Antibody specifically binding to porcine parvovirus VP2 protein or antigen binding fragment thereof and application thereof

Abstract

The invention relates to the technical field of immunology and veterinary diagnosis, and particularly provides an antibody specifically bound with porcine parvovirus VP2 protein or an antigen binding fragment thereof and application of the antibody or the antigen binding fragment. The antibody or the antigen binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3, and has high specificity and affinity to PPV VP2 protein. The invention also provides a nucleic acid molecule for coding the antibody, an expression cassette, a recombinant vector and a recombinant cell. The coincidence rate of a porcine parvovirus blocking ELISA antibody detection kit and a blocking protein chip antibody detection kit established on the basis of the antibody exceeds 95% compared with a traditional hemagglutination inhibition method, and the porcine parvovirus blocking ELISA antibody detection kit and the blocking protein chip antibody detection kit have good specificity, repeatability and stability, are suitable for high-flux and rapid detection of the porcine parvovirus antibody, and have wide application prospects. And an effective technical tool is provided for epidemic disease diagnosis and vaccine immune effect evaluation.

Description

Technical Field

[0001] This application relates to the fields of immunology and veterinary diagnostic technology, specifically providing an antibody or antigen-binding fragment thereof that specifically binds to porcine parvovirus VP2 protein and its application. Background Technology

[0002] Porcine parvovirus (PPV) belongs to the family Parvoviridae and the genus Parvovirus. It is a non-enveloped, single-stranded DNA virus. The viral genome is approximately 5 kb in length and contains two major open reading frames (ORFs) encoding the structural proteins VP1, VP2, and VP3. Among them, the VP2 protein is a major component of the viral capsid and a key protein determining the spatial conformation of the viral particle. Studies have shown that the VP2 protein has good immunogenicity and self-assembly capabilities, and can form virus-like particles in in vitro expression systems. Its conformational epitopes can effectively mimic natural viral particles, inducing high levels of neutralizing antibodies in animals. Therefore, the VP2 protein is considered a core target antigen for the development of PPV diagnostic reagents and the design of subunit vaccines.

[0003] Currently, laboratory diagnostic methods for PPV mainly include virus isolation, hemagglutination inhibition assay, immunofluorescence assay, and enzyme-linked immunosorbent assay (ELISA). While virus isolation is considered the "gold standard," it is complex, time-consuming, and requires cell culture, making it unsuitable for rapid clinical screening. The hemagglutination inhibition assay is a commonly used serological method, but it relies on guinea pig erythrocytes, involves cumbersome serum pretreatment steps, is susceptible to subjective interpretation, and struggles with high-throughput detection. Furthermore, while polyclonal antibody-based ELISA methods have improved detection efficiency, they still suffer from low antibody specificity, significant batch-to-batch variability, and susceptibility to cross-reactivity with other porcine viruses, affecting the accuracy and reliability of test results.

[0004] Therefore, developing highly specific and high-affinity monoclonal antibodies against the PPV VP2 protein, and establishing standardized and scalable immunoassay methods based on these antibodies, has become an urgent need for the development of PPV diagnostic technology. Summary of the Invention

[0005] One of the purposes of this application is to provide antibodies that specifically bind to porcine parvovirus VP2 protein or antigen-binding fragments thereof and their applications, so as to provide monoclonal antibodies with high affinity that specifically bind to PPV VP2 protein and their derivative tools.

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

[0007] An antibody or antigen-binding fragment thereof that specifically binds to porcine parvovirus VP2 protein, said antibody or antigen-binding fragment comprising: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO.1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.3.

[0008] Furthermore, the antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment.

[0009] Furthermore, the antibody is a monoclonal antibody 3C3, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

[0010] Furthermore, the antibody is a single-chain antibody, wherein the heavy chain variable region and the light chain variable region are linked by a linker peptide.

[0011] Biological materials related to the antibodies or antigen-binding fragments of this application, wherein the biological materials are any 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 eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c);

[0016] (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0017] Furthermore, the nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.2; and / or

[0018] The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.4.

[0019] The application of this application is in the preparation of products for detecting porcine parvovirus;

[0020] Optionally, the product is a blocking ELISA antibody detection kit or a blocking protein chip antibody detection kit.

[0021] A porcine parvovirus blocking ELISA antibody detection kit includes: a support medium coated with PPV VP2 protein, 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.

[0022] Furthermore, the coating concentration of the PPV VP2 protein is 0.4~0.8 μg / mL;

[0023] Optionally, the support medium is a microtiter plate;

[0024] Optionally, the enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0025] Optionally, the detection reagent includes a colorimetric solution and a stop solution.

[0026] A porcine parvovirus blocking protein chip antibody detection kit includes: a chip substrate spotted with PPV VP2 protein and quality control protein, enzyme-labeled reagent, washing solution, colorimetric solution and result interpretation system, wherein the enzyme-labeled reagent is an enzyme-labeled antibody of this application or its antigen-binding fragment;

[0027] Optionally, the chip substrate is a nanofilm;

[0028] Optionally, the concentration of the PPV VP2 protein used for spotting is 0.8~1.2 mg / mL, and the spotting volume is 15~25 nL / spot.

[0029] Optionally, the quality control protein is goat anti-mouse IgG;

[0030] Optionally, the working concentration of the enzyme-labeled antibody in the enzyme-labeled reagent is 1:15000 to 1:25000.

[0031] The technical effects of this application are as follows:

[0032] This application provides a high-affinity antibody or its antigen-binding fragment against the VP2 protein of porcine parvovirus (PPV), exhibiting significant comprehensive technical advantages in the serological detection of PPV. It specifically recognizes key antigenic epitopes on the PPV VP2 protein, has strong binding ability to viral particles, and shows no cross-reactivity with other common porcine viruses, laying the foundation for high-accuracy detection. The blocking ELISA and blocking protein chip detection methods established based on this antibody or its antigen-binding fragment benefit from the excellent blocking performance of the antibody or its antigen-binding fragment, achieving highly sensitive and specific detection of specific antibodies in PPV-infected serum. Compared with traditional methods such as the hemagglutination inhibition (HI) test, the detection protocol of this application eliminates the dependence on guinea pig erythrocytes, significantly simplifies the operation process, facilitates standardization, and is more suitable for large-scale, high-throughput screening of clinical samples. The developed kit has good reproducibility and batch-to-batch stability, and the detection results are objective and reliable, with high concordance with classical methods, providing an efficient and reliable new diagnostic tool for the epidemiological investigation, vaccination efficacy evaluation, and population eradication of porcine parvovirus. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0037] Unless otherwise specified, all percentage concentrations mentioned in this application are mass / volume percentages (w / v) or volume / volume percentages (v / v), and the concentration ranges include endpoint values ​​and all subranges within that range.

[0038] "Antibody" refers to an immunoglobulin molecule that can specifically bind to antigens. Its basic structural unit consists of two heavy chains and two light chains linked by disulfide bonds, and each chain contains a variable region and a constant region. This term encompasses complete antibody molecules, their functional fragments (such as antigen-binding fragments), derivatives (such as chimeric and humanized antibodies), and polypeptides containing antigen-binding domains.

[0039] "Monoclonal antibody" refers to a group of substantially homologous antibodies produced by a single B cell clone, whose amino acid sequences are essentially identical. The monoclonal antibody of this application specifically refers to an antibody against the porcine parvovirus VP2 protein, and more particularly to an antibody secreted by hybridoma cell line 3 that has specific variable region sequences (SEQ ID NO. 1 and 3).

[0040] "Heavy chain variable region (VH)" and "light chain variable region (VL)" refer to the N-terminal domains responsible for antigen recognition in the antibody heavy and light chains, respectively. VH and VL together constitute the antigen binding site through their respective complementarity-determining regions. In this application, the VH sequence shown in SEQ ID NO.1 and the VL sequence shown in SEQ ID NO.3 are specifically referred to.

[0041] "Antigen-binding fragments" refer to antibody fragments that contain the complete variable region (VH and / or VL) of the antibody, capable of specifically binding to antigens but lacking some or all of the constant region. Examples include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single-chain antibodies. These fragments can be obtained by enzymatic digestion of complete antibodies or recombinant DNA technology.

[0042] Fab fragment: A monovalent antigen-binding fragment consisting of a light chain (containing a variable region and a constant region) and the N-terminal portion of a heavy chain (containing the heavy chain variable region and the first constant region CH1).

[0043] Fab' fragment: A monovalent antigen-binding fragment that includes a portion of the heavy chain hinge region sequence on the Fab basis, containing one or more cysteine ​​residues from the hinge region.

[0044] F(ab')2 fragment: A bivalent antigen-binding fragment formed by two Fab' fragments linked by disulfide bonds in the hinge region, containing two antigen-binding sites.

[0045] scFv fragment: Single-chain variable region fragment, a single polypeptide chain formed by directly linking the heavy chain variable region and the light chain variable region through an artificially designed linker peptide, retaining the antigen-binding specificity of the complete antibody.

[0046] Fv fragment: The smallest antigen-binding unit formed by the non-covalent binding of the heavy chain variable region and the light chain variable region.

[0047] "Linker peptides" are short peptide sequences used to link the heavy chain variable region and the light chain variable region in single-chain antibody forms such as scFv. They are usually composed of repeating units of flexible amino acid residues (such as glycine and serine) to provide sufficient degrees of freedom for the two domains.

[0048] "Nucleic acid molecule" refers to deoxyribonucleic acid or ribonucleic acid encoding the antibody or its antigen-binding fragment (especially its heavy chain variable region and / or light chain variable region) described in this application, including but not limited to cDNA, synthetic DNA or optimized gene sequences.

[0049] The “expression cassette” comprises the nucleic acid molecule described in this application and a DNA construct operatively linked thereto with regulatory sequences (such as promoters, terminators, enhancers, signal peptide sequences, polyadenylation signals) necessary to control its transcription and / or translation.

[0050] "Recombinant vector" refers to a recombinant nucleic acid molecule obtained by inserting the nucleic acid molecule or expression cassette of this application into a suitable vector backbone (such as a plasmid, bacteriophage, or viral vector) for the delivery and expression of the nucleic acid sequence.

[0051] "Recombinant host cells" are eukaryotic or prokaryotic cells introduced into the nucleic acid molecules, expression cassettes, or recombinant vectors of this application through transfection, transformation, or transduction, and can be used to produce the antibodies or their antigen-binding fragments.

[0052] "Blocking ELISA Antibody Detection Kit" refers to an enzyme-linked immunosorbent assay (ELISA) system based on the principle of antigen-antibody competitive binding. It contains essential components such as solid-phase coated antigen and enzyme-labeled antibody. In this method, the target antibody in the test sample competes with the enzyme-labeled antibody to bind to the solid-phase coated antigen. The degree of signal inhibition is positively correlated with the content or activity of the target antibody in the test sample; that is, the higher the content of the target antibody, the lower the colorimetric signal.

[0053] The "Blocking Protein Chip Antibody Detection Kit" is a solid-phase microarray detection system based on the competitive blocking immunoassay principle, used to detect porcine parvovirus (PPV) specific antibodies in samples. The kit uses a chip (e.g., a nanomembrane) containing PPV VP2 protein and a quality control protein (such as goat anti-mouse IgG) as a solid-phase carrier. Serum samples are simultaneously incubated with enzyme-labeled monoclonal antibodies (e.g., monoclonal antibody 3C3), allowing the sample antibody to compete with the enzyme-labeled antibody for binding to the VP2 protein sites on the chip. Higher serum antibody concentrations result in higher blocking rates and weaker colorimetric signals. The grayscale values ​​of the detection and control points are read using a microdisk chip imager, and the blocking rate is calculated (the formula is [1 - grayscale value of the detection point / average grayscale value of the control point] × 100%). A blocking rate ≥ 50% is considered antibody positive, and < 50% is considered negative. This technology platform combines the high throughput and low sample volume advantages of protein chips with the specificity and objectivity of blocking methods, making it suitable for antibody level screening and disease epidemiological investigations in large-scale pig farms.

[0054] The antibody or antigen-binding fragment thereof that specifically binds to porcine parvovirus VP2 protein provided in this application contains a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3.

[0055] QVQLQQSGAELVSPGTSVKLSCKTSGYTFTSYWIHWVKQRSGQGLEWIARIFPGTDTIQYTEKFDKVTLTADKSSSTAYMQLSSLTSEDSAVYFCARFYGDTGIVYWGQGTLVTVSA (SEQ ID NO. 1).

[0056] QIVLQSPAIMSASPGEKVTMTCSASSGVSYMYWYQQKPGSSPRLLIYATSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQWTYYPFTFGGGTE (SEQ ID NO. 3).

[0057] In some embodiments, the antibody or its antigen-binding fragment is a Fab, Fab', F(ab')2, scFv, or Fv fragment. In some embodiments, the antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 antibody or a fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is a monoclonal antibody or its antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody.

[0058] In a preferred embodiment, the antibody is monoclonal antibody 3C3 obtained in the embodiments of this application. The heavy chain subclass of monoclonal antibody 3C3 is IgG1, and the light chain subclass is kappa.

[0059] Antibodies are immunoglobulins (Igs) used by the immune system to recognize and neutralize foreign substances such as bacteria and viruses. Immunoglobulins are mainly classified into five types: IgA, IgD, IgE, IgG, and IgM. IgG antibodies have four subtypes: IgG1, IgG2, IgG3, and IgG4, with differences in the position and number of disulfide bonds between different subtypes. The light chain of immunoglobulin (Ig) is divided into two subtypes: κ (kappa) and λ (lambda). Experimental testing and identification have confirmed that the monoclonal antibody 3C3 protected in this application belongs to the IgG1 heavy chain subtype and the kappa light chain subtype.

[0060] In a preferred embodiment, the antibody is the single-chain antibody 3C3 obtained in the embodiments of this application, and the heavy chain variable region and the light chain variable region of the single-chain antibody 3C3 are linked by a linker peptide.

[0061] Here, the heavy chain variable region and light chain variable region of the single-chain antibody 3C3 can be linked using linker peptides commonly used in the art.

[0062] This application also provides biological materials related to the antibody or its antigen-binding fragment of this application, wherein the biological material is any one of the following:

[0063] (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.

[0064] (b) Expression cassette containing the nucleic acid molecules in (a).

[0065] (c) A recombinant vector containing either the nucleic acid molecule in (a) or the expression cassette in (b).

[0066] (d) Recombinant eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0067] (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

[0068] In a preferred embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.2; preferably, the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4.

[0069] CAGGTCCAGCTGCAGCAGTCTGGAGCTGAACTGGTGAGTCCTGGGACTTCAGTGAAGCTGTCCTGCAAGACTTCTGGCTACACTTTCACCAGCTACTGGATTCACTGGGTAAAACAGAGGTCTGGACAGGGCCTTGAGTGGATTGCAAGGATTTTTCCTGGAACTGATACTATTCAG TACACTGAGAAGTTCGACAAGGTCACACTGACTGCAGACAAATCCTCCAGCACTGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGATTCTACGGCGATACCGGGATTGTTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO.2).

[0070] CAAATTGTTCTCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAGGTGTAAGTTACATGTACTGGTATCAGCAGAAGCCAGGATCCTCCCCAGACTCCTGATTTATGCCACATCCAAC CTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGACTTATTACCCGTTCACGTTCGGAGGGGGGACCGAG (SEQ ID NO.4).

[0071] This application also provides the use of the above-mentioned antibodies or their antigen-binding fragments or biological materials in the preparation of products for detecting porcine parvovirus.

[0072] In some embodiments, the product is a reagent or kit. The heavy chain variable region and light chain variable region provided in this application can specifically bind to porcine parvovirus. Therefore, porcine parvovirus can be detected using this property and commonly used detection methods in the art, and the product can be prepared accordingly. In a preferred embodiment, the kit is a porcine parvovirus blocking ELISA antibody detection kit or a blocking protein chip antibody detection kit. From the perspective of convenient detection and easy interpretation of detection results, the VP2 protein in the kit is pre-coated as an antigen onto a support medium. The specific support medium can be reasonably designed as needed, such as an ELISA plate (e.g., polystyrene material), a membrane carrier (e.g., nitrocellulose membrane, glass cellulose membrane, or nylon membrane), or microspheres.

[0073] This application also provides a porcine parvovirus blocking ELISA antibody detection kit, which includes: a support medium coated with PPV VP2 protein, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an enzyme-labeled antibody or its antigen-binding fragment provided in this application.

[0074] A blocking ELISA antibody detection kit constructed based on this antibody uses the VP2 protein expressed by baculovirus as a solid-phase antigen and an enzyme-labeled monoclonal antibody as a competing reagent. By optimizing the coating concentration and reaction system, effective detection of serum neutralizing antibodies was achieved. Compared with the traditional hemagglutination inhibition assay, this method eliminates the need for guinea pig red blood cell preparation, simplifying the sample pretreatment process. The detection results are objectively read by an ELISA reader, avoiding subjective errors from visual interpretation. The throughput is significantly improved, allowing for the simultaneous testing of at least 92 samples, making it suitable for disease monitoring and antibody screening in large-scale pig farms. The overall concordance rate with the hemagglutination inhibition method exceeds 95%, indicating that it can serve as a reliable alternative to existing gold standard methods.

[0075] In a preferred embodiment, the enzyme-labeled reagent is an enzyme-labeled monoclonal antibody 3C3.

[0076] In a preferred embodiment, the coating concentration of PPV VP2 protein is 0.4-0.8 μg / mL, preferably 0.5 μg / mL. Preferably, the supporting medium is a microtiter plate; preferably, the enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase.

[0077] It should be noted that the coating concentration of PPV VP2 protein can be, but is not limited to, 0.40 μg / mL, 0.41 μg / mL, 0.42 μg / mL, 0.43 μg / mL, 0.44 μg / mL, 0.45 μg / mL, 0.46 μg / mL, 0.47 μg / mL, 0.48 μg / mL, 0.49 μg / mL, 0.50 μg / mL, 0.51 μg / mL, 0.52 μg / mL, 0.53 μg / mL, 0.54 μg / mL, 0.55 μg / mL, 0.56 μg / mL, 0.57 μg / mL, 0.58 μg / mL, 0.59 μg / mL, etc. mL, 0.60μg / mL, 0.61μg / mL, 0.62μg / mL, 0.63μg / mL, 0.64μg / mL, 0.65μg / mL, 0.66μg / mL, 0.67μg / mL, 068μg / mL, 0.69μg / mL, 0.70 μg / mL, 0.71μg / mL, 0.72μg / mL, 0.73μg / mL, 0.74μg / mL, 0.75μg / mL, 0.76μg / mL, 0.77μg / mL, 0.78μg / mL, 0.79μg / mL or 0.80μg / mL.

[0078] In a preferred embodiment, the detection reagent includes a colorimetric solution and a stop solution. Preferably, the colorimetric solution includes colorimetric solution A and colorimetric solution B, wherein colorimetric solution A contains 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid, and 0.03% w / v urea peroxide, and colorimetric solution B contains 0.02% w / v tetramethylbenzidine and 10% v / v anhydrous ethanol. Preferably, the stop solution is a 2M H2SO4 solution.

[0079] In a preferred embodiment, the kit further includes at least one of a positive control, a negative control, a washing buffer, and a sample diluent. Preferably, the positive control is positive serum from VP2 protein-immunized pigs; preferably, the negative control is a PBS solution containing 20% ​​v / v newborn calf serum and 0.05% v / v Proclin 300; preferably, the washing buffer is phosphate buffer; preferably, the sample diluent is a PBS solution containing 20% ​​v / v newborn calf serum and 0.1% v / v Proclin 300.

[0080] This application also provides a porcine parvovirus blocking protein chip antibody detection kit, comprising: a chip substrate spotted with PPVVP2 protein and quality control protein, enzyme-labeled reagent, washing solution, colorimetric solution and result interpretation system, wherein the enzyme-labeled reagent is an enzyme-labeled antibody of this application or its antigen-binding fragment.

[0081] The protein chip detection kit further leverages the high specificity and high affinity of antibodies. By spotting micro-samples of VP2 protein onto a nanomembrane substrate and combining microfluidics and chip scanning technology, the detection system is miniaturized and integrated. This technology platform significantly reduces reagent and sample usage and shortens reaction time while maintaining detection sensitivity and specificity, and has higher potential for throughput expansion, providing a new technological pathway for high-throughput, rapid on-site detection.

[0082] Both detection kits exhibited good repeatability and stability, with intra- and inter-batch coefficients of variation both below 10%. They effectively detected positive sera at different dilutions, with detection titers comparable to hemagglutination inhibition methods. Furthermore, no cross-reactivity was observed in positive sera from various swine diseases, and the specificity met the quality requirements for diagnostic reagents. Overall, the antibodies and detection kits presented in this application provide a simple, sensitive, specific, and standardized technical tool for the clinical diagnosis of porcine parvovirus, evaluation of vaccine efficacy, and epidemiological investigations, demonstrating significant application value.

[0083] 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.

[0084] Example 1: Obtaining hybridoma cells

[0085] 1. Mouse immunization

[0086] Using PPV HN-2011 strain (disclosed in Chinese patent application CN102886043A) as a template, the PPV VP2 gene was amplified, and protein expression was performed using a baculovirus expression system. The protein was purified using affinity chromatography. The purified PPVVP2 protein was used as an immunogen to immunize 5-6 week old Balb / c mice via subcutaneous injection at multiple sites on the back, with each mouse receiving 100 μg. The initial immunization used Freund's complete adjuvant, followed by booster immunizations using Freund's incomplete adjuvant, with each immunization occurring two weeks apart. One week after the third immunization, mouse serum was collected to detect antibody titers (using indirect immunofluorescence). When the antibody titer was not lower than 1:6400, a pulse immunization was performed. To further improve antibody titers and increase the likelihood of obtaining positive clones, the pulse immunization involved tail vein injection of inactivated PPV HN-2011 strain antigen (with a pre-inactivation viral load of 10). 7.0 TCID 50 0.5 ml ( / ml) was administered as a shock immunization, and three days afterward, the spleen of the mouse was used for fusion with myeloma cells.

[0087] 2. Cell fusion

[0088] Mouse spleens were obtained under aseptic conditions and ground on a sterile 200-mesh sieve to obtain a spleen cell suspension. This suspension was mixed with myeloma cells at a ratio of 10:1. At 37°C, 1 ml of PEG 1500 was added within 1 minute, and the reaction was allowed to proceed for 1 minute. Then, 1 ml of preheated 37°C DMEM culture medium was added to terminate the reaction, and this process was repeated within 1 minute, followed by another 1 minute of incubation. This process was repeated 5 times (the fusion tube was shaken throughout the fusion process). The volume was then increased to 20 ml with preheated DMEM culture medium, and the mixture was incubated at 37°C for 15 minutes, followed by centrifugation at 800 rpm for 7 minutes. The supernatant was discarded, and the cells were resuspended in fetal bovine serum culture medium (with HAT selective medium). The cells were then added to 96-well plates at 50 μL per well and incubated at 37°C in a 5% CO2 incubator. On days 3 and 6, the medium was partially replaced with 20% fetal bovine serum culture medium (with HAT selective medium). When the hybridoma cells grew to approximately 1 / 5 of the bottom of the well, the supernatant was collected to detect specific antibodies.

[0089] 3. Screening hybridoma cells

[0090] Positive clones were screened using the PPV indirect immunofluorescence assay (IFA). The antigen plate preparation method for IFA detection is as follows: ST cells were seeded at 22,000-25,000 cells / well in a 96-well plate and cultured at 37°C in a 5% CO2 incubator until a basic monolayer was formed. Each well was then inoculated with 300 TCID50 of PPV HN-2011 virus strain. 50 100 μl / well was added to each PPV antigen plate, and the cells were cultured until the lesions reached 60%–80%. The cells were then fixed with 80% ice-cold acetone and stored at -20°C. After the PPV antigen plate was brought to room temperature, the hybridoma cell supernatant was examined. Hybridoma cells in wells showing specific fluorescence (positive wells) were cultured in 20% fetal bovine serum medium (with HT selective medium, Sigma), counted, and adjusted to 3–5 cells / ml. 100 μl of diluted cells was added to each well, followed by 100 μl of HT selective medium. The wells were then incubated at 37°C with 5% CO2. On days 3 and 6, the medium was partially changed to 20% fetal bovine serum medium (with HT selective medium, Sigma), and cell colony formation was observed. Antibody activity was promptly detected. After obtaining positive monoclonal cells from subcloning, they were passaged and expanded, and cultured at 2 × 10⁶ cells / well. 6 Each cell line was preserved in liquid nitrogen. Seven hybridoma cell lines were finally screened out and named hybridoma cell 1, hybridoma cell 2, hybridoma cell 3, hybridoma cell 4, hybridoma cell 5, hybridoma cell 6, and hybridoma cell 7, respectively.

[0091] Example 2: Preparation and Identification of Monoclonal Antibodies

[0092] 1. Preparation of monoclonal antibody ascites

[0093] Seven- to eight-week-old Balb / c mice were sensitized with sterile liquid paraffin. 0.5 ml per mouse was injected intraperitoneally over 10 to 18 days (approximately 3.0 × 10⁻⁶ ml). 6 Hybridoma cells (cells / mL) were injected. The mice's abdomen was gently massaged daily after injection until it became noticeably swollen laterally, at which point ascites was collected. The ascites was centrifuged at 10,000 rpm for 10 minutes, the supernatant was collected, aliquoted, and stored at -70°C.

[0094] 2. Hybridoma cell ascites titer determination

[0095] The prepared PPV antigen plates were brought to room temperature, and the titers of the prepared monoclonal antibodies 1, 2, 3, 4, 5, 6, and 7 were measured, yielding values ​​of 1:6400, 1:12800, 1:12800, 1:12800, 1:6400, 1:12800, and 1:3200, respectively. This demonstrates that monoclonal antibodies 1, 2, 3, 4, 5, 6, and 7 exhibit good reactivity with the virus and can be used to develop diagnostic reagents.

[0096] 3. Purification and content determination of monoclonal antibodies

[0097] Ascites fluid from mice containing hybridoma cells 1, 2, 3, 4, 5, 6, and 7 was purified by affinity chromatography. The specific procedure was as follows: The frozen ascites fluid sample was thawed, centrifuged at 10,000 rpm at 4°C for 10 min, and the clear liquid was aspirated. The solution was diluted with 3 column volumes of loading buffer (binding buffer formulation: 20 mM Na₂HPO₄, 0.15 M NaCl, pH 8.0) and purified using a Protein G affinity chromatography column. The column eluent was at pH 2.5. The eluted antibodies were immediately adjusted to neutral using a neutralization buffer (1M Tris-HCl, pH 8.5) in 0.1M glycine buffer, followed by SDS-PAGE gel analysis and protein content determination. The results showed that the purified monoclonal antibodies 1, 2, 3, 4, 5, 6, and 7 all had a purity greater than 90%, and protein contents of 3.8 mg / ml, 3.3 mg / ml, 3.5 mg / ml, 3.2 mg / ml, 3.6 mg / ml, 3.1 mg / ml, and 3.3 mg / ml, respectively, which meet the requirements for clinical application.

[0098] Example 3: Labeling and Activity Detection of Monoclonal Antibodies

[0099] 1. Horseradish peroxidase (HRP) labeling of monoclonal antibodies

[0100] Horseradish peroxidase (HRP) labeling was performed using the sodium periodate method, with the following steps: 2 mg HRP was dissolved in 0.5 ml of triple-distilled water; 0.5 ml of freshly prepared 0.06 mol / L NaIO4 solution was added to the supernatant and incubated at 4°C in the dark for 30 min; 0.5 ml of 160 mmol / L ethylene glycol was added to the supernatant and incubated at room temperature for 30 min; 2 mg of PPV monoclonal antibody was added to the supernatant, mixed well, and transferred to a treated dialysis bag. Dialysis was performed in 1000 ml of 0.05 mmol / L sodium carbonate buffer at 4°C overnight; the dialysate was transferred to a 10 ml centrifuge tube, 0.25 ml of freshly prepared 5 g / L NaBH4 solution was added, mixed well, and incubated at 4°C for 2 h; an equal volume of saturated ammonium sulfate solution was added, and the mixture was incubated at 4°C for 30 min, followed by centrifugation at 3000 r / min for 25 min at 4°C, and the supernatant was discarded; the precipitate was dissolved in 1.5 ml of 0.02 mol / L pH 7.4 solution. In PBS, aspirate into a dialysis bag and dialyze in 0.02 mol / L pH 7.4 PBS overnight at 4°C. Transfer the dialysate to a microcentrifuge tube and centrifuge at 10,000 r / min for 30 min at 4°C. Aspirate the supernatant, add an equal volume of glycerol, mix well, and store at -20°C for later use.

[0101] 2. Enzyme-labeled antibody paired detection

[0102] Positive sera immunized with PPV VP2 protein were selected and tested with different enzyme-labeled antibodies. The results are shown in Table 1.

[0103] Table 1 Monoclonal antibody pairing and activity assay

[0104] Note: + indicates positive, - indicates negative.

[0105] The results showed that enzyme-labeled monoclonal antibodies 3, 4, and 5 could be detected positively, with monoclonal antibody 3 showing the highest detected titer. Monoclonal antibody 3 (mAb 3) was named 3C3.

[0106] Example 4 Identification of Monoclonal Antibodies

[0107] 1. Subclass identification

[0108] The subclasses of monoclonal antibody 3C3 were identified using a monoclonal antibody subclass identification kit. The results showed that the heavy chain subclass of 3C3 was IgG1, and the light chain subclass was kappa.

[0109] 2. Western blot identification

[0110] First, PPV VP2 protein was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a membrane, Western blot was performed using a 3C3 dilution of monoclonal antibody as the primary antibody and a dilution of HRP-labeled goat anti-mouse IgG as the secondary antibody. The results showed that both the monoclonal antibody and PPV VP2 protein produced specific bands, indicating that the monoclonal antibody can recognize PPV VP2 protein.

[0111] 3. Specificity identification

[0112] Classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus, porcine circovirus type 2, porcine epidemic diarrhea virus, and porcine transmissible gastroenteritis virus were prepared into IFA antigen plates. After fixation with 80% cold acetone, the plates were air-dried and stored at -20°C for later use. Before use, each antigen plate was warmed to room temperature, washed once with PBS, and then diluted with monoclonal antibody 3C3. The plates were then detected using the standard IFA method. Results showed that no specific fluorescence was observed in the cell wells inoculated with different viruses containing monoclonal antibody 3C3, indicating that monoclonal antibody 3C3 did not react with other porcine viruses and exhibited good specificity.

[0113] Example 5: Preparation and Identification of a Blocking ELISA Antibody Detection Kit

[0114] 1. Preparation of the blocking ELISA antibody detection kit

[0115] Using purified PPV VP2 protein expressed by baculovirus as the coating antigen, an ELISA method for blocking PPV monoclonal antibody 3C3 labeled with HRP was established. The working concentration of PPV VP2 protein was determined to be 0.5 μg / mL by checkerboard titration, the optimal working concentration of enzyme-labeled monoclonal antibody 3C3 was 1:2000, and the optimal serum dilution was 1:10.

[0116] Coating: PPV VP2 protein was added at 0.5 μg / mL, gently shaken to mix, and coated overnight at 2-8°C;

[0117] Sealing: Discard the liquid in the plate, wash 3-5 times, pat dry, add 100μl of 1.5% BSA to each well, gently shake to mix, seal the plate with sealing film and incubate at 37℃ for 2h;

[0118] Sample addition: Discard the liquid in the plate, wash 3-5 times, pat dry, add 100 μl of negative control (repeat 2 wells), 100 μl of positive control (repeat 2 wells), and 100 μl of serum to be tested diluted 1:10, gently shake to mix, seal the plate with sealing film and incubate at 37°C for 1 h;

[0119] Add enzyme-labeled antibody: Discard the liquid in the plate, wash 3-5 times, pat dry, add 100 μl PPV monoclonal antibody 3C3 (1:2000) to each well, gently shake to mix, seal the plate with sealing film and incubate at 37°C for 1 h;

[0120] Color development: Discard the liquid in the plate, wash 3-5 times, pat dry, add 50 μl of color development solution A (containing 1.47% w / v disodium hydrogen phosphate, 0.93% w / v citric acid and 0.03% w / v urea peroxide) and color development solution B (0.02% w / v tetramethylbenzidine and 10% v / v anhydrous ethanol) to each well, and incubate at 37°C in the dark for 10-15 min;

[0121] Termination: Add 50 μl of stop solution (2M H2SO4) to each well to terminate the reaction in the order of substrate addition, and read the OD value at a wavelength of 450-630 nm using a microplate reader.

[0122] Result determination: Blocking rate PI (%) = 1 - (OD of serum sample / mean OD of negative control) × 100%

[0123] A positive result is defined as a blocking rate ≥ 50%, while a negative result is defined as a blocking rate < 50%.

[0124] Trial criteria: Negative control OD value must be ≥1, positive control blockade rate >60%.

[0125] 2. Specificity identification of ELISA kits

[0126] Three consecutive batches of kits were used to test five PPV-negative porcine serum samples (1#~5#) and five PPV-negative guinea pig serum samples (6#~10#) to evaluate their specificity. The results are shown in Table 2.

[0127] Table 2 Results of ELISA kit detection of negative serum

[0128] Other serum samples that were positive for classical swine fever virus antibody, porcine reproductive and respiratory syndrome virus antibody, porcine circovirus type 2, pseudorabies virus, PEDV, and baculovirus were tested. The results are shown in Table 3.

[0129] Table 3 Specificity test results of ELISA kit

[0130] The kits from three consecutive batches all tested negative for 10 negative serum samples, proving that their specificity meets the requirements; the kits from three consecutive batches also tested negative for other common swine virus positive serum samples, indicating that the ELISA kits in this application do not cross-react with other viruses and that the kits have good specificity.

[0131] 3. Sensitivity determination of ELISA kits

[0132] PPV-positive swine serum was diluted 10-fold and then serially diluted 2-fold, and tested using the blocking kit prepared in this application to determine the sensitivity of the kit to positive control serum. The results are shown in Table 4.

[0133] Table 4 Sensitivity test for positive serum

[0134] The results showed that the kit achieved a detection titer of 1:320 when detecting serially diluted positive serum.

[0135] 4. Repeatability test of ELISA kit

[0136] One PPV-positive serum sample and one PPV-negative serum sample were taken. Three different batches of ELISA plates were used. Intra-batch replication assays were performed three times on one plate, and inter-batch replication assays were performed on different plates. OD450-630nm values ​​were measured, and the coefficient of variation (CV) was calculated. A CV < 10% indicates good repeatability and stability of the kit. Results are shown in Table 5. The formula for calculating the coefficient of variation (CV) is: CV (%) = (Standard deviation of OD450-630nm / Mean of OD450-630nm) × 100%.

[0137] Table 5 Repeatability test results

[0138] The results showed that the intra-batch and inter-batch detection results of different batches of ELISA plates were consistent, with coefficients of variation all <10%, indicating that the kit had good reproducibility.

[0139] 5. Comparison experiment between ELISA kit and PPV HI detection method

[0140] Eighty serum samples that tested positive for PPV HI and 25 serum samples that tested negative were compared using the blocking kit prepared in this application. The results are shown in Table 6.

[0141] Table 6. Comparison of detection results between ELISA and PPV HI detection methods.

[0142] The results showed that the ELISA method had a 96.2% concordance rate with the HI method, and could replace the HI method for detection.

[0143] Example 6: Preparation and Identification of Protein Chips

[0144] 1. Preparation of the microarray reagent kit

[0145] The microarray kit was prepared using purified PPV VP2 protein expressed with baculovirus, HRP-labeled PPV monoclonal antibody 3C3, and goat anti-mouse IgG. After screening, the optimal spotting volume was determined to be 20 nL / spot, the optimal spotting concentration of PPV VP2 protein was 1.0 mg / ml, the optimal spotting concentration of goat anti-mouse IgG was 0.2 mg / ml, the optimal working concentration of HRP-3C3 was 1:20000, and the optimal serum dilution factor was 10-fold.

[0146] Activation of nanofilms: Place the nanofilm in an activation tank containing an activation solution, ensuring that the entire surface of the nanofilm is immersed in the activation solution without the generation of bubbles. After soaking for 30 minutes, rinse three times with ultrapure water and then blow dry.

[0147] Spotting: Dilute PPV VP2 protein to 1 mg / ml with spotting diluent to prepare the detection spotting solution, and dilute goat anti-mouse IgG to 0.2 mg / ml with spotting diluent to prepare the quality control spotting solution. Use a spotting instrument to spot the samples onto the nanofilm at a spotting volume of 20 nL / spot according to the set matrix. The spotting environment temperature is 20-25℃ and the humidity is 40%-70%.

[0148] Assembly: Take out the spotted nanofilm, place it in the middle of the chip fixture base, cover it with the fixture cover, install the clamping strips on both sides, and assemble it into a PPV antibody detection chip.

[0149] Sealing: After the chip is assembled, add sealing solution, 200µl / well, and seal at 20-25℃ for 1 hour. Discard the sealing solution and pat dry on absorbent paper.

[0150] Sample addition: Add 300µl of washing buffer to each well, allow to stand at room temperature for 3 minutes to fully soak, then discard the washing buffer and pat dry on absorbent material. Pre-dilute the serum sample to be tested with sample diluent by 2 times, and add 50µl of the diluted sample to each well.

[0151] Add enzyme: Add 50 µl of enzyme-labeled reagent to each well. Incubate the microplate at 37°C and 1000 rpm for 30 minutes using a constant temperature shaker.

[0152] Washing: Vertically discard the reaction solution, add 300 μl of washing solution to each well, soak for 10–20 seconds, and discard the washing solution. Repeat this washing process 3 times consecutively, and on the last wash, wipe the plate as dry as possible on absorbent material.

[0153] Color development: Add 100µl of TMB substrate solution to each well of the chip and incubate in a microplate constant temperature oscillator at 37°C and 1000rpm for 15 minutes.

[0154] Measurement: Vertically discard the reaction solution, removing as much liquid as possible. Remove the chip cover and lay a lint-free paper flat on the surface of the chip nanofilm. Gently press and dab until there are no water marks on the surface of the nanofilm (do not rub the surface of the chip nanofilm back and forth with the lint-free paper). Measure the results using a microporous chip imager within 10 minutes.

[0155] Result determination

[0156] Test validity determination: The gray value of the quality control point should be between 15000 and 35000; otherwise, the test of that well is invalid.

[0157] Calculation method

[0158] The average gray value of each test well's quality control point needs to be calculated separately. The average gray value of the quality control point is calculated as follows: (gray value of quality control point 1 + gray value of quality control point 2 + gray value of quality control point 3) / 3.

[0159] Blocking rate = (1 - gray value of detection point / average gray value of quality control point) × 100%.

[0160] Judgment method: A blocking rate of ≥50% is considered as antibody positive.

[0161] A blocking rate of less than 50% is considered an antibody-negative result.

[0162] 2. Specificity identification of the microarray reagent kit

[0163] Three consecutive batches of the chip kit were used to detect five PPV-negative porcine serum samples (1#~5#) and five PPV-negative guinea pig serum samples (6#~10#) to evaluate their specificity. The results are shown in Table 7.

[0164] Table 7 Results of negative serum tests using the chip kit

[0165] Other serum samples that were positive for classical swine fever virus antibody, porcine reproductive and respiratory syndrome virus antibody, porcine circovirus type 2, pseudorabies virus, PEDV, and baculovirus were tested. The results are shown in Table 8.

[0166] Table 8. Specificity test results of the chip kit

[0167] All 10 negative serum samples tested by the three batches of kits were negative, proving that their specificity meets the requirements; all positive serum samples tested by the three batches of kits for other common swine virus diseases were negative; indicating that the protein chip kit of this application has no cross-reactivity with other viruses and the kit has good specificity.

[0168] 3. Sensitivity determination of chip reagent kits

[0169] PPV-positive swine serum was diluted 10-fold and then serially diluted 2-fold, and measured using the chip kit prepared in this application to determine the sensitivity of the kit to positive control serum. The results are shown in Table 9.

[0170] Table 9 Sensitivity test for positive serum

[0171] The results showed that the kit achieved a detection titer of 1:320 when detecting serially diluted positive serum.

[0172] 4. Repeatability test of the chip reagent kit

[0173] One sample each of PPV-positive and PPV-negative serum were collected. Inter-batch and intra-batch repeatability tests were performed using protein chips prepared from three different batches. The blocking rate was measured, and the coefficient of variation (CV) was calculated. A CV < 10% indicates good reproducibility and stability of the kit. The results are shown in Table 10.

[0174] Table 10 Repeatability Test Results

[0175] The results showed that the intra-batch and inter-batch detection results of different batches of protein chip kits were consistent, with coefficients of variation all <10%, indicating that the kits had good reproducibility.

[0176] 5. Comparison test between chip reagent kit and PPV HI detection method

[0177] Eighty serum samples that tested positive for PPV HI and 25 serum samples that tested negative were compared using the prepared chip kit. The results are shown in Table 11.

[0178] Table 11 Comparison of detection results between the chip kit and the PPV HI detection method

[0179] The results showed that the concordance rate between the chip kit and the HI method was 95.2%, and the chip kit can replace the HI method for detection.

[0180] Example 7: Determination of the variable region sequence of a monoclonal antibody

[0181] Based on the sequence characteristics of murine monoclonal antibodies, primer sequences for the heavy chain variable region were designed:

[0182] P1:5'-ACTAGTTGACGTGGTCTCTAGGGTCACTTAGTTTTCCT-3' (SEQ ID NO.5);

[0183] P2:5'-CGGAAGCTTCCAGCGRCCARKCCATATACIGRTGG-3' (SEQ ID NO.6);

[0184] Design primer sequences for the light chain variable region:

[0185] P3:5'-GCCATCTCATGRAGWCATTKWCYCAAGTCTTT-3' (SEQ ID NO.7);

[0186] P4:5'-CGGACGCTTACTGCCTGGTAAGAAGATGGA-3' (SEQ ID NO.8);

[0187] In the primer sequences above, "R" represents the base "A or G", "K" represents the base "G or T", "I" represents inosine nucleoside, "Y" represents the base "C or T", and "W" represents the base "A or T".

[0188] mRNA was extracted from hybridoma cells 3C3, reverse transcribed into cDNA, and then amplified by high-fidelity PCR using universal primers for the variable region. The PCR product was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for DNA sequencing. DNA sequencing results: The gene encoding the monoclonal antibody 3C3 was identified. The DNA sequence of the heavy chain variable region encoding gene is shown in SEQ ID No. 2 of the sequence listing, and the DNA sequence of the light chain variable region encoding gene is shown in SEQ ID No. 4 of the sequence listing. The amino acid sequences are shown in SEQ ID No. 1 and SEQ ID No. 3, respectively.

[0189] Example 8 Preparation and Identification of Single-Chain Antibody 3C3

[0190] The heavy chain variable region (VH) and light chain variable region (VL) genes of the monoclonal antibody were amplified, and after being transferred into a linker peptide, they were ligated into the prokaryotic expression vector pET-32a(+). Recombinant plasmids were constructed, transformed into BL21 competent cells for expression, and the fusion protein was obtained. The corresponding single-chain antibody 3C3 was prepared using the variable region sequence of the monoclonal antibody 3C3 as described in Example 7. The ELISA titer of 3C3 was determined according to Example 5. The results showed that the ELISA titer of the single-chain antibody was ≥1:12800, indicating good reactivity. IFA detection using a PPV antigen plate was positive, indicating that the single-chain antibody 3C3 can recognize PPV.

[0191] The results above show that the variable region sequences shown in SEQ ID No. 1, SEQ ID No. 3 or SEQ ID No. 2, SEQ ID No. 4 can be used for the preparation of porcine parvovirus genetically engineered antibodies.

[0192] 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.

[0193] 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. An antibody or antigen-binding fragment thereof that specifically binds to porcine parvovirus VP2 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.1 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO.

3.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is Fab, Fab', F(ab')2, scFv, or Fv.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody is a monoclonal antibody 3C3, with the heavy chain subclass being IgG1 and the light chain subclass being kappa.

4. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a single-chain antibody, wherein the heavy chain variable region and the light chain variable region are linked by a linker peptide.

5. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 4, 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 eukaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c); (e) Recombinant prokaryotic cells containing the nucleic acid molecule in (a), the expression cassette in (b), or the recombinant vector in (c).

6. The biomaterial according to claim 5, characterized in that, The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.2; and / or The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.

4.

7. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, or the biological material according to claim 5 or 6, in the preparation of products for detecting porcine parvovirus; Optionally, the product is a blocking ELISA antibody detection kit or a blocking protein chip antibody detection kit.

8. A porcine parvovirus blocking ELISA antibody detection kit, characterized in that, include: The accompanying contents include a support medium coated with PPV VP2 protein, an enzyme-labeled reagent, and a detection reagent, wherein the enzyme-labeled reagent is an antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

9. The reagent kit according to claim 8, characterized in that, The coating concentration of the PPV VP2 protein was 0.4~0.8 μg / mL; Optionally, the support medium is a microtiter plate; Optionally, the enzyme labeled is horseradish peroxidase, alkaline phosphatase, or β-D-galactosidase. Optionally, the detection reagent includes a colorimetric solution and a stop solution.

10. A porcine parvovirus blocking protein chip antibody detection kit, characterized in that, include: The chip substrate containing PPV VP2 protein and quality control protein, enzyme-labeled reagent, washing solution, colorimetric solution and result interpretation system are used for spotting. The enzyme-labeled reagent is an antibody or its antigen-binding fragment as described in any one of claims 1 to 4. Optionally, the chip substrate is a nanofilm; Optionally, the concentration of the PPV VP2 protein used for spotting is 0.8~1.2 mg / mL, and the spotting volume is 15~25 nL / spot. Optionally, the quality control protein is goat anti-mouse IgG; Optionally, the working concentration of the enzyme-labeled antibody in the enzyme-labeled reagent is 1:15000 to 1:25000.

Images