Porcine reproductive and respiratory syndrome virus nucleocapsid protein antibody, fusion protein and detection kit and method

Abstract

The invention discloses a rabbit-derived single B cell antibody of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein, a fusion protein, a detection kit and a detection method. The invention provides two rabbit-derived single B cell antibodies T-11 and T-12, wherein amino acid sequences of variable regions of a heavy chain and a light chain of the rabbit-derived single B cell antibodies T-11 and T-12 are respectively shown as SEQ ID No.1-4; also provided are PRRSV-1 type and type 2 nucleocapsid protein fusion proteins BiN-2 (SEQ ID No.5) and antibody compositions comprising T-11 and T-12. Based on the antibody and the fusion protein, a competitive ELISA detection method and a kit are established, T-11 is used for capturing BiN-2 to construct an indirect coating antigen, biotin-labeled T-12 is used as a competitive antibody, and a judgment result is detected through an enzymatic reaction and a light absorption value. The method can be used for simultaneously detecting the PRRSV-1 type and the PRRSV-2 type antibodies, the sensitivity and the specificity both reach 97% or above, the method has no cross reaction with other porcine virus antibodies, the operation is simple and convenient, the stability is good, and a reliable tool is provided for accurate prevention and control of PRRS and epidemiological monitoring.

Description

Antibodies, fusion proteins, and detection kits and methods for porcine reproductive and respiratory syndrome virus nucleocapsid protein Technical Field

[0001] This invention belongs to the field of biological and viral detection technology, specifically relating to broad-spectrum reactive rabbit-derived single B-cell antibodies T-11 and T-12 of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein, the fusion protein BiN-2 of PRRSV genotype 1 and genotype 2 nucleocapsid proteins, and a competitive ELISA kit and detection method for detecting PRRSV antibodies constructed based on the above antibodies and fusion proteins. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV), which seriously threatens the healthy development of the global pig industry. The disease is characterized by reproductive disorders in pregnant sows (abortion, stillbirth, mummified fetuses) and respiratory symptoms in pigs of all ages. Since its first outbreak in the late 1980s, it has caused enormous economic losses to the global pig industry and has become one of the core diseases restricting the large-scale and healthy development of the pig farming industry.

[0003] PRRSV is an enveloped, single-stranded, positive-sense RNA virus belonging to the genus *Arteritisvirus* of the family Arteritisviridae. Its most prominent biological characteristics are high genetic variability and antigenic diversity. Based on gene sequence differences, PRRSV is mainly divided into two genotypes: PRRSV-1 (European type) and PRRSV-2 (American type). In my country's pig farming industry, PRRSV-2 infection has long been the dominant genotype. In recent years, PRRSV-1 has also emerged as an epidemic in some areas, creating a complex situation of co-circulation of both genotypes, posing a significant challenge to disease prevention and monitoring.

[0004] Serological diagnosis is a core technical means for assessing the PRRSV infection status and immunization efficacy in swine herds, and its detection performance directly depends on the quality of specific antigens and antibodies. The nucleocapsid protein (N protein) of PRRSV is the structural protein with the highest expression level after viral infection, possessing strong immunogenicity and high conservation across different PRRSV genotypes, making it an ideal target for serological detection. Currently, most commercially available PRRSV antibody detection kits use indirect ELISA methods, but they generally suffer from two major technical drawbacks: first, they struggle to simultaneously cover both PRRSV-1 and PRRSV-2 genotypes, failing to meet the detection needs in scenarios where both genotypes coexist; second, the direct coating of antigens results in poor antigen stability, leading to poor reproducibility and affecting the reliability of the test results.

[0005] Monoclonal antibodies, with their advantages of high specificity, good uniformity, and scalable production, have become the core material for serological detection technologies. However, monoclonal antibodies prepared using traditional hybridoma techniques suffer from limitations in immunogenicity and insufficient broad-spectrum activity, making them unsuitable for detection scenarios with diverse PRRSV genotypes. Single-B-cell antibody technology, which eliminates the need for cell fusion, allows for the direct screening of specific B cells from peripheral blood mononuclear cells of immunized animals and the cloning of their antibody genes. This enables the rapid preparation of broad-spectrum monoclonal antibodies targeting conserved antigenic epitopes, providing a new technological approach to solving the aforementioned technical challenges.

[0006] Based on this, the present invention screens two broad-spectrum reactive rabbit-derived single B-cell antibodies against PRRSV N protein using single B-cell antibody technology, constructs a fusion antigen of PRRSV-1 and type 2 N proteins, and then establishes a competitive ELISA method and kit with high specificity and sensitivity that can simultaneously detect two genotypes of PRRSV antibodies, providing technical support for the precise prevention and control and epidemiological monitoring of PRRS. Summary of the Invention

[0007] The purpose of this invention is to provide broad-spectrum reactive rabbit-derived single B-cell antibodies T-11 and T-12 against porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid proteins, exhibiting high binding specificity for both PRRSV genotype 1 and 2 nucleocapsid proteins. Another purpose of this invention is to provide a fusion protein BiN-2 of PRRSV genotype 1 and 2 nucleocapsid proteins as a broad-spectrum detection antigen. A further purpose of this invention is to provide a competitive ELISA method and kit based on the aforementioned antibodies and fusion protein, achieving accurate and efficient detection of PRRSV-1 and 2 antibodies, and solving the problems of incomplete genotype coverage and poor stability in existing detection technologies.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: 1. Rabbit-derived single B-cell antibodies T-11 and T-12. The present invention provides a rabbit-derived single B-cell antibody T-11 of porcine reproductive and respiratory syndrome virus nucleocapsid protein, the amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of its light chain variable region is shown in SEQ ID No. 2. This antibody can specifically capture the prokaryotically expressed PRRSV fusion protein BiN-2, and is suitable for preparing BiN-2 antigen indirectly coated ELISA plates.

[0009] This invention also provides a rabbit-derived monoclonal B-cell antibody, T-12, against porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein. The amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 3, and the amino acid sequence of its light chain variable region is shown in SEQ ID No. 4. This antibody specifically recognizes the nucleocapsid proteins of PRRSV-1 and PRRSV-2, recognizing conserved antigenic epitopes between types, making it suitable as a competitive antibody for establishing competitive ELISA methods.

[0010] 2. Fusion Protein BiN-2 This invention provides a fusion protein BiN-2 of porcine reproductive and respiratory syndrome virus (PRRSV) genotype 1 and 2 nucleocapsid proteins, the amino acid sequence of which is shown in SEQ ID No. 5. This sequence includes the N protein sequence (amino acids 11-133) of PRRSV-1 / LV strain (GenBank: A26843.1), the N protein sequence (amino acids 138-265) of PRRSV-2 / GSWW / 2018 strain (GenBank: OP764591.1), a middle linker sequence (GPGPG), and 6×His tag sequences at the N and C ends, which can be used as a broad-spectrum detection antigen in the development of ELISA kits.

[0011] 3. Antibody Composition This invention provides an antibody composition for porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein, comprising the aforementioned rabbit-derived single B cell antibodies T-11 and T-12. T-11 serves as a coating antibody to capture BiN-2 protein and prepare an antigen-indirectly coated ELISA plate; T-12 serves as a detection antibody, biotin-labeled for establishing a competitive ELISA detection mode. Both antibodies recognize different antigenic epitopes, effectively distinguishing between PRRSV-infected porcine N protein antibody-negative and positive sera, exhibiting good sensitivity and specificity.

[0012] 4. Competitive ELISA Method This invention provides a competitive ELISA method for detecting PRRSV antibodies, using the above-mentioned antibody composition and fusion protein BiN-2, comprising the following steps: (1) Preparation of antigen-indirectly coated reaction plate: ELISA plate is coated with rabbit-derived single B cell antibody T-11, washed, and then fusion protein BiN-2 is added for capture. After washing again, the plate is blocked to obtain an antigen-indirectly coated reaction plate. The coating concentration of rabbit-derived single B cell antibody T-11 is 0.1~2.0 μg / mL, preferably 1.0 μg / mL; the capture concentration of fusion protein BiN-2 is 0.2~2.0 μg / mL, preferably 1.0 μg / mL. Blocking is performed using PBS buffer containing 5% sucrose and 1% BSA at 37°C for 1 h.

[0013] (2) Sample incubation: The serum sample to be tested, negative control serum, positive control serum and biotin-labeled rabbit mono-B cell antibody T-12 were mixed and added to the antigen-coated reaction plate for incubation; the working concentration of biotin-labeled rabbit mono-B cell antibody T-12 was 0.1~1.0 μg / mL, preferably 0.2 μg / mL. The incubation conditions were 37℃ for 1 h.

[0014] (3) Enzymatic reaction: After washing, add horseradish peroxidase (HRP) labeled avidin for incubation, wash again, and then add substrate solution for color development; the dilution ratio of HRP labeled avidin is 1:50000~1:60000, the incubation conditions are 37℃ for 15 min, the substrate solution is TMB substrate, and the color development time is 10~15 min.

[0015] (4) Result determination: Detect absorbance value and calculate the blocking rate PI. When PI>40%, it is judged as antibody positive, and when PI≤40%, it is judged as antibody negative.

[0016] 5. Competitive ELISA Kit This invention provides a competitive ELISA kit for detecting PRRSV antibodies. The core components are an ELISA plate coated with T-11 antibody and capturing BiN-2 protein, and biotin-labeled T-12 antibody; the auxiliary components include 100× concentrated HRP-labeled avidin, 25× concentrated PBST washing buffer, serum diluent, TMB substrate solution, stop solution, positive control serum, and negative control serum.

[0017] The 25× concentrated PBST washing solution contains NaCl, KCl, Na2HPO4·12H2O, KH2PO4, and Tween 20, with a pH of 7.4; the serum diluent is a phosphate buffer containing 1.0% goat serum, 1.0% rabbit serum, 2.0% bovine serum albumin, and 0.02% Proclin-300 preservative; the stop solution is a 0.3~1.0 mol / L H2SO4 solution.

[0018] The preferred method for preparing 25× concentrated washing solution is as follows: Add 200g NaCl, 5g KCl, 72.5g Na2HPO4•12H2O, 5g KH2PO4, and 12.5 mL Tween 20 to every 500 mL of ultrapure water, bring the volume to 1000 mL, adjust the pH to 7.4, autoclave, and store at room temperature for later use.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. Strong antibody broad spectrum: The T-12 antibody of the present invention can specifically recognize the common conserved epitopes of PRRSV-1 and PRRSV-2 nucleocapsid proteins. The BiN-2 fusion protein contains the N protein sequences of both genotypes. The combination of the two enables the detection method to cover both genotypes of PRRSV at the same time, which solves the problem of incomplete genotype coverage of existing kits and is suitable for the prevention and control of mixed PRRSV epidemics in my country.

[0020] 2. Excellent detection performance: The method employs an indirect antigen coating mode, using T-11 antibody to specifically capture BiN-2 protein, significantly improving antigen stability and detection repeatability. Experimental verification shows that the sensitivity of this method for detecting PRRSV-1 and PRRSV-2 positive sera is 100% and 95%, respectively, and the specificity for detecting negative sera is 97.9%, with an area under the ROC curve of 0.9978, indicating extremely high accuracy.

[0021] 3. High specificity: The competitive ELISA method of this invention has no cross-reactivity with sera infected with foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), pseudorabies virus (PRV), and Seneca virus (SVA), which can effectively avoid false positive results and is suitable for the specific diagnosis of PRRS.

[0022] 4. Simple operation and controllable cost: All components of the reagent kit have been optimized and standardized. The detection process is short (about 3 hours in total) and requires no special instruments or equipment. It can meet the batch testing needs of grassroots veterinary stations, pig farms and third-party testing institutions, and has broad application prospects. Attached Figure Description

[0023] Figure 1. SDS-PAGE electrophoresis to verify the expression and purification of rabbit-derived mono-B cell antibody; Figure 2. Indirect immunofluorescence assay to verify the reactivity of rabbit-derived mono-B cell antibody with PRRSV; Figure 3. Indirect ELISA to verify the reactivity of rabbit-derived mono-B cell antibody with PRRSV N protein; Figure 4. SDS-PAGE detection results of BiN-2 protein expression and purification; Figure 5. Competitive ELISA results of PRRSV-infected pig serum and PRRSV-negative pig serum; Figure 6. ROC curve analysis of the sensitivity and specificity of competitive ELISA detection; Figure 7. Ratio of antibody levels to N protein in 10 PRRSV-1-infected pigs detected by competitive ELISA; Figure 8. Specificity of competitive ELISA detection of FMDV, CSFV, PRV, and SVA. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0025] Example 1: Preparation and Identification of Rabbit-Derived Monoclonal B-Cell Antibodies T-11 and T-12. The heavy and light chain genes of rabbit-derived T-11 and T-12 antibodies were derived from PRRSV-1 N protein (Euro-N). A monoclonal B-cell antibody gene library was established after immunizing rabbits with Euro-N antigen. Rabbits were immunized three times with a 21-day interval. Four days after immunization, 100 mL of anticoagulated blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated. Resuspend PBMCs in 3 mL of 1640 medium in a centrifuge tube, add mouse anti-rabbit CD4, CD8, and mouse anti-human CD14 antibodies, and incubate on ice for 30 minutes; wash twice, then add goat anti-mouse IgG magnetic beads and incubate on ice for 30 minutes; after negative selection of B cells using an LD column, incubate the enriched B cells with biotin-labeled Euro-N antigen at 4°C for 20 minutes, then add Anti-Rabbit IgM-FITC antibody and Mouse anti-biotin APC according to the instructions and incubate for 20 minutes; resuspend the cells in 1640 medium and incubate according to Euro-N-APC. + / IgM-FITC -Signal sorting was performed. Target cells were sorted into single B cells using a BD Fusion sorter and transferred to flow cytometry tubes containing 1640 medium. Single B cells were isolated by constructing GEMs using a microfluidic "double cross" cross-linking system based on 10x Genomics technology. The GEMs were collected and reverse transcribed in a PCR instrument to obtain cDNA with 10x barcode and UMI information. Then, using the cDNA as a template, nested PCR amplification was performed using rabbit B cell receptor (BCR) gene amplification primers to obtain the full-length rabbit BCR V(D)J gene. Finally, the library was sequenced using an Illumina sequencing platform to obtain paired BCR sequence information. The library data was quality controlled and analyzed using an R language program.

[0026] Following the above procedure, a PRRSV-specific rabbit BCR library was established, from which fully expressed rabbit antibodies were screened. The heavy and light chain variable region gene sequences of the T-11 and T-12 antibodies were synthesized by Genewiz (www.genewiz.com) and codon optimized, then cloned into the rabbit antibody heavy and light chain expression vectors (CH-pcDNA3.4 and CL-pcDNA3.4), respectively. Recombinant expression plasmids were extracted and purified using an endotoxin-free plasmid extraction kit (Tiangen). The light and heavy chain expression plasmids were mixed at a molar ratio of 3:2 and then co-transfected into HEK293F cells using liposomes. Feed was added 18 hours after transfection. The antibody-containing culture supernatant was collected 7 days after transfection.

[0027] The expressed B-cell antibodies were purified using a histidine-tagged protein affinity chromatography column, and the expression of the antibodies was verified by SDS-PAGE. The results showed that rabbit-derived IgG molecules T-11 and T-12 were successfully expressed, with a heavy chain molecular weight of approximately 50 kDa and a light chain molecular weight between 20 and 25 kDa. The light chain molecular weight of the T-11 antibody was larger than that of the T-12 antibody, and the purity reached over 95% (Figure 1).

[0028] T-11 Heavy chain variable region amino acid sequence (SEQ ID No. 1): METGLRWLLLVAVLKGVQCQSVEESGGRLVAPGTPLTLTCTASGFDISEYVMSWVRQAPGKGLEWIGVINIYGTTWYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCVRDLYGAYTTIWGPGTLVTVSST-11 Light chain variable region amino acid sequence (SEQ ID No. 2): MDTRAPTQLLGLLLLWLPGARCALVMTQTPSPVSAAVGGTVTINCQASESIISRYLSWYQQKPGQRPKLLIYSASTLASGVPSRFKGSGSGTEYTLTISDLECDDAATYYCAGYKNYNNDDNGFGGGTEVVVKT-12 Heavy chain variable region amino acid sequence (SEQ ID No. 2) No. 3): METGLRWLLLVAVLKGVQCQSVEESGGRLVKPDESLTLTCTASGFSLSSYAMSWVRQAPGKGLEWIGVMDNDDSAYYATWVNGRFTISRTSTTVDLQMTSLTTEDTATYFCVRDVYISDSELWGPGTLVTVSST-12 light chain variable region amino acid sequence (SEQ ID) No. 4) MDTRAPTQLLGLLLLWLPGATFALVMTQTPSSTSEPLGGTVTIKCQASQSIGSYLSWYQQKPGQPPKLLIYGASTLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGVYGYSSDDGRAFGGGTEVVVR Example 2 Antibody Reactivity Verification 1. Indirect Immunofluorescence Assay (IFA) Marc-145 cells grown in 24-well cell culture plates were infected with PRRSV-1 and PRRSV-2, respectively. When obvious cytopathic effects appeared in the cells, pre-cooled methanol-acetone solution (volume ratio 1:1) was added, and the cells were fixed at 4°C for 15 minutes. After washing with PBS buffer, 200 µl of T-11 and T-12 antibodies with a working concentration of 5 µg / ml were added to each well, and the cells were incubated at 37°C for 1 hour. After washing with PBS buffer, goat anti-rabbit IgG (H+L) fluorescent antibody diluted 1:200 was added, and the mixture was incubated at 37°C in the dark for 1 hour. After washing three times with PBS, the images were observed using an EVOS FL fluorescence imaging system (Life Technology).

[0029] IFA detection results showed that T-11 and T-12 bound to the N protein in Marc-145 cells infected with PRRSV-1 and PRRSV-2, respectively, and showed specific green fluorescence. Among them, the positive signal of T-12 in IFA detection was stronger than that of T-11. This indicates that T-11 and T-12 can specifically recognize the N protein of PRRSV-1 and PRRSV-2, and are broad-spectrum reactive antibodies against PRRSV (Figure 2).

[0030] 2. Indirect ELISA Validation: Laboratory-prepared PRRSV-1 and PRRSV-2 N proteins (Euro-N, America-N), and their fusion protein BiN-2, were coated into 96-well plates at 100 ng / well and incubated overnight at 4°C. The plates were then washed three times with PBS buffer (PBST) containing 0.05% Tween and blocked for 2 hours at 37°C with PBS solution containing 1% BSA and 5% sucrose. After three more washes, T-11 and T-12 antibodies at concentrations of 0.003-20 μg / mL were added, with PBS used as a negative control, and incubated at 37°C for 1 hour. 100 µL of goat anti-rabbit IgG-HRP antibody (1:30000 dilution) was added to each well, and the plates were incubated at 37°C for 30 min. TMB substrate was added, and the plates were incubated at 37°C for 15 min. After stopping the reaction, the absorbance (OD) was measured at 450 nm using a microplate reader. 450 When the antibody concentration is diluted to 5 µg / mL, a P / N ratio ≥ 2.1 is considered positive (P is the OD value for two replicate wells per dilution of the antibody). 450 The average value, N is the OD of all wells in the PBS control group. 450 (average of values).

[0031] Indirect ELISA results showed that at antibody concentrations of 5 µg / mL and above, under the same antibody concentration conditions, the OD values ​​of T11 and T-12 were... 450 All values ​​were higher than the cut-off value, which is the CO (positive cut-off value), CO = 2.1 × N (N is the negative control OD). 450 (Value). The results showed that T-11 and T-12 could specifically react with Euro-N, America-N and BiN-2 antigens, and were broad-spectrum reactive antibodies, as shown in Figure 3.

[0032] Example 3: Expression and Purification of BiN-2 Protein. The BiN-2 expression plasmid was designed and preserved in our laboratory. The amino acid sequence of the BiN-2 protein is shown in SEQ ID No. 5, including the histidine tag sequences at both ends (HHHHHH), the linker sequence in the middle (GPGPG), the upstream PRRSV-1 N protein sequence (amino acids 11-133), and the downstream PRRSV-2 sequence (amino acids 138-265). The synthesized sequence was inserted into the pET-28a vector for protein expression. The BiN-2 expression plasmid was transformed into E. coli BL21 competent cells and plated on solid LB agar plates containing 50 mg / L kanamycin, and cultured at 37°C for 12 h. Single colonies were picked and inoculated into liquid LB agar containing 50 mg / L kanamycin, and cultured at 37°C and 220 r / min for 2-4 h. When the bacterial OD... 600 When the value reaches about 0.6 to 0.8, BiN-2 protein is expressed by optimizing conditions such as induction temperature, IPTG induction concentration, and expression time.

[0033] By optimizing the induction conditions of BiN-2 expressing bacteria, BiN-2 protein was successfully obtained at 37℃, 0.5 mmol / L IPTG, and for 6 h of induction. The inclusion bodies were purified, and after denaturation, the protein was purified by nickel column affinity chromatography. The protein was then refolded using conventional dialysis methods, and the refolded protein was used to establish an ELISA antibody detection method. SDS-PAGE results showed that the BiN-2 protein was approximately 35 kDa, with a single band and high purity, as shown in Figure 4.

[0034] Fusion protein BiN-2 amino acid sequence (SEQ ID No.5):MGHHHHHHAAAPNNNGRQQNKKKGDGQPVNQLCQMLGKIIAQQRQSKGRGPGKKNKNKNLEKPHFPLATEDDVRHHFTPSERQLCLSSIRTAFNQGAGTCTLSDSGRISYTVEFSLPTHHTVRLIRVTTSPSA GPGPGAGKNQRQKKKENTAPMGNGQPVNQLCQLLGKMMKSQRQQSKRGQTKRKRPEKPHFPLAAEDDIRHHLTQTERSLCLQSIQTAFNQGAGAASLSSPGKVSFQVEFMLPVAQTVRLIRVTSTSAGQGANHHHHHHH.

[0035] Example 4: Establishment of an Antibody Competitive ELISA Method. Through screening, it was determined that T-11 and T-12 could form antibody pairs for establishing a competitive ELISA method. By optimizing the working concentrations of T-11, BiN-2 antigen, and biotin-labeled T-12 antibody, and by maximizing the ratio of absorbance values ​​of negative serum to positive serum, the incubation time and blocking buffer system were optimized. The final competitive ELISA reaction conditions were as follows: The ELISA plate was coated overnight at 4°C with a final concentration of 1.0 μg / mL T-11 antibody (dissolved in 100 mL of pH 9.6 carbonate buffer); after washing three times with PBST (PBS containing 0.05% Tween), 1.0 μg / mL BiN-2 antigen (diluted in PBS solution) was added and the plate was captured at room temperature for 2 h, followed by washing three times with PBST; then, blocking buffer (PBS containing 5% sucrose and 1% BSA) was added and the plate was blocked at 37°C for 1 h. The supernatant was discarded, the ELISA plate was dried, and the antigen-indirectly coated reaction plate was prepared. When testing samples, add 30 μL of serum diluent to each well of the serum dilution plate, then add 30 μL of the test sample, negative and positive control sera sequentially, and set up two blank control wells (60 μL of serum diluent). Next, add 60 μL of biotin-labeled T-12 antibody (0.2 μg / mL), transfer 100 μL to the ELISA plate, gently vortex to mix, seal with sealing film, and incubate at 37°C for 1 h. After washing five times with PBST (PBS containing 0.05% Tween), add 100 μL of HRP-labeled avidin (GenScript) diluted 1:60000, and incubate at 37°C for 15 min. After washing five times with PBST, blot dry, add 100 mL of TMB substrate to each well, and develop at 37°C for 10–15 min. Finally, add 100 mL of stop solution (1.0 mol / L H2SO4) to each well to terminate the reaction, and use a microplate reader to detect the absorbance value (OD) at a wavelength of 450 nm. 450 Calculate the blocking rate (PI) between control serum and the same test sample, PI = (OD of blank control) / (OD of test sample). 450 -Sample OD 450 ) / Blank control OD 450 The cutoff value for competitive ELISA was determined based on the detection results of PRRSV-infected and uninfected swine serum with clear backgrounds.

[0036] Example 5: Critical Value and Cross-Reactivity Validation. Ninety-six PRRSV-negative sera were tested using an optimized competitive ELISA assay. Each sample was tested twice using the optimized cELISA method, and the PI value for each sample was calculated. The mean (X) and standard deviation (SD) of the PI values ​​for the 96 samples were calculated. The critical value was determined as the mean PI value for negative samples + 2 times the standard deviation. PI ≥ (X + 2SD) was considered positive; PI < (X + 2SD) was considered negative. ROC curve analysis was performed using sera from 132 PRRSV-positive animals and 96 PRRSV-negative animals to determine the critical value, sensitivity, and specificity of the cELISA. Statistical analysis and data visualization were performed using GraphPad Prism software.

[0037] Animal experiments were conducted using 10 piglets (numbered 22, 23, 24, 25, 26, 27, 28, 31, 32, and 33). Blood samples were collected on days 0, 3, 5, 15, 21, 28, 35, and 42 after PRRSV-1 infection. Serum was separated and serum antibody levels were detected using the established ELISA method. Additionally, 112 PRRSV-1 positive serum samples and 120 PRRSV-2 positive serum samples stored in the laboratory were analyzed to evaluate the sensitivity of the method.

[0038] Statistical analysis of the test results showed that the average PI of the 96 negative serum samples was 22.5%, the variance was 8.4%, and X+2SD=39.3%. Therefore, the cutoff value was determined to be 40%. A PI greater than or equal to 40% was considered positive, and a PI less than 40% was considered negative. Using this criterion, the positive detection rates for PRRSV-1 and PRRSV-2 infected swine serum samples already available in the laboratory were 100% (112 / 112) and 95% (114 / 120), respectively, demonstrating good sensitivity. The specificity for detecting 96 PRRSV-uninfected swine serum samples was 97.9% (94 / 96) (Figure 5). ROC analysis showed that the area under the curve (AUC) reached 0.9978 (95% confidence interval: 0.9950-1.00) (Figure 6), indicating that the detection results of this method have good accuracy. The serum antibody fluctuation pattern of 10 piglets immunized with PRRSV-1 was detected. The results showed that antibody positivity could be detected 15 days after the first immunization and remained stable until day 42 (Figure 7).

[0039] Example 6. Specificity Evaluation: The established cELISA method was used to detect positive sera from pigs infected or immunized with Foot-and-Mouth Disease Virus (FMDV) (n=5), Classical Swine Fever Virus (CSFV) (n=3), Pseudorabies Virus (PRV) (n=3), and Seneca Virus (SVA) (n=5) stored in our laboratory. Each sample was tested in duplicate wells, and PRRSV-1 (n=5) and PRRSV-2 (n=5) positive controls were included to verify the specificity of the cELISA. The results showed that this method did not cross-react with FMDV, CSFV, PRV, and SVA antibodies; all results were negative, indicating that the method has excellent specificity (Figure 8).

Claims

1. A rabbit-derived single-cell B-cell antibody T-11 against porcine reproductive and respiratory syndrome virus nucleocapsid protein, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of its light chain variable region is shown in SEQ ID No.

2.

2. A rabbit-derived single-cell B-cell antibody T-12 against porcine reproductive and respiratory syndrome virus nucleocapsid protein, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID No. 3, and the amino acid sequence of its light chain variable region is shown in SEQ ID No.

4.

3. A fusion protein BiN-2 of porcine reproductive and respiratory syndrome virus nucleocapsid protein, characterized in that, Its amino acid sequence is shown in SEQ ID No.

5.

4. An antibody composition for porcine reproductive and respiratory syndrome virus nucleocapsid protein, characterized in that, It includes the rabbit-derived single B cell antibody T-11 as described in claim 1 and the rabbit-derived single B cell antibody T-12 as described in claim 2.

5. The use of the rabbit-derived single B-cell antibody of claim 1 or 2, the fusion protein of claim 3, or the antibody composition of claim 4 in the preparation of reagents or kits for detecting porcine reproductive and respiratory syndrome virus N protein or antibodies thereof.

6. A competitive ELISA method for detecting antibodies against porcine reproductive and respiratory syndrome virus, characterized in that, Using the antibody composition of claim 3 and the fusion protein BiN-2 of claim 4, the method includes the following steps: (1) preparing an antigen-coated reaction plate: coating an ELISA plate with rabbit-derived single B cell antibody T-11, washing it, adding fusion protein BiN-2 for capture, washing it again, and blocking it to obtain an antigen-coated reaction plate; (2) sample incubation: mixing the serum sample to be tested, negative control serum, positive control serum with biotin-labeled rabbit-derived single B cell antibody T-12, and adding it to the antigen-coated reaction plate for incubation; (3) enzymatic reaction: after washing, adding HRP-labeled avidin for incubation, washing it again, and adding substrate solution for color development; (4) result determination: detecting the absorbance value, calculating the blocking rate PI, judging the antibody positive when PI > 40%, and judging the antibody negative when PI ≤ 40%.

7. The method according to claim 8, characterized in that, In step (1), the coating concentration of the rabbit-derived single B cell antibody T-11 is 0.1~2.0 μg / mL, and the capture concentration of the fusion protein BiN-2 is 0.2~2.0 μg / mL; in step (2), the working concentration of the biotin-labeled rabbit-derived single B cell antibody T-12 is 0.1~1.0 μg / mL.

8. A competitive ELISA kit for detecting antibodies against porcine reproductive and respiratory syndrome virus, characterized in that, The product comprises: an enzyme-labeled plate coated with the rabbit mono-B cell antibody T-11 of claim 1 and capturing the fusion protein BiN-2 of claim 4; and biotin-labeled rabbit mono-B cell antibody T-12 of claim 2.

9. The reagent kit according to claim 8, characterized in that, It also includes one or more of the following components: enzyme-labeled avidin, washing solution, serum diluent, substrate solution, stop solution, positive control serum and negative control serum.

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