared in Example 4.2 was tested, and the results were: 1:16 dilution was positive and 1:32 dilution was negative.

[0185] The kit prepared in Example 4.2 was used for detection. Twenty positive serum samples from protein-immunized pigs were obtained, and all were positive.

[0186] 4.4.3 Clinical Application

[0187] The kit prepared in Example 4.2 was used for detection. Twenty-one serum samples from three pigs were immunized with protein. Results: Two-thirds of the samples turned positive two weeks after immunization, and all of them turned positive three weeks after immunization.

[0188] The kit prepared in Example 4.2 was used in clinical applications to test 20 ASF-positive swine serum samples and 1000 negative swine serum samples collected before 2018. The results showed that the S / N values ​​of the 20 ASF-positive swine serum samples were all <0.5, indicating that they were all positive; the S / N values ​​of the 1000 negative swine serum samples collected before 2018 were all >0.5, indicating that they were all negative.

[0189] Example 5: Sequencing of the variable region of the ASFV pB602L protein monoclonal antibody 3A3

[0190] Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed:

[0191] F: 5'-ACTAGTCGACATGAAATGCAGCT-3' (SEQ ID No. 6);

[0192] R: 5'-CCAGGGRCCARKGGATARACN-3' (SEQ ID No. 7);

[0193] Design primer sequences for the light chain variable region:

[0194] F: 5'-ACTAGTCGACATGGAGWCAGACA-3' (SEQ ID No. 8);

[0195] R: 5'-CCCAAGCTTACTGGATGGTG-3' (SEQ ID No. 9);

[0196] “R” represents A or G, “K” represents G or T, “N” represents any one of A, C, G or T, and “W” represents A or T.

[0197] Hybridoma cells were collected, RNA was extracted and reverse transcribed to serve as a template, and the variable region sequence was amplified using the primers described above. The amplified product was sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. Results: The amino acid sequences of the heavy chain variable region and light chain variable region of the monoclonal antibody 3A3 were determined as shown in SEQ ID No. 2 and SEQ ID No. 4, respectively, and the gene sequences are shown in SEQ ID No. 3 and SEQ ID No. 5, respectively.

[0198] SEQ ID No. 2:

[0199] EVQLQQSGAELVKPGASVKLSCSVSGFNIKDTYVHWVKQRPEQGLEWIGRIDPAIGDTKFAPNFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARDYDWYFDVWGAGTTVTVSS.

[0200] SEQ ID No. 3:

[0201] GAGGTTCAGCTGCAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCTCAGTTTCTGGCTTCAACATTAAAGACACCTATGTACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGATTGGTGATACTAAATTTGCCCCGAACTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTTCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGGGATTACGATTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。

[0202] SEQ ID No.4:

[0203] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHNYGAYTFGGGTKLEIK。

[0204] SEQ ID No.5:

[0205] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACAATTATGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA。

[0206] Unless otherwise defined, all technical and scientific terms used throughout this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. In case of any inconsistency, the meaning as stated in this application or derived from the content described herein shall prevail. Furthermore, the terminology used in this description is for the purpose of describing embodiments of this application only and is not intended to limit this application.

[0207] Note that the above are merely preferred embodiments and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the technical concept of this application, all of which fall within the scope of protection of this application.

Claims

1. A truncated fragment of the pB602L protein of African swine fever virus, named Its characteristics are, The truncated fragment has an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO.

1.

2. An antibody or antigen-binding fragment thereof that specifically binds to the African swine fever virus pB602L protein, characterized in that, The antibody or its antigen-binding fragment comprises: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.2 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

4.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment; Optionally, the antibody is a monoclonal antibody 3A3, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

4. A biomaterial relating to the antibody or its antigen-binding fragment as described in claim 2 or 3, characterized in that, The biomaterial is any one of the following: (a) A nucleic acid molecule containing a sequence encoding a heavy chain variable region and / or a light chain variable region encoding the antibody or an antigen-binding fragment thereof; (b) Expression cassette containing the nucleic acid molecules in (a); (c) A recombinant vector containing the nucleic acid molecule in (a) or the expression cassette in (b); (d) Recombinant host cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

5. The biomaterial according to claim 4, characterized in that, The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.3; and / or the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.

5.

6. The application of the truncated fragment of claim 1, the antibody of claim 2 or 3, or its antigen-binding fragment, or the biological material of claim 4 or 5 in the preparation of African swine fever virus ELISA antibody detection products; Optionally, the product is an African swine fever virus indirect ELISA antibody detection kit or an African swine fever virus blocking ELISA antibody detection kit.

7. An indirect ELISA antibody detection kit for African swine fever virus, characterized in that, The kit comprises: a support medium coated with the truncated fragment of claim 1, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled anti-pig IgG antibody.

8. The reagent kit according to claim 7, characterized in that, The The coating concentration is 0.6-1.0 μg / mL, preferably 0.8 μg / mL; Optionally, the support medium is a microtiter plate; Optionally, the enzyme labeled in the enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase. Optionally, the detection reagent includes a colorimetric solution and a stop solution; Optionally, the kit may further include at least one of a positive control, a negative control, a washing solution, and a sample diluent.

9. An African swine fever virus blocking ELISA antibody detection kit, characterized in that, The kit comprises: a support medium coated with the truncated fragment of claim 1, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody or its antigen-binding fragment as described in claim 2 or 3.

10. The reagent kit according to claim 9, characterized in that, The enzyme-labeled reagent is an enzyme-labeled monoclonal antibody 3A3; Optionally, the The coating concentration is 1.2-1.8 μg / mL, preferably 1.6 μg / mL; Optionally, the support medium is a microtiter plate; Optionally, the enzyme labeled in the enzyme labeling reagent is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase. Optionally, the detection reagent includes a colorimetric solution and a stop solution; Optionally, the kit may further include at least one of a positive control, a negative control, a washing solution, and a sample diluent.

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