Monoclonal antibody specifically combined with peste des petits ruminants virus N protein and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a monoclonal antibody specifically combined with peste des petits ruminants virus N protein and application thereof. The sequence of the monoclonal antibody CDR1-H is as shown in SEQ ID NO: 1; the sequence of the CDR2-H is as shown in SEQ ID NO: 2; the sequence of the CDR3-H is as shown in SEQ ID NO: 3; the sequence of the CDR1-L is as shown in SEQ ID NO: 4; the sequence of the CDR2-L is LMS; the sequence of the CDR3-L is as shown in SEQ ID NO: 5. The monoclonal antibody can be subjected to specific reaction with peste des petits ruminants virus or N protein of the peste des petits ruminants virus; the enzyme-labeled monoclonal antibody is used as a detection antibody, a double-antibody sandwich ELISA method for detecting the peste des petits ruminants virus is established, the method is good in specificity and high in sensitivity, and the coincidence rate with a commercial kit is as high as 93.75%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a monoclonal antibody specifically binding to N protein of Peste des Petits Ruminants Virus and application thereof. BACKGROUND

[0002] Peste des Petits Ruminants Virus (PPRV) belongs to Paramyxoviridae Morbillivirus, and infects animals to cause Peste des Petits Ruminants (PPR), a highly contagious disease. PPRV not only infects small ruminants, but also infects large ruminants such as camels, endangered wild animals and other animals of the order Artiodactyla with latent infection, which poses a serious threat to biodiversity.

[0003] PPRV is a single-stranded negative-strand RNA virus, and its genome encodes 6 structural proteins (N, P, M, F, H and L) and 2 non-structural proteins (C and V). The N protein is the most abundant and conserved structural protein, and has high immunogenicity and expression level. The main role of the N protein is to encapsulate the viral genome to form a helical ribonucleoprotein, so as to protect the RNA from degradation by host RNA enzymes. The replication of PPRV in the host body mainly depends on the replication complex formed by N protein, P protein and L protein. The molecular size of N protein is 58 kDa, and it is the first protein expressed in the replication process of PPRV. The core domain of the N protein is responsible for RNA encapsulation and formation of the helical nucleocapsid of the virus. The N protein promotes the assembly of the virus together with the M protein, and promotes the packaging of the viral genome RNA. PPRV N protein binds to the M1 domain of protein kinase R (PKR) activation protein (PACT), enhances the interaction between PACT and PKR, and thus promotes the activation of PKR and subsequent eIF2α phosphorylation and SG formation, i.e. the N protein regulates the host PKR / eIF2α / SG axis to promote viral replication.

[0004] Monoclonal antibodies have been widely used in life science research and clinical practice due to their strong specificity, high affinity and low preparation cost. The binding capacity of antibodies and antigens depends on the variable region of each pair of light / heavy chains, and the complementarity determining region (CDR) is the most important determinant. ELISA has been widely used in basic research, disease diagnosis and biopharmaceuticals since its development in the 1970s, due to its high detection sensitivity, antigen-antibody specific recognition ability and good repeatability. Therefore, it is of great significance to develop a specific and sensitive virus detection ELISA kit using specific monoclonal antibodies for PPR prevention and control. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody that can specifically bind to the PPRV N protein, and to establish a double-antibody sandwich ELISA method using a rabbit-derived polyclonal antibody against the PPRV N protein as a capture antibody and the monoclonal antibody as a detection antibody, and to optimize and develop a PPRV detection kit.

[0006] The present invention specifically includes the following: In a first aspect, the present invention provides a monoclonal antibody that specifically binds to the PPRV N protein, wherein its variable region comprises three heavy chain complementarity-determining regions and three light chain complementarity-determining regions, wherein: (a) The amino acid sequence of CDR1-H (representing heavy chain CDR1 in this specification) is shown in SEQ ID NO:1(GYNFTDFP); (b) The amino acid sequence of CDR2-H (representing heavy chain CDR2 in this specification) is shown in SEQ ID NO:2(INTETGEP); (c) The amino acid sequence of CDR3-H (representing heavy chain CDR3 in this specification) is shown in SEQ ID NO:3 (ARLWDF); (d) The amino acid sequence of CDR1-L (representing the light chain CDR1 in this specification) is shown in SEQ ID NO:4(KSLLYKDGK TY); (e) The amino acid sequence of CDR2-L (representing the light chain CDR2 in this specification) is LMS; (f) The amino acid sequence of CDR3-L (represented in this specification as light chain CDR3) is shown in SEQ ID NO:5(QQLVEYPFT).

[0007] Furthermore, the monoclonal antibody is a single-chain antibody or an antigen-binding fragment, wherein the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

[0008] Secondly, the present invention provides a monoclonal antibody that specifically binds to the PPRV N protein, comprising a heavy chain variable region and a light chain variable region, wherein: (a) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6 (QIQLVQSGPELKKPAETVKISCKASGYNFTDFPMHWVKQAPGKGLKWVGWINTETGEPTYADDFKGRFAFSLETSATTAYLQINNLKNEDTATYFCARLWDFWGQGTTLIVSS); (b) The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:7 (DIVLTQDEFSNPVTSGESVSISCRSSKSLLYKDGKTYLSWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPFTFGSGTKLEIK).

[0009] The aforementioned monoclonal antibodies exhibit good reactivity with both PPRV and recombinant PPRV N protein.

[0010] Thirdly, the present invention provides nucleotides encoding the monoclonal antibody. The nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

[0011] Fourthly, the present invention provides biological materials comprising the aforementioned nucleotides, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

[0012] Fifthly, the present invention provides the use of the monoclonal antibody, the nucleotide, or the biological material in the preparation of a detection reagent, kit, or test strip for detecting peste des petits ruminants virus.

[0013] Preferably, the kit is a double-antibody sandwich ELISA kit, and the test strip is a test strip based on the double-antibody sandwich principle, such as a colloidal gold immunochromatographic test strip.

[0014] Sixthly, the present invention provides a double-antibody sandwich ELISA kit for detecting peste des petits ruminants (PPR) virus. The kit includes a capture antibody and a detection antibody. The capture antibody is a polyclonal antibody that specifically binds to the PPR N protein, and the detection antibody is a monoclonal antibody that specifically binds to the PPR N protein. The kit also includes an ELISA plate, a positive control, a negative control, a coating buffer, a sample dilution buffer, a blocking buffer, a chromogenic buffer, a stop buffer, and a washing buffer.

[0015] Preferably, the polyclonal antibody is a rabbit-derived polyclonal antibody.

[0016] Preferably, the polyclonal antibody is diluted with a coating buffer, which is a 0.05 mol / L sodium carbonate-sodium bicarbonate buffer at pH 9.6.

[0017] Preferably, the blocking solution is a 0.1 mol / L PBS solution containing 1% casein.

[0018] Preferably, the colorimetric solution is a TMB colorimetric solution.

[0019] Preferably, the terminating solution is a 1 mol / L H2SO4 solution.

[0020] Preferably, the washing solution is 1×PBST.

[0021] In a seventh aspect, the present invention provides a double-antibody sandwich ELISA detection method for detecting peste des petits ruminants virus, the method comprising the following steps: (1) Coating the microplate: Coat the microplate with rabbit polyclonal antibody diluted with coating solution, and then seal the plate for reaction; (2) Blocking the ELISA plate: Wash the ELISA plate with washing solution, add blocking solution, and seal the plate for reaction; (3) Add the test sample and control: Wash the enzyme-labeled plate with washing solution, add the test sample or positive and negative control, and seal the plate for reaction; (4) Add enzyme-labeled monoclonal antibody: Wash the enzyme-labeled plate with washing buffer, add horseradish peroxidase-labeled monoclonal antibody working solution to each well, and seal the plate for reaction; (5) Color development: Wash the microplate with washing solution, add color development solution to each well, and react in the dark; (6) Termination: Add an equal amount of termination solution to each well and read the OD. 450 The light absorption value; (7) Result determination: OD 450 ≥0.227, antigen positive; OD 450 <0.227, antigen negative.

[0022] Preferably, the coating amount of the polyclonal antibody is 0.2 μg / well.

[0023] Preferably, step (1) is: diluting the rabbit polyclonal antibody to 2 μg / mL with coating buffer, 100 μL per well, and coating overnight at 4°C; Preferably, step (2) is as follows: wash the microplate treated in step (1) three times with 1×PBST, add 120 μL of 1% casein buffer to each well, and react at 37°C for 1 h; Preferably, step (3) is as follows: wash the enzyme-labeled plate after step (2) with 1×PBST 3 times, and add 100μL / well of positive control, negative control and sample to be tested to positive control well, negative control well and sample to be tested well respectively, and react at 37℃ for 1h; Preferably, the positive control is an inactivated PPRV virus solution diluted 1:32 with sample dilution solution, and the negative control is Vero cell culture medium.

[0024] Preferably, step (4) is as follows: wash the enzyme-labeled plate after step (3) with 1×PBST 3 times, add horseradish peroxidase-labeled monoclonal antibody 100 μL per well, and react at 37°C for 1 h; Preferably, the enzyme-labeled antibody is a horseradish peroxidase-labeled monoclonal antibody diluted 1:6000.

[0025] Preferably, step (5) is as follows: wash the microplate after step (4) with 1×PBST, add 100 μL of substrate solution to each well, and react at 37°C in the dark for 15 min; Preferably, step (6) is: take the enzyme-labeled plate after step (5) and add 50 μL of 1 mol / L sulfuric acid solution to each well to terminate the reaction.

[0026] The beneficial effects of this invention are: This invention establishes a double-antibody sandwich ELISA method for detecting PPR (Pelteobril Disease) virus (PPR) based on the principle of double-antibody sandwich ELISA. The method utilizes a monoclonal antibody that specifically binds to the N protein of PPR as the detection antibody. The established double-antibody sandwich ELISA method exhibits high specificity and sensitivity, with a high concordance rate (93.75%) with commercially available kits. It is simple to operate, low in cost, and provides stable and reliable results, making it suitable for import / export quarantine and screening of large batches of samples from livestock farms. This provides new technical support for PPR prevention and control. Attached Figure Description

[0027] Figure 1 The results of subtype identification of the monoclonal antibody that specifically binds to the PPRV N protein in this invention; Figure 2 The purification results of the monoclonal antibody that specifically binds to the PPRV N protein in this invention; Figure 3 The physicochemical properties of the monoclonal antibody that specifically binds to the PPRV N protein in this invention are as follows: A: Acid stability of monoclonal antibody 10⁻³; B: Alkali stability of monoclonal antibody 10⁻³; C: Thermal stability of monoclonal antibody 10⁻³. Figure 4 The results of the reactivity identification of the monoclonal antibody that specifically binds to the PPRV N protein in this invention; Figure 5 Stacked map of sequencing sample abundance of the monoclonal antibody that specifically binds to the PPRV N protein in this invention; Figure 6 The present invention contains the amino acid (SEQ ID NO: 6) and nucleotide sequence (SEQ ID NO: 8) of the heavy chain variable region of a monoclonal antibody that specifically binds to the PPRV N protein. Figure 7 The present invention contains the amino acid (SEQ ID NO:7) and nucleotide sequence (SEQ ID NO:9) of the light chain variable region of a monoclonal antibody that specifically binds to the PPRV N protein. Figure 8 The results of the optimized reaction conditions for the double-antibody sandwich ELISA described in this invention; Figure 9 The results of threshold determination for the double-antibody sandwich ELISA described in this invention; Figure 10 The specificity test results of the double-antibody sandwich ELISA described in this invention; Figure 11 The results of the sensitivity test of the double-antibody sandwich ELISA described in this invention. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] The antigen used in the following examples is the recombinant small ruminant virus N protein expressed in prokaryotes, prepared by: through restriction enzyme cleavage sites. Eco RI and Xho I cloned the gene encoding the N protein of small ruminant virus (SEQ ID NO:10) into the pET-30a(+) vector to construct the recombinant plasmid pET-30a(+)-N; the recombinant plasmid was transformed into... E .coli BL21 competent cells were screened in LB medium containing kanamycin and induced to express recombinant N protein at 28°C for 12 h with a final IPTG concentration of 0.6 mmol / L. The recombinant N protein was mainly expressed in a soluble form. The recombinant peste des petits ruminants virus (PPR) N protein was purified using a nickel agarose affinity chromatography column. The concentration of the purified N protein was determined to be 1.17 mg / mL using the Pierce® BCA Protein Assay Kit.

[0030] The Nigeria75 / 1 vaccine strain PPRV (a gift from Researcher Zhi Haibing of the China Institute of Veterinary Drug Control), Sp2 / 0 myeloma cells, and PPRV positive serum used in the following examples were all preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center); clean-grade 8-10 week old female BALB / c mice were provided by the Laboratory Animal Center of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (Lanzhou Branch of the China Animal Health and Epidemiology Center).

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Example 1: Screening, identification and preparation of monoclonal antibodies against PPRV N protein 1. Construction of hybridoma cell lines Female BALB / c mice aged 8–10 weeks were immunized with recombinant PPRV N protein, 100 μg / mouse, three times at 14-day intervals. Spleen cells from the immunized mice were fused with Sp2 / 0 cells, and hybridoma cells were screened using selective HAT medium. Positive wells were screened using indirect ELISA, and the selected positive hybridoma cells were subcloned using limiting dilution, with a total of three subclonings. The cells were then expanded and cultured, and finally, OD (endogenous oxidative stress) cells were preserved. 450 The highest value was found in 10⁻³ hybridoma cells within the cell pore.

[0033] 2. Determination of monoclonal antibody subtypes Monoclonal antibodies in hybridoma cell culture supernatant were identified using the Isostrip Mouse Monoclonal Antibody Isotyping Kit: 20 μL of hybridoma cell supernatant was diluted with 180 μL of pH 7.2 PBS; 150 μL of this diluted solution was added to a test tube containing blue powder from the kit, and gently vortexed until the blue powder was completely dissolved; the Isotrip colloidal gold test strip was inserted into the bottom of the tube, and the results were observed within 5–10 minutes. The results showed that the monoclonal antibody subtype secreted by 10⁻³ hybridoma cell lines was identified as Ig2b, with a light chain type of κ (…). Figure 1 ).

[0034] 3. Preparation, purification, and titer detection of monoclonal antibodies 3.1 Preparation of ascites Female BALB / c mice aged 8–10 weeks were intraperitoneally injected with liquid paraffin, 0.2 mL per mouse. Seven days later, 1 × 10⁻⁶ g of paraffin was injected intraperitoneally. 6 10⁻³ hybridoma cells in good growth condition were used. After inoculation, the abdomen of the mice was observed daily. When the abdomen of the mice was significantly distended and they had difficulty moving, they were euthanized by cervical dislocation. The peritoneal fluid of the mice was centrifuged at 3000 r / min for 20 min using a sterile syringe. The clear supernatant was collected, aliquoted, labeled, and stored at -80℃ for later use.

[0035] 3.2 Purification of Ascites Fluid Ascites fluid was crudely purified by saturated ammonium sulfate precipitation, followed by affinity chromatography using a Cytive HiTrap Protein GHP antibody purification column, and identified by SDS-PAGE electrophoresis. Figure 2 It can be concluded that the heavy chain of the monoclonal antibody obtained after purification of ascites fluid is about 55KD, and the light chain is about 25KD.

[0036] 3.3 Valence Testing The purified monoclonal antibody 10⁻³ was serially diluted with PBST from 1:1000 to a total of 11 dilutions. Indirect ELISA was performed by coating ELISA plates with recombinant PPRV N protein (0.2 μg / well) and Nigeria 75 / 1 vaccine strain PPRV (1:50 dilution), respectively. The specific criterion was that the highest dilution with an S / N > 2.0 was considered the ELISA titer of the monoclonal antibody. The results are shown in Table 1. When recombinant PPRV N protein was used as the coating antigen, the highest titer of monoclonal antibody 10⁻³ reached 2.56 × 10⁻³. 5 When PPRV is used as the coating antigen, the titer of the monoclonal antibody 10⁻³ can reach 6.4 × 10⁻³. 4 .

[0037] Table 1. Titer test results of purified monoclonal antibody 10⁻³ 4. Identification of the stability of monoclonal antibodies Monoclonal antibody 10⁻³ was diluted 1:10 with HCl solution (pH 2.2), carbonate buffer (pH 9.6), and PBS buffer, respectively. The HCl and carbonate buffer diluted antibody 10⁻³ were stored at 4°C, while the PBS buffer diluted antibody 10⁻³ was heat-treated in a 56°C water bath. Samples treated with acid, alkali, and heat were collected at different time points (4, 8, 12, 24, and 48 hours), and the stability of monoclonal antibody 10⁻³ was assessed using indirect ELISA. Results are as follows: Figure 3 The results showed that the monoclonal antibody 10⁻³ exhibited a continuous decreasing trend upon acid treatment. Figure 3 A) shows a significant decreasing trend immediately after alkali treatment. Figure 3 (B) When subjected to heat treatment, the activity of monoclonal antibodies decreases rapidly at 10⁻³, with subsequent activity fluctuations being relatively small. Figure 3 C).

[0038] 5. Western blotting detection Western blotting was used to detect the reactivity of monoclonal antibody 10⁻³ with Nigeria 75 / 1 vaccine strain PPRV and recombinant PPRV N protein (PPRV N). The results showed that monoclonal antibody 10⁻³ exhibited good reactivity with both PPRV and recombinant PPRV N protein. Figure 4 ).

[0039] Example 2: Determination of the 10⁻³ variable region sequence of monoclonal antibody specifically binding to the N protein of peste des petits ruminants virus. 1. Obtaining the variable region gene of the heavy and light chains of monoclonal antibodies Expand the culture of hybridoma cells that secrete 10⁻³ monoclonal antibodies. Once the cells reach the logarithmic growth phase, count and collect the hybridoma cells until they reach 5 × 10⁻³ cells. 6 Total RNA was extracted using the HiPure RNA Mini Columns (Magen) kit; detailed instructions are provided in the product manual. RNA was dissolved in RNase-free water, and the concentration and integrity of total RNA were assessed using NanoDrop and nucleic acid electrophoresis. Reverse transcription was performed using SMART Scribe Reverse Transcriptase (Takara) and its oligo-dT and template switcholigo (TSO); detailed instructions are provided in the product manual. The obtained double-stranded cDNA was used as a template for amplification. The upstream primer was anchored to TSO, and the downstream primer bound to the constant region of either the heavy or light chain. The 5' ends of the upstream and downstream primers were labeled with P5 and P7 adapters, respectively. Heavy and light chain fragments were amplified independently in the first round of PCR. The first-round PCR product was purified using magnetic beads, and a second round of PCR was performed using the purified product as a template. In this stage, index primers were ligated to both ends of the first-round PCR product to form a TruSeq dual-indexed library. The library was purified using magnetic beads and quantified using Qubit; the sequence was determined using an Illumina MiSeq PE300.

[0040] The 10 sequences with the highest abundance were selected for abundance analysis. The abundance packing map is shown below. Figure 5 Select the most productive gene sequence as the target gene sequence.

[0041] 2. Analysis of gene sequencing results of the variable domains of the heavy and light chains of monoclonal antibodies Based on sequencing results, the gene specifically binding to the variable domain of the heavy and light chains of monoclonal antibodies against the PPRV N protein was analyzed using NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ). The analysis results showed that the VH nucleotide (SEQ ID NO:8) and amino acid (SEQ ID NO:6) sequences of the variable region of the heavy chain of the 10⁻³ antibody in hybridoma cell line were as follows: Figure 6 As shown, the heavy chain variable region gene is 339 bp in length, encoding 113 amino acid residues, and includes the highly variable regions CDR1-H (SEQ ID NO:1), CDR2-H (SEQ ID NO:2), and CDR3-H (SEQ ID NO:3); the light chain variable region VL nucleotide (SEQ ID NO:9) and amino acid (SEQ ID NO:7) sequences are as follows. Figure 7As shown, the variable region gene is 336 bp in length, encoding 112 amino acid residues, and includes highly variable regions CDR1-L (SEQ ID NO:4), CDR2-L (LMS) and CDR3-L (SEQ ID NO:5).

[0042] Example 3: Small Ruminant Disease Virus Double Antibody Sandwich ELISA Detection Kit and its Usage Method 1. Commercially available (ID Vet) double-antibody sandwich ELISA kit 1.1 Operating Procedures S1: Dilute the control and test samples by 2 times with diluent No. 13. Set up 2 wells for each of the negative and positive controls. Add 50 μL of the diluted control to each well. Add 50 μL of the diluted test sample to each of the remaining wells. Seal the plate and incubate at 37°C for 45 minutes.

[0043] S2: Discard the liquid in the well and wash the plate 6 times with 1× detergent.

[0044] S3: Dilute the concentrated enzyme conjugate (10×) 10 times (1×) with dilution buffer No. 19, add 100 μL to each well, seal the plate with a sealing membrane, and incubate at 21°C for 30 minutes.

[0045] S4: Wash as above, add 100 μL of substrate solution to each well, and incubate at 21°C in the dark for 15 minutes.

[0046] S5: Add 100 μL of stop solution to each well to terminate the reaction, and measure the OD of each well. 450 value.

[0047] 1.2 Result Determination Calculate the S / P% of each sample using the formula: S / P% = [(OD 样本 -OD NC ) / (OD PC -OD NC )] ×100%; Experimental criteria: Average OD value of positive control (OD) PC The value is greater than 0.500, and the average OD value of the control and the average OD value of the negative control (OD) NC The ratio of S / P% to P% is greater than 3. Result interpretation: S / P% ≥ 20%, positive; S / P% < 20%, negative.

[0048] 2. The monoclonal antibody 10⁻³ of this invention is used in a double-antibody sandwich ELISA method. 2.1 Screening of optimal capture antibody and detection antibody pairs Six-week-old New Zealand white rabbits were immunized with recombinant PPRV N protein (500 μg / rabbit), with a booster immunization 14 days later. Rabbit serum was separated 14 days after the second immunization to obtain rabbit polyclonal antibodies specifically binding to PPRV N protein. An orthogonal experiment was used to screen for the optimal antibody pair using the rabbit polyclonal antibody specifically binding to PPRV N protein and the monoclonal antibody 10⁻³ prepared in this invention. The results are shown in Table 2. Rabbit polyclonal antibody No. 2 (5.98 mg / mL) showed the best performance as the capture antibody and monoclonal antibody 10⁻³ as the detection antibody.

[0049] Table 2. Screening results of the best antibody pairs 2.2 Establishment of a double-antibody sandwich ELISA method Follow these steps to screen the coating amount, blocking buffer, working solution dilution of enzyme-labeled monoclonal antibody (HRP-labeled mouse monoclonal antibody 10⁻³), and sample dilution buffer for rabbit polyclonal antibody No. 2.

[0050] S1: Dilute rabbit polyclonal antibody No. 2 to a certain concentration with carbonate buffer (0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH 9.6), 100 μL per well. Coat overnight at 4°C.

[0051] S2: Wash the plate three times with 1×PBST washing buffer at a rate of 300 μL / well, add 120 μL of blocking buffer to each well, and incubate at 37°C for 60 minutes.

[0052] S3: Wash as above. Add 100 μL of Nigeria 75 / 1 vaccine strain PPRV (P) diluted 1:32 to the positive control well and add 100 μL of normal Vero cell supernatant (N) to the negative control well. Incubate at 37°C for 60 minutes.

[0053] S4: Wash as above, add 100 μL of HRP-labeled mouse monoclonal antibody 10⁻³ to each well, and incubate at 37°C for 60 minutes.

[0054] S5: Wash as above, add 100 μL of substrate TMB to each well, and react at 37°C in the dark for 15 minutes.

[0055] S6: Add 50 μL of stop solution (1 mol / L H2SO4 solution) to each well to terminate the reaction, and measure the OD using a microplate reader. 450 value.

[0056] The results showed that among the three coating amounts of rabbit polyclonal antibody No. 2, the P / N value (5.303) was the highest when the coating amount was 0.2 μg / well. Figure 8A); Among the blocking solutions 1% BSA, 1% bovine serum, and 1% casein buffer (0.1 mol / L PBS solution containing 1% casein), the P / N value (6.295) was the highest when blocked with 1% casein buffer. Figure 8 B); Among the 10⁻³ dilutions of enzyme-labeled monoclonal antibodies, 1:4000, 1:6000, and 1:8000, the P / N value was highest at a dilution of 1:6000 (17.385). Figure 8 C); Among the sample diluents RIPA, TRITON-100, and PBS, RIPA had the highest P / N value (3.478) as the sample diluent. Figure 8 D). Therefore, the optimized reaction conditions for obtaining the double-antibody sandwich ELISA are as follows: the coating amount of rabbit polyclonal antibody No. 2 is 0.2 μg / well, the enzyme-labeled monoclonal antibody is diluted at 1:6000, and 1% casein buffer and RIPA lysis buffer are used as blocking buffer and sample dilution buffer, respectively.

[0057] 2.3 Determination of the cutoff value for the double-antibody sandwich ELISA detection method Ninety-six negative sheep tissue samples (previously tested negative using a commercially available double-antibody sandwich ELISA kit) were analyzed using the double-antibody sandwich ELISA method established in section 2.2. The cutoff value was based on the OD values ​​of the 96 negative tissue samples. 450 The mean was calculated by adding three times the standard deviation (SD). Results showed that OD values ​​were found in 96 negative tissue samples. 450 The mean was 0.122, and the standard deviation was 0.035. Based on the critical value, the critical value for the double-antibody sandwich ELISA was calculated to be 0.227 (0.122 ± 3 SD). Figure 9 Therefore, when OD 450 A result of ≥0.227 is considered positive.

[0058] 2.4 Specificity Test The double-antibody sandwich ELISA method established in section 2.2 was used to detect Nigeria 75 / 1 vaccine strain PPRV, argali parainfluenza virus type 5 (PIV5), ovine orthopnea virus (ORFV), sheep pox virus (CaPV), foot-and-mouth disease virus (FMDV), and bovine nodular dermatosis virus (LSDV). The test results are as follows: Figure 10 As shown, except for the PPRV sample, the OD values ​​of the other viruses... 450 The values ​​were all less than 0.227, indicating that the established double-antibody sandwich ELISA has good specificity and does not cross-react with other common pathogens infecting sheep.

[0059] 2.5 Sensitivity Test The double-antibody sandwich ELISA method established in 2.2 was used to detect serially diluted Nigeria75 / 1 vaccine strain PPRV (8000 TCID50 to 7.8125 TCID50) and recombinant PPRVN protein (100 μg / mL to 0.03 μg / mL). Figure 11 The results showed that when the recombinant PPRV N protein was diluted to 0.195 μg / mL ( Figure 11 A) PPRV diluted to 250 TCID50 ( Figure 11 The test result was still positive at step B). This indicates that the research method has good sensitivity.

[0060] 2.6 Compliance Test The double-antibody sandwich ELISA method established in section 2.2 was used to simultaneously test 240 clinical samples with a commercially available kit. The concordance rate and Kappa value were used to evaluate their compatibility. The concordance rate was calculated as [(a+b) / n]×100%, and the Kappa value was calculated as PA=(a+b) / n; PC=[(a+b)(a+c)+(c+d)(b+d)] / n²; Kappa=(PA-PC) / (1-PC) (Kappa≥0.75 indicates good concordance; 0.75>Kappa≥0.4 indicates moderate concordance; Kappa<0.4 indicates poor concordance). The results showed that the concordance rate between the double-antibody sandwich ELISA method established in this invention and the ID Vet kit was 93.75%, and the Kappa value was 0.86, indicating that the established double-antibody sandwich ELISA method is reliable.

[0061] Table 3. Comparison Results Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody that specifically binds to the N protein of peste des petits ruminants virus, said monoclonal antibody comprising three heavy chain complementarity-determining regions CDR1-H, CDR2-H, and CDR3-H and three light chain complementarity-determining regions CDR1-L, CDR2-L, and CDR3-L; characterized in that, The amino acid sequence of CDR1-H is shown in SEQ ID NO:1; The amino acid sequence of CDR2-H is shown in SEQ ID NO:2; The amino acid sequence of CDR3-H is shown in SEQ ID NO:3; The amino acid sequence of CDR1-L is shown in SEQ ID NO:4; The amino acid sequence of CDR2-L is LMS; The amino acid sequence of CDR3-L is shown in SEQ ID NO:

5.

2. The monoclonal antibody that specifically binds to the N protein of peste des petits ruminants virus according to claim 1, characterized in that, The monoclonal antibody is a single-chain antibody or an antigen-binding fragment, wherein the antigen-binding fragment is a Fab fragment, an F(ab')2 fragment, or a single-chain Fv fragment.

3. The monoclonal antibody that specifically binds to the N protein of peste des petits ruminants virus according to claim 1, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region being shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region being shown in SEQ ID NO:

7.

4. A nucleotide encoding the monoclonal antibody according to any one of claims 1-3.

5. The nucleotide according to claim 4, characterized in that, The nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO:8 and SEQ ID NO:9, respectively.

6. A biological material comprising the nucleotide of claim 4 or 5, wherein the biological material is an expression cassette, transposon, plasmid vector, viral vector, or host cell.

7. The use of the monoclonal antibody according to any one of claims 1-3, the nucleotide according to claim 4 or 5, or the biological material according to claim 6 in the preparation of a detection reagent, kit, or test strip for detecting peste des petits ruminants virus.

8. The application according to claim 7, characterized in that, The kit is a double-antibody sandwich ELISA kit, and the test strip is a colloidal gold immunochromatographic test strip.

9. A double-antibody sandwich ELISA kit for detecting peste des petits ruminants virus, characterized in that, The kit includes a capture antibody and a detection antibody, wherein the capture antibody is a polyclonal antibody that specifically binds to the N protein of peste des petits ruminants virus, and the detection antibody is a monoclonal antibody as described in any one of claims 1-3.

10. The reagent kit according to claim 9, characterized in that, The kit also includes an ELISA reaction plate, a positive control, a negative control, a coating solution, a sample diluent, a blocking solution, a colorimetric solution, a stop solution, and a washing solution.

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