Specific monoclonal antibody against n protein of peste des petits ruminants virus and application thereof

Abstract

The application belongs to the technical field of biology, and particularly relates to a specific monoclonal antibody against N protein of Peste des petits ruminants virus and application thereof. The application discloses a monoclonal antibody 6E8 against N protein of Peste des petits ruminants virus, wherein the monoclonal antibody 6E8 comprises a heavy chain variable region shown in SEQ ID NO. 6 and a light chain variable region shown in SEQ ID NO. 7; the monoclonal antibody 6E8 can specifically react with Peste des petits ruminants virus and N protein thereof; further, the application establishes a blocking ELISA method for detecting Peste des petits ruminants virus antibody by taking HRP-labeled monoclonal antibody 6E8 as a blocking antibody, and the method has good specificity and high sensitivity, and the coincidence rate with a commercial kit is 92.31%. The application provides a new material for developing a diagnostic preparation, and provides new technical support for prevention and control of Peste des petits ruminants and implementation of a global elimination plan.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a specific monoclonal antibody against the N protein of small ruminant virus and its application. Background Technology

[0002] Peste des petits ruminants (PPR) is an acute, highly contagious, and rapidly spreading transnational disease caused by the peste des petits ruminants virus (PPRV). Its main clinical symptoms include fever, diarrhea, enteritis, and pneumonia. It is listed as a notifiable animal disease by the OIE and is classified as a Class A animal disease in my country. PPRV primarily infects small domestic ruminants such as goats and sheep, but it can also infect large ruminants such as camels, endangered wild animals, and even-toed ungulates that may be latently infected, posing a serious threat to biodiversity. In 2012, my country explicitly included PPR in its list of 13 key invasive animal diseases for prevention. In 2015, the FAO and OIE adopted and launched the Global Strategy for the Control and Eradication of Peste des Petits Ruminants, designating PPR as the next global eradication target.

[0003] PPRV is a single-stranded, negative-sense RNA virus whose genome encodes six structural proteins (N, P, M, F, H, and L) and two non-structural proteins (C and V). The replication complex formed by the nucleocapsid protein N, along with P and L proteins, is the key molecular basis for viral replication. The N protein, together with the M protein, promotes viral assembly, facilitating the encapsulation of the viral genomic RNA. As the first protein expressed during PPRV replication, the N protein is the most abundant and conserved structural protein among viral proteins, exhibiting high immunogenicity. Animals infected with PPRV rapidly produce high-titer antibodies against the N protein. Although the N protein does not induce neutralizing antibodies, it can induce a strong cell-mediated immune response.

[0004] Monoclonal antibodies are antibodies secreted by a single B cell that specifically recognize only one antigenic epitope. Due to their high specificity, high affinity, and low preparation cost, they have been widely used in life science research and clinical applications. The variable regions of the antibody heavy and light chain pairs determine its binding ability to the antigen, with the complementarity-determining region (CDR) being the most important determining factor. Since its development in the 1970s, ELISA has been widely used in basic research, clinical diagnostics, and biopharmaceuticals due to its advantages of ease of operation, high sensitivity, high specificity, low cost, and suitability for batch testing. Therefore, developing specific and sensitive ELISA kits using screened specific monoclonal antibodies is of great significance for disease prevention and control. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention prepared a monoclonal antibody that specifically binds to the PPRV N protein. The complementarity-determining region and variable region sequences of this monoclonal antibody were obtained through sequencing. Using this monoclonal antibody as the blocking antibody, a blocking ELISA method was established, and an optimized small ruminant plague virus antibody detection kit was developed. Specifically, it includes the following:

[0006] In a first aspect, the present invention provides a specific monoclonal antibody against the N protein of peste des petits ruminants virus, wherein the variable region CDR of the antibody heavy chain is composed of CDR1 as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; and the variable region CDR of the antibody light chain is composed of CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence of YAS, and CDR3 with an amino acid sequence as shown in SEQ ID NO.5.

[0007] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.7.

[0008] In a second aspect, the present invention provides a nucleic acid that encodes the antibody heavy chain and antibody light chain of the monoclonal antibody described in the first aspect above.

[0009] Preferably, the nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.9.

[0010] Thirdly, the present invention provides a biomaterial comprising the nucleic acid described in the second aspect above.

[0011] Preferably, the biological material includes an expression cassette, transposon, plasmid vector, viral vector, or host cell.

[0012] Fourthly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the preparation of a detection reagent for detecting peste des petits ruminants virus (PPR), or in the detection of natural infectivity of PPR for non-disease diagnosis purposes, or in the study of the function of the PPR N protein.

[0013] Preferably, the reagent is a test strip or a reagent kit.

[0014] Preferably, the test strip is a colloidal gold immunochromatographic test strip, and the kit is a blocking ELISA kit.

[0015] Fifthly, the present invention provides an enzyme conjugate, the enzyme conjugate comprising:

[0016] (i) Monoclonal antibodies as described in the first aspect above;

[0017] (ii) and horseradish peroxidase conjugated with the monoclonal antibody in (i).

[0018] In a sixth aspect, the present invention provides a blocking ELISA kit for detecting antibodies against peste des petits ruminants virus, the kit comprising the monoclonal antibody described in the first aspect above.

[0019] Preferably, the kit further includes a coating antigen, wherein the coating antigen is a recombinant PPRV N protein.

[0020] Preferably, the coating antigen is a recombinant PPRV N protein with a his tag.

[0021] Preferably, the kit further 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.

[0022] Preferably, the coating solution is a 0.05 mol / L sodium carbonate-sodium bicarbonate buffer solution with a pH of 9.6.

[0023] Preferably, the blocking solution is a 0.1 mol / L PBS solution containing 2% bovine serum.

[0024] Preferably, the colorimetric solution is a TMB solution.

[0025] Preferably, the terminating solution is a 1 mol / L H2SO4 solution.

[0026] Preferably, the washing solution is 1×PBST.

[0027] In a seventh aspect, the present invention provides a blocking ELISA detection method for detecting antibodies against peste des petits ruminants virus, the method comprising the following steps:

[0028] (1) Coating of ELISA plates: Coating ELISA plates with recombinant small ruminant virus N protein diluted with coating solution, and then sealing the plates for reaction;

[0029] (2) Blocking the ELISA plate: Wash the ELISA plate with washing solution, add blocking solution, and seal the plate for reaction;

[0030] (3) Add the test sample and control: Add the test sample or positive and negative controls, and seal the plate for reaction;

[0031] (4) Add enzyme-labeled monoclonal antibody: Wash the enzyme-labeled plate with washing buffer, add working solution of the monoclonal antibody described in the first aspect above labeled with horseradish peroxidase (HRP) to each well, and seal the plate for reaction;

[0032] (5) Color development: Wash the microplate with washing solution, add substrate solution to each well, and react in the dark;

[0033] (6) Termination: Add stop solution to each well and read OD. 450 The light absorption value;

[0034] (7) Result determination: S / N%≤43.3%, antibody positive; S / N%>43.3%, antibody negative.

[0035] Preferably, the coating amount of the recombinant PPRV N protein is 0.005 μg / well.

[0036] Preferably, step (1) is: diluting the recombinant PPRV N protein to 0.05 μg / mL with coating solution, 100 μL / well, and coating at 4°C for more than 16 hours;

[0037] Preferably, step (2) is as follows: wash the enzyme-labeled plate treated in step (1) three times with 1×PBST, add 2% bovine serum buffer, 120 μL / well, and react at 37°C for 1 h;

[0038] Preferably, step (3) is: pat the enzyme-labeled plate after step (2) dry, add positive control, negative control and test sample, 100 μL / well, and react at 37℃ for 45 min;

[0039] Preferably, the positive control, negative control, and test sample are all diluted 1:1 with serum diluent.

[0040] Preferably, step (4) is as follows: wash the enzyme-labeled plate after step (3) with 1×PBST 3 times, add horseradish peroxidase HRP-labeled monoclonal antibody as described in the first aspect above, 100 μL / well, and react at 37°C for 30 min;

[0041] Preferably, the horseradish peroxidase (HRP)-labeled monoclonal antibody described in the first aspect above is diluted 1:4000.

[0042] Preferably, step (5) is: wash the enzyme-labeled plate after step (4) with 1×PBST, add substrate solution, 100 μL / well, and react at 37°C in the dark for 15 min;

[0043] Preferably, step (6) is: take the enzyme-labeled plate after step (5) and add 50 μL of 1 mol / L H2SO4 solution to each well to terminate the reaction.

[0044] The beneficial effects of this invention are: ① This invention provides a monoclonal antibody that specifically binds to the N protein of peste des petits ruminants (PPR); ② The monoclonal antibody exhibits good reactivity with both PPR and the PPR N protein; ③ Based on the principle of blocking ELISA, a blocking ELISA method for detecting PPR antibodies was established using the monoclonal antibody that specifically binds to the N protein of PPR as described in this invention. The established blocking ELISA method has good specificity and high sensitivity, and a high concordance rate (92.31%) with commercially available kits. It is suitable for disease detection, monitoring, and epidemiological investigation, providing new technical support for PPR prevention and control. Attached Figure Description

[0045] Figure 1 The purification results of the monoclonal antibody of this invention.

[0046] Figure 2 This is a stacked map of the abundance of sequencing samples of the monoclonal antibody described in this invention.

[0047] Figure 3 The results of determining the blocking ELISA threshold described in this invention. Detailed Implementation

[0048] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0049] The antigen used in the following examples is the recombinant small ruminant virus N protein (KY628761.1) expressed in prokaryotes. The preparation method is as follows: the recombinant plasmid pET-30a(+)-N expressing the N protein is transformed into E. coli BL21 competent cells, screened in LB medium containing kanamycin, and induced to express for 18 h at 18℃ with a final IPTG concentration of 0.8 mmol / L. The recombinant N protein is mainly expressed in a soluble form. The recombinant N protein is purified using a nickel agarose affinity chromatography column. The concentration of the purified recombinant N protein is determined to be 0.79 mg / mL using the Pierce® BCA Protein Assay Kit.

[0050] The Nigeria 75 / 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).

[0051] Unless otherwise specified, all experimental reagents used in the following examples are commercially available or can be synthesized according to the methods described in the text or known methods; and unless otherwise specified, all experimental methods used in the following examples are conventional methods.

[0052] Example 1: Preparation of monoclonal antibody 6E8 against peste des petits ruminants virus N protein

[0053] 1. Construction of hybridoma cell lines that stably secrete monoclonal antibodies

[0054] BALB / c mice were immunized with the expressed recombinant N protein, 100 μg / mouse, three times at 14-day intervals. Hybridoma cells were screened using cell fusion technology and selective HAT medium. Positive hybridoma cell wells were screened using indirect ELISA, and then subcloned using limiting dilution, a total of three subclonings. Three positive hybridoma cell lines were finally selected, and their OD values ​​were preserved after expansion culture. 450 The hybridoma cell with the highest value was named 6E8.

[0055] 2. Determination of monoclonal antibody subtypes

[0056] The supernatant from hybridoma cell culture medium 6E8 was collected, and the monoclonal antibody isotype was identified using a mouse monoclonal antibody Ig class / subclass / isotype identification kit (BIODRAGON), following the kit's instructions. The results showed that the secreted monoclonal antibody 6E8 had a heavy chain isotype of IgG1 and a light chain isotype of κ.

[0057] 3. Preparation of enzyme-labeled monoclonal antibodies

[0058] 3.1 Preparation of ascites

[0059] Each 8–10 week old female BALB / c mouse was intraperitoneally injected with 0.2 mL of liquid paraffin, followed by an intraperitoneal injection of 5 × 10⁻⁶ mol / L of paraffin 7 days later. 56E8 hybridoma cells in good growth condition were collected. The abdomen of the mice was observed daily after inoculation. When the abdomen of the mice became significantly distended and they had difficulty moving, the mice were euthanized by cervical dislocation. The dark red liquid was aspirated from the abdomen of the mice, centrifuged at 3000 r / min for 20 min, and the clear supernatant was collected, aliquoted, labeled and stored at -20℃ for later use.

[0060] 3.2 Purification of ascites fluid

[0061] Ascites fluid-type monoclonal antibodies were purified using a combination of saturated ammonium sulfate precipitation and Protein G chromatography, and identified by SDS-PAGE electrophoresis. Figure 1 It can be concluded that the heavy chain of the purified monoclonal antibody 6E8 is approximately 55 kDa, and the light chain is approximately 25 kDa.

[0062] 3.3 Labeling and titer determination of monoclonal antibody 6E8

[0063] HRP-6E8 was obtained by labeling the monoclonal antibody 6E8 using the HRP Labling-NH2 kit (DOJINDO Research Institute, Japan). The specific operation was performed according to the instructions.

[0064] The titer of HRP-6E8 was detected by indirect ELISA using Nigeria 75 / 1 vaccine strain PPRV (1:50) and recombinant PPRV N protein (0.2 μg / well) as coating antigens, respectively. sp2 / 0 cell supernatant and normal mouse ascites fluid were used as negative controls, and anti-PPRV positive serum was used as a positive control. The specific criterion was that the highest dilution of ascites fluid with a P / N ratio > 2.1 was taken as the ELISA titer of HRP-6E8. Table 1 shows that when PPRV was used as the coating antigen, the titer of the monoclonal antibody 6E8 reached 3.2 × 10⁻⁶. 4 When recombinant PPRV N protein is used as the coating antigen, the HRP-6E8 titer can reach a maximum of 1.28 × 10⁻⁶. 5 .

[0065] Table 1. ELISA titer test results of HRP-6E8

[0066] Example 2: Determination of the variable region sequence of the monoclonal antibody 6E8 against the N protein of peste des petits ruminants virus (PPR).

[0067] 1. Obtaining the variable region gene of the heavy and light chains of monoclonal antibodies

[0068] Hybridoma cells that stably secrete the monoclonal antibody 6E8 were cultured on a large scale. Once the cells reached the logarithmic growth phase, they were collected, counted, and their concentration was adjusted to 5 × 10⁻⁶. 6Total RNA was extracted using the HiPure RNA Mini Columns (Magen) kit. RNA was dissolved in RNase-free water, and the concentration and integrity of total RNA were detected by NanoDrop and nucleic acid electrophoresis. Reverse transcription was performed using SMARTScribe Reverse Transcriptase (Takara) and its oligo-dT and template switch oligo (TSO). 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 the purified product was used as a template for a second round of PCR. In this stage, index primers were ligated to both ends of the first-round PCR product to form a TruSeq dual-index library. The library was purified using magnetic beads and quantified using Qubit, with sequencing performed using an Illumina MiSeq PE300.

[0069] The 10 sequences with the highest abundance were selected for abundance analysis, and the abundance packing plot is shown below. Figure 2 Select the most productive gene sequence that ranks highest as the target gene sequence.

[0070] 2. Analysis of gene sequencing results of the variable domains of the heavy and light chains of monoclonal antibodies

[0071] Based on the sequencing results, the genes of the variable domains of the heavy and light chains of the monoclonal antibody 6E8 were analyzed using NCBI-IgBLAST (v1.17.0) and the IMGT database (http: / / www.imgt.org / ). The analysis showed that the full-length gene of the variable region of the heavy chain of the monoclonal antibody 6E8 is 372 bp (SEQ ID NO.8), encoding 124 amino acid residues (SEQ ID NO.6), and includes the variable regions CDR1-H (SEQ ID NO.1), CDR2-H (SEQ ID NO.2), and CDR3-H (SEQ ID NO.3); the full-length gene of the variable region of the light chain is 321 bp (SEQ ID NO.9), encoding 107 amino acid residues (SEQ ID NO.7), and includes the variable regions CDR1-L (SEQ ID NO.4), CDR2-L (YAS), and CDR3-L (SEQ ID NO.5).

[0072] The specific sequence is as follows:

[0073] CDR1-H: GYTFTNYV;

[0074] CDR2-H: FNPYSDVI;

[0075] CDR3-H: ARAGNYFDYDGPHVMDY;

[0076] CDR1-L: QSIGTN;

[0077] CDR2-L: YAS;

[0078] CDR3-L: QQSNSWPFT;

[0079] Heavy chain variable region: EFQLQQSGPELVTPGASVRMSCKASGYTFTNYVIHWVKQKPGQGLEWIGYFNPYSDVIKYNEKFKGRATLTSDKSSSTAYMELSSLTSEDSAVYYCARAGNYFDYDGPHVMDYWGQGTSVTVSS;

[0080] Light chain variable region: DILLTQSPVILSVSPGERVSLSCRARQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNSWPFTFGSGTKLEIK;

[0081] Heavy chain variable region gene: GAGTTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAACGCCTGGGGCTTCAGTGAGGATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTATCCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACAGTGATGTTATTAAGTATAATGAGAAGTTCAAAGGCAGGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGCGGGCAACTACTTTGATTACGACGGACCCCATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA;

[0082] Light chain variable region gene: GACATCTTGCTGACTCAGTCACCAGTCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTCTCCTGCAGGGCCAGACAGAGCATTGGCAAAACATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTG AGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA.

[0083] Example 3: Small Ruminant Disease Virus Antibody Blocking ELISA Detection Kit and its Use

[0084] 1. Instructions for using commercially available (ID Vet) cELISA kits

[0085] 1.1 Solution Preparation

[0086] (1) Washing solution: Dilute 20× concentrated washing solution 20 times with deionized water.

[0087] (2) Enzyme conjugate: Dilute the 10× concentrated enzyme conjugate 10 times with diluent No. 4.

[0088] 1.2 Operating Procedures

[0089] (1) Add 25 μL of diluent No. 13 to each well, then add 25 μL of PPRV positive control (PC) to each well A1 and B1, add 25 μL of PPRV negative control (NC) to each well C1 and D1, and add 25 μL of the sample to be tested to the remaining wells;

[0090] (2) Gently shake to mix, and incubate at 37±3°C for 45±4 minutes;

[0091] (3) Add 300 μL of washing solution to each well and wash 3 times;

[0092] (4) Add 100 μL of enzyme conjugate to each well and incubate at room temperature (21±5°C) for 30±3 minutes;

[0093] (5) Add 300 μL of washing solution to each well and wash 3 times;

[0094] (6) Add 100 μL of substrate solution to each well and incubate at room temperature (21±5°C) in the dark for 15±2 minutes;

[0095] (7) Add 100 μL of stop solution to each well to terminate the colorimetric reaction;

[0096] (8) Measure the OD value at a wavelength of 450 nm, and calculate the blocking rate (S / N%) of each sample according to the formula: S / N% = (OD 样品 / Average OD NC )×100; Experimental criteria: average OD NC >0.7, average OD PC / Average OD NC <0.3. Result interpretation: S / N%>60%, antibody negative; S / N%≤50%, antibody positive; 50%<S / N%≤60%, suspicious.

[0097] Six positive and three negative serum samples from our laboratory were selected using commercially available cELISA kits for use in establishing a subsequent blocking ELISA detection method.

[0098] 2. The monoclonal antibody 6E8 described in this invention is used for the establishment and detection of blocking ELISA.

[0099] Based on the principle of blocking ELISA, this invention uses recombinant PPRV N protein as the coating antigen, and the enzyme-labeled monoclonal antibody HRP-6E8 against PPRV N protein prepared in Example 1 of this invention as the detection antibody. Positive and negative sera were detected respectively, and the detection effect of enzyme-labeled monoclonal antibody HRP-6E8 prepared in Example 1 of this invention for blocking ELISA was compared.

[0100] 2.2 Optimal antigen-antibody reaction concentration

[0101] (1) Coating: Dilute the recombinant protein PPRV-N with coating solution to 0.1, 0.05, 0.025, 0.0125 μg / mL, add 100 μL / well to the enzyme-labeled reaction plate, and incubate at 4℃ for more than 16 h.

[0102] (2) Washing: Discard the coating solution and wash 3 times with PBST.

[0103] (3) Blocking: Add 120 μL / well blocking solution, incubate at 37°C for 60 min, discard the blocking solution, and dry.

[0104] (4) Adding samples: Add 100 μL of control serum or test serum diluted 1:1 to wells and incubate at 37°C for 45 min.

[0105] (5) Washing: Discard the reaction solution and wash 3 times with PBST.

[0106] (6) Add HRP-6E8 diluted in different proportions (1:1000, 1:2000, 1:4000, 1:8000), 100 μL / well, gently shake to mix, and incubate at 37°C for 30 min.

[0107] (7) Washing: Discard the reaction solution and wash 3 times with PBST.

[0108] (8) Color development: Add 100 μL of TMB substrate per well and incubate at 37°C for 10 min.

[0109] (9) Termination: Add 50 μL of stop solution per well and measure the absorbance OD value. 450 Calculate the S / N% value according to the formula.

[0110] S / N% = (Sample OD) 450 / Negative control OD 450 ) × 100%

[0111] The results showed that when the P / N value was the minimum and the blocking ELISA principle was met, the optimal coating concentration of recombinant protein PPRV-N was 0.05 μg / mL (Table 2), and the optimal working concentration of HRP-6E8 was 1:4000 (Table 3).

[0112] Table 2 Screening results of antigen coating concentration

[0113] Table 3. Screening results for working concentrations of enzyme-labeled monoclonal antibodies

[0114] 2.3 Screening of blocking solutions

[0115] Blocking solutions of 1% BSA, 1% casein, 0.8% gelatin, and 2% bovine serum were added at a rate of 120 μL per well for blocking. Two replicate assays were performed using negative and positive sera to measure OD. 450 The P / N ratio was calculated to screen the optimal blocking solution. The results showed that 2% bovine serum solution had the best blocking effect (Table 4).

[0116] Table 4 Screening results of blocking fluids

[0117] 2.4 Determination of Critical Values

[0118] Based on the determined optimal antigen coating amount, blocking buffer, sample volume, working concentration of enzyme-labeled monoclonal antibody, reaction time, and substrate, an ELISA antibody detection method for blocking peste des petits ruminants virus (PPR) was established. This method and an ID-Vet cELISA kit were used to detect PPR in 225 sheep serum samples with clear backgrounds. The accuracy and critical values ​​of the established method were analyzed using MedCalc statistical software. Statistical analysis showed that the area under the ROC curve for the established blocking ELISA method was 0.985 (…). Figure 3 When the Youden index is at its maximum (0.916), the S / N% is ≤43.3%, corresponding to a sensitivity of 97.6% and a specificity of 93.9%. Figure 3 According to the criteria in section B, the critical value S / N% for determining whether an antibody is positive or negative is 43.3%, that is, S / N% ≤ 43.3% indicates antibody positivity, and S / N% > 43.3% indicates antibody negative.

[0119] 2.6 Specificity test

[0120] The specificity of the monoclonal antibody in blocking the ELISA reaction was determined by detecting positive sera for PPRV, sheep pox virus (CaPV), foot-and-mouth disease virus type O (FMDV-O), and bluetongue virus (BTV) and anti-pET30a / BL21 antibody under optimized conditions. The results showed that the blocking ELISA method was negative for FMDV-O, BTV, SPPV positive sera, and pET30a / BL21 antibody (Table 5), consistent with the results of the ID-Vet kit. This indicates that the blocking ELISA has good specificity.

[0121] Table 5 Specific detection results

[0122] 2.7 Sensitivity Experiment

[0123] PPRV-positive serum was diluted 2, 4, 8, 16, 32, and 64 times, and detected using the established blocking ELISA. The results showed that the blocking ELISA method was still positive for 8-fold diluted PPRV-positive control serum (Table 6), indicating that the established blocking ELISA has good sensitivity.

[0124] Table 6 Sensitivity test results

[0125] 2.8 Comparative Experiment

[0126] The established blocking ELISA and ID-Vet kits were used to simultaneously test 221 serum samples, and the concordance rate between the blocking ELISA and ID-Vet kits was compared. The results showed that the two methods yielded consistent results for 204 serum samples, with a concordance rate of 92.31% between the blocking ELISA and ID-Vet kits (Table 7).

[0127] Table 7 Comparison and detection results

[0128] 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 specific monoclonal antibody against the N protein of peste des petits ruminants virus, said monoclonal antibody comprising an antibody heavy chain and an antibody light chain, characterized in that, The variable region CDR of the antibody heavy chain consists of CDR1 as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; The variable region CDR of the antibody light chain consists of CDR1 with an amino acid sequence as shown in SEQ ID NO.4, CDR2 with an amino acid sequence of YAS, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

5.

2. The monoclonal antibody as described in claim 1, characterized in that, The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.6, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

7.

3. A nucleic acid, characterized in that, The nucleic acid encodes the antibody heavy chain and antibody light chain of the monoclonal antibody according to any one of claims 1-2.

4. The nucleic acid as described in claim 3, characterized in that, The nucleotide sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.8, and the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

9.

5. A biological material comprising the nucleic acid of claim 3 or 4, said biological material comprising an expression cassette, transposon, plasmid vector, viral vector, or host cell.

6. The application of the monoclonal antibody as described in claim 1 or 2 in the preparation of a reagent for detecting peste des petits ruminants virus (PPR), or in the detection of natural infectivity of PPR for non-disease diagnosis purposes, or in the study of the function of the PPR N protein.

7. The application as described in claim 6, characterized in that, The reagent is a test strip or a reagent kit.

8. The application as described in claim 7, characterized in that, The test strip is a colloidal gold immunochromatographic test strip, and the kit is a blocking ELISA kit.

9. An enzyme conjugate, characterized in that, The enzyme conjugate includes: (i) The monoclonal antibody as described in claim 1 or 2; (ii) and horseradish peroxidase conjugated with the monoclonal antibody in (i).

10. A blocking ELISA kit for detecting antibodies against peste des petits ruminants virus, characterized in that, The kit includes the monoclonal antibody as described in claim 1 or 2.

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