An antibody or antigen-binding fragment thereof targeting a conserved region of the GP5 protein of porcine reproductive and respiratory syndrome virus and its application.

Abstract

This invention relates to an antibody or antigen-binding fragment thereof targeting a conserved region of the porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein. The conserved region is selected from amino acids 32–101 and / or 125–199. The antibody comprises 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: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4. This antibody or its antigen-binding fragment comprises a heavy chain and a light chain and can specifically bind to the GP5 protein, and can be used to detect the presence or content of PRRSV GP5 protein in a sample.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an antibody or antigen-binding fragment thereof targeting a conserved region (32–101 aa and / or 125–199 aa) of the GP5 protein of porcine reproductive and respiratory syndrome virus, and its application. Background Technology

[0002] Porcine Reproductive and Respiratory Syndrome (PRRS), commonly known as blue ear disease in pigs, is a highly contagious disease caused by the PRRS virus (PRRSV). This virus can infect pigs of all ages through multiple routes, including contact and airborne transmission, but it is particularly harmful to pregnant sows and piglets. Clinical manifestations are mainly reproductive disorders in sows (such as abortion, stillbirth, mummified fetuses, and weak piglets) and respiratory symptoms in piglets, significantly increasing piglet mortality. PRRSV has significant immunosuppressive properties, targeting and destroying alveolar macrophages, leading to decreased immunity and susceptibility to secondary bacterial or viral infections, creating a vicious cycle of "immunosuppression—multiple infections." This causes significant economic losses to the global pig industry and severely restricts the sustainable development of intensive pig farming. Currently, PRRS prevention and control mainly relies on comprehensive measures centered on "vaccination + monitoring and eradication," with accurate and rapid diagnostic technology being key to effective monitoring and epidemic tracing.

[0003] While various diagnostic methods exist for porcine reproductive and respiratory syndrome (PRRS), each has its limitations. Virus isolation, considered the "gold standard" for etiological detection, yields accurate results, but its cumbersome and time-consuming process makes it difficult to meet the needs of rapid screening at the grassroots level. In molecular biology testing, techniques such as RT-PCR, real-time quantitative PCR, and loop-mediated isothermal amplification (LAMP), while possessing high sensitivity and specificity, rely on sophisticated instruments and specialized operation, making them unsuitable for on-site application in pig farms. Among serological tests, indirect immunofluorescence assays and virus neutralization assays, while specific, are limited by low throughput, high subjectivity, or the delayed production of neutralizing antibodies (1–2 months post-infection), hindering early diagnosis. In contrast, enzyme-linked immunosorbent assay (ELISA) has become the mainstream technology for PRRSV serological detection due to its high throughput, low cost, and ease of operation. However, current ELISA kits still face challenges such as single-target targeting, difficulty in distinguishing between wild-type virus infection and vaccine immunization, and reliance on imported core reagents, restricting their accurate application at the grassroots level. Therefore, developing PRRSV monoclonal antibodies with independent intellectual property rights can not only provide core raw materials for establishing detection methods such as sandwich ELISA and blocking ELISA with high sensitivity and specificity, but also is expected to break through existing technical bottlenecks in distinguishing between wild-type and vaccine strains and covering multiple genotype strains, providing key support for the development of domestically produced and precise rapid on-site detection technology for PRRSV.

[0004] PRRSV belongs to the genus *Pteroviravina* of the family *Pteroviravidae*. It is a single-stranded positive-sense RNA virus with a genome length of approximately 15 kb. The virus is mainly divided into two genotypes: PRRSV-1 (European type) and PRRSV-2 (American type). Currently, PRRSV-2 is the predominant strain in my country, and its strains can be further divided into several lineages, with lineage 1 (NADC30-like strains) being the currently dominant circulating strain. GP5 protein is one of the main structural proteins of PRRSV, encoded by ORF5 in the viral genome. It is a glycosylated envelope protein with a molecular weight of approximately 25 kDa. It plays a crucial role in viral infection, virulence, and the induction of neutralizing immune responses. The protein structure includes an N-terminal signal peptide, an extracellular region (containing a variable number of glycosylation sites), a hydrophobic transmembrane region (containing three transmembrane domains), and a C-terminal hydrophilic region. The glycosylation sites are important targets for inducing neutralizing antibodies during viral infection; the resulting antibodies can block the binding of the virus to host cell receptors (such as CD163), thereby inhibiting viral invasion. Although the GP5 protein has high genetic variability, with only 60%–80% amino acid homology between different genotypes and even subtypes, making detection methods targeting GP5 alone susceptible to false negatives due to strain variations, this protein still plays a key role in PRRSV-induced neutralizing antibodies, and the neutralizing epitopes it carries have a central position in antiviral immunity.

[0005] Therefore, GP5 protein remains a key indicator for assessing the body's antiviral immune response and has irreplaceable value in constructing multi-target detection systems and achieving "neutralization activity-related detection". Summary of the Invention

[0006] The present invention aims to provide an antibody or antigen-binding fragment thereof targeting a conserved region of the porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein; the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain and is capable of specifically binding to the GP5 protein, and can be used to detect the presence or content of PRRSV GP5 protein in a sample.

[0007] In a first aspect, to achieve the above objectives, the present invention provides an antibody or antigen-binding fragment thereof targeting a conserved region of the porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein, wherein the conserved region is selected from amino acids 32–101 and / or 125–199; the antibody comprises a heavy chain variable region and a light chain variable region, wherein 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.

[0008] Preferably, as an improvement, the antibody is a monoclonal antibody.

[0009] Preferably, as an improvement, the heavy chain subclass of the antibody is IgG1, and the light chain is κ type.

[0010] In a second aspect, the present invention provides a nucleic acid molecule encoding the antibody described in the first aspect, the nucleic acid molecule comprising a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 1; and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 3.

[0011] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0012] Preferably, as an improvement, the vector is a pET-32a(+) expression vector.

[0013] Fourthly, the present invention provides a host cell comprising the recombinant expression vector described in the third aspect.

[0014] Fifthly, the present invention provides a cell line capable of producing the antibody.

[0015] In a sixth aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment in the preparation of reagents or kits for detecting porcine reproductive and respiratory syndrome virus GP5 protein.

[0016] The beneficial effects of this invention are: This invention relates to antibodies or antigen-binding fragments targeting conserved regions (amino acids 32–101 and / or 125–199) of the porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein; these antibodies or antigen-binding fragments can specifically bind to the GP5 protein. The antibodies or antigen-binding fragments can be used to detect the presence or content of PRRSV GP5 protein in samples. The selected region exhibits high conservation across different PRRSV strains, thereby improving the stability and consistency of detection results and making it suitable for constructing GP5 protein-based immunoassay methods and related applications. Attached Figure Description

[0017] Figure 1 The image shows the SDS-PAGE results of imidazole eluted samples collected at different times in Example 1; where M is the protein molecular weight standard (Marker), 1 is the total protein sample of bacteria without induced expression, 2 is the total protein sample of bacteria after induced expression, and 3-9 are eluted samples collected at different times with a concentration of 250 mM imidazole. Figure 2 This is a graph showing the results of serum titer detection in mice after immunization in Example 2; Figure 3 This is a graph showing the results of ascites titer detection in mice after injection of GP5 cell line in Example 3; Figure 4 This is the result of SDS-PAGE of the purified GP5 monoclonal antibody in Example 3; where M is the protein molecular weight standard (Marker) and 1-3 are the purified GP5 monoclonal antibodies at different elution times. Figure 5 This is a graph showing the results of the GP5 monoclonal antibody specificity detection in Example 3. Detailed Implementation

[0018] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0019] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0020] Example 1: Obtaining recombinant protein of porcine reproductive and respiratory syndrome virus GP5-C1 In this embodiment, GP5-C1 recombinant protein was prepared. The specific preparation method includes the following steps: 1. Construction of recombinant expression vectors This study designed antigens based on the GP5 protein sequence of the porcine reproductive and respiratory syndrome virus (PRRSV) NADC30-like strain (GenBank accession number: QIC53138.1). Analysis showed that the GP5 protein is 199 amino acids (aa) in length, with a 31-amino acid signal peptide sequence at its N-terminus and three transmembrane domains, exhibiting high overall hydrophobicity.

[0021] Given that the full-length ORF5 gene sequence is not conducive to soluble expression when directly used in prokaryotes, this invention, based on epitope prediction results, truncates the GP5 protein sequence by removing the N-terminal signal peptide, the first transmembrane region (1–31 aa), and the last transmembrane region (102–124 aa). The antigenically active segments 32 aa–101 aa and 125 aa–199 aa are linked using a linker peptide (GGSGGS). Codon optimization is then performed on this sequence to obtain the target gene sequence, named GP5-C1. The GP5-C1 gene sequence was synthesized by Anshengda Biotechnology Co., Ltd., and cloned into the pET-32a(+) expression vector to construct the recombinant expression vector pET-32a-GP5-C1.

[0022] 2. Expression and purification of GP5-C1 protein After confirming the correct BL21(DE3) clone containing the recombinant expression vector pET-32a-GP5-C1, plasmids were extracted and transformed using standard methods. Single colonies were then picked and cultured in LB medium containing ampicillin resistance until the bacterial concentration reached A... 600 When the concentration was 0.5, expression was induced for 18 h with 0.1 mM IPTG at 200 rpm and 16℃. The cells were collected by centrifugation at 4℃ and 4000 rpm for 20 min, resuspended and washed with 20 mL PBS, and then sonicated in an ice bath with Binding buffer at 30 W. The sonication was repeated for 2 s with a 2 s interval until the bacterial solution was relatively clear. The supernatant was collected by centrifugation at 4℃ and 12000 rpm for 20 min for purification.

[0023] The target protein (GP5-C1 recombinant protein) was purified by nickel column affinity chromatography: A 50% NI-NTA column was packed, washed with 4 mL of deionized water, and then equilibrated with 5 mL of binding buffer. The lysate containing the GP5-C1 recombinant protein was added to the column, and the permeate was collected and re-loaded. Unbound proteins were washed away with binding buffer, followed by 20 mL of washing buffer. Proteins were then eluted with 250 mM imidazole elution buffer. The purity of the eluted sample was determined by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown: GP5-C1 recombinant protein with high purity was obtained by affinity chromatography. The concentration of purified GP5-C1 recombinant protein was determined by BCA method. The purified recombinant protein was cleaved using thrombin to remove the fusion tag, and then purified again by nickel column affinity chromatography. SDS-PAGE electrophoresis was used to detect the cleavage and purification effects. Figure 1 As shown, the content was determined.

[0024] Example 2: Establishment of a GP5-C1 recombinant protein monoclonal antibody cell line This embodiment prepared a GP5-C1 recombinant protein monoclonal antibody cell line. The specific preparation method includes the following steps: 1. Mouse immunization The GP5-C1 recombinant protein obtained in Example 1 was used as an immunogen to immunize Balb / c mice. For the first immunization (day 1 of the first immunization), 100 μg of the immunogen was emulsified with Freund's complete adjuvant at a 1:1 volume ratio, and injected subcutaneously into five immunization sites on the back and abdomen of each mouse. A second immunization was performed on day 15, using Freund's incomplete adjuvant emulsified with the immunogen at a 1:1 volume ratio, at a dose of 50 μg of the immunogen, following the same immunization method. A third immunization was performed on day 29, following the same method as the second immunization. On day 36, a small amount of tail blood was collected for ELISA testing; if the antibody titer was greater than 1:10000, a pulse immunization of 100 μg of the immunogen was administered intraperitoneally three days before cell fusion.

[0025] 2. Mouse serum titer detection (1) Antigen coating: The GP5-C1 recombinant protein obtained in Example 1 was adjusted to 1 μg / mL in the coating buffer. 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C.

[0026] (2) Washing: On the second day, discard the liquid in the well, pat dry, and wash twice with PBST in a plate washer.

[0027] (3) Blocking: Add 200 μL of blocking solution to each well and incubate at 37℃ for 1 h.

[0028] (4) Preparation of serum (primary antibody): Blood was collected from the tail of mice, and serum from unimmunized mice was used as a negative control.

[0029] (5) Add primary antibody: Use blocking solution to serially dilute the serum samples to be tested. Use non-immunized mouse serum diluted 1:2500 as a negative control and blocking solution as a blank control. Incubate at 37℃ for 1 h, discard the liquid in the well, pat dry, wash twice with PBST, and pat dry.

[0030] (6) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody (HRP-goat anti-mouse) 5000 times with blocking buffer, 100 μL / well, incubate at 37℃ for 1 h, discard the liquid in the well, and pat dry.

[0031] (7) Color development and measurement: Add 50 μL of TMB chromogenic solution to each well, incubate at 37℃ in the dark for 15 min, then add 100 μL of 2 M sulfuric acid to terminate the reaction. Measure the A value at 450 nm using a microplate reader. Calculation: The highest antiserum dilution factor when the ratio of the A value of the test well to the A value of the negative control well (P / N) is ≥2.1 is taken as the serum titer. Mice with a dilution greater than 1:10000 are prepared for the next fusion step. The results are as follows: Figure 2 As shown, the reaction between recombinant GP5-C1 protein and immune serum was identified by indirect ELISA, with a mouse serum titer of 1:64000.

[0032] 3. Cell fusion Aseptically, spleen cells from immunized mice were mixed with myeloma cells from SP2 / 0 mice at a ratio of approximately 5:1 in a 50 mL centrifuge tube. After washing twice with culture medium, the supernatant was discarded. Over 50 seconds, 0.9 mL of preheated PEG-1500 was slowly added to disperse the cells as evenly as possible. The mixture was allowed to stand for 1 min. 20 mL of preheated serum-free DMEM culture medium at 37°C was slowly added dropwise, 2 mL over the first two minutes and 18 mL over the next two minutes, all within 4 minutes. The mixture was allowed to stand for 3 minutes, then centrifuged at 800 rpm for 5 minutes and the supernatant was discarded. Preheated FBS and HAT culture medium were added, and the mixture was gently pipetted to mix. The mixture was then transferred to 96-well plates at a density of 200 μL per well and incubated.

[0033] 4. Screening of positive hybridoma cells Ten days after cell fusion, when the fused cells filled more than 50% of the wells, hybridoma cells were screened using an indirect ELISA method.

[0034] 5. Subcloning of positive hybridoma cells Subcloning was performed on the positive wells using a limiting dilution method. The number and location of cell clusters in the positive wells were observed under an inverted microscope. Cell clusters were then aspirated using a 200 μL pipette tip in a clean bench, and the cell count was diluted to 1-2 cells per 100 μL. Prepared feeder cells were then added to 96-well plates (100 μL per well), labeled, and incubated at 37°C for 9 days using a 5% CO2 incubator. After three subcloning cycles until one cell was found per well, the titer of the cell supernatant was measured using an indirect ELISA method. If the positive rate reached 100%, the culture was expanded, preserved, and labeled as the GP5 cell line.

[0035] Example 3: Preparation and Identification of Monoclonal Antibodies Against GP5-C1 Protein In this embodiment, a monoclonal antibody against the GP5-C1 protein was prepared and its subclass was identified. The specific steps are as follows: 1. Preparation of ascites fluid and determination of ascites titer using GP5-C1 protein monoclonal antibody 12-16 week old female BALB / c mice were intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Ten days later, each mouse was injected with 0.5 mL of a GP5 cell suspension preserved in Example 2 (5 × 10⁻⁶ cells). 5 (Each mouse was injected with the cell suspension, and the day of injection was counted as day 1). On day 7, after significant abdominal distension, ascites fluid was collected, centrifuged at 3000 rpm for 20 min, adipose tissue was removed, and the supernatant was collected and stored at -20℃ for later use. The titer of the ascites fluid was determined by indirect ELISA, and the results are as follows: Figure 3 As shown: Ascites antibody titer 1:1280000.

[0036] 2. Purification of monoclonal antibodies Ascites fluid was collected, and GP5 monoclonal antibody was purified using the caprylic acid-ammonium sulfate precipitation method, as follows: Ascites fluid was collected and centrifuged at 12000 rpm for 5 min at 4°C. The supernatant was collected, and 2 volumes of 0.06 M acetate buffer (pH 4.0) were added to adjust the pH to 4.5. 33 μL of caprylic acid was added per mL of ascites fluid, and the mixture was stirred at room temperature for 30 min. The mixture was then incubated at 4°C for 1 h to allow for complete precipitation of contaminating proteins. The mixture was centrifuged at 1000 g for 30 min at 4°C. 0.277 g of ammonium sulfate powder was added per mL of the supernatant. The beaker was placed on a magnetic stirrer and stirred for 1 h. The mixture was then centrifuged at 10000 g for 20 min at 4°C. The supernatant was discarded, and the precipitate was dissolved in PBS. SDS-PAGE was used to identify the purity of the monoclonal antibody. The results are shown below. Figure 4 As shown: High-purity GP5 monoclonal antibody was obtained.

[0037] 3. Identification of Monoclonal Antibody Types and Subclasses The mouse subtype identification kit was used for experimental procedures. The GP5 monoclonal antibody was identified as IgG1, kappa light chain (k type).

[0038] Meanwhile, GENEWIZ (Suzhou Genewiz Biotechnology Co., Ltd.) was commissioned to sequence the monoclonal antibody produced by the GP5 cell line preserved in Example 2 (i.e., the GP5 monoclonal antibody purified in step 2 of Example 3). The results are as follows: The nucleotide sequence of the heavy chain variable region of the GP5 monoclonal antibody is: 5'-CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACGTTCACCAGCTACTGGATGAACTGGGTTAAGCAGAGGCCTGAGCAAGGCCTTGAGTGGATTGGAAGGATTGATCCTTACGAT AGTGAAACTCACTACAATCAAAAGTTCAAGGACAAGGCCATATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGCTCTATTACTGTGCAAGACGGTTAGCGTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA-3' (SEQ ID NO: 1), The corresponding amino acid sequence is: QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWMNWVKQRPEQGLEWIGRIDPYDSETHYNQKFKDKAILTVDKSSSTAYMQLSSLTSEDSALYYCARRLAFDYWGQGTTLTVSS (SEQ ID NO: 2); The nucleotide sequence of the light chain variable region is: 5'-GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGCTATATGAACTGGTACCAACAGAAACCAGGACAGTCACCCAAACTCCTCATCTATGTT GCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA-3' (SEQ IDNO: 3), The corresponding amino acid sequence is: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQSPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPRTFGGGTKLEIK (SEQ ID NO: 4).

[0039] 4. Specificity detection of monoclonal antibodies The antigens used were porcine reproductive and respiratory syndrome virus (PRRSV) GP5 (GP5), PRRSV N (2–87 aa) protein (N), PRRSV Nsp7 protein (NSP), porcine pseudorabies virus gE (52–260 aa) protein (gE-52–260), porcine pseudorabies virus gB (59–337 aa) protein (gB-59–337), porcine pseudorabies virus gB (507–734 aa) protein (gB-507–734), porcine foot-and-mouth disease virus 3ABC protein (3ABC), African swine fever virus I226R protein (I226R), African swine fever virus CD2v protein (CD2v), African swine fever virus p30 protein (p30), and African swine fever virus p72 protein (p72). All the antigens used in the tests were obtained by constructing expression vectors in the laboratory and inducing prokaryotic expression.

[0040] The ELISA plate was coated under the same conditions, using the GP5 monoclonal antibody purified in step 2 of Example 3 as the primary antibody and HRP-labeled goat anti-mouse IgG as the secondary antibody. A values ​​were read on the ELISA reader. 450The values ​​were calculated by taking the average of three replicates for each sample. The specificity of the monoclonal antibody was detected using an indirect ELISA method (refer to Example 2). The results are as follows: Figure 5 As shown, the monoclonal antibody produced by the GP5 cell line preserved in Example 2 (i.e., the GP5 monoclonal antibody purified in step 2 of Example 3) has good specificity and can be used for qualitative or quantitative detection of porcine reproductive and respiratory syndrome virus GP5 in the test sample.

[0041] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An antibody or antigen-binding fragment thereof targeting a conserved region of the GP5 protein of porcine reproductive and respiratory syndrome virus (PRRSV), characterized in that: The conserved region is selected from amino acids 32–101 and / or 125–199; the antibody 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: 2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. The antibody or antigen-binding fragment thereof targeting the conserved region of the GP5 protein of porcine reproductive and respiratory syndrome virus according to claim 1, characterized in that: The antibody is a monoclonal antibody.

3. An antibody or antigen-binding fragment thereof targeting a conserved region of the GP5 protein of porcine reproductive and respiratory syndrome virus according to claim 2, characterized in that: The heavy chain subclass of the antibody is IgG1, and the light chain is κ type.

4. A nucleic acid molecule encoding the antibody according to any one of claims 1 to 3, characterized in that: The nucleic acid molecule contains a nucleotide sequence encoding the heavy chain variable region as shown in SEQ ID NO: 1; and a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO:

3.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. The recombinant expression vector for nucleic acid molecules according to claim 5, characterized in that: The vector is the pET-32a(+) expression vector.

7. A host cell comprising the recombinant expression vector of claim 5 or 6.

8. A cell line, characterized in that: The cell line is capable of producing the antibody as described in any one of claims 1 to 3.

9. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of reagents or kits for detecting porcine reproductive and respiratory syndrome virus GP5 protein.

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