Kit for detecting inner-Robis sheep disease virus

Abstract

The invention relates to a kit for detecting an internally-running sheep disease virus, and belongs to the technical field of internally-running sheep disease virus detection. The invention aims to solve the problem of accurately detecting the inner-Robis sheep disease virus. The invention provides application of a monoclonal antibody of an internally-running sheep disease virus in preparation of a kit for detecting the internally-running sheep disease virus. The kit comprises the monoclonal antibody of the internally-running sheep disease virus, and the heavy chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO.8. The invention also provides application of the monoclonal antibody of the internally-running sheep disease virus in preparation of the kit for detecting the internally-running sheep disease virus. The light chain amino acid sequence of the monoclonal antibody is as shown in SEQ ID NO. 9. The kit is mainly used for detecting whether the sheep body is infected with the internally-running sheep disease virus or not.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Nairobi sheep disease virus detection, and particularly relates to a kit for detecting Nairobi sheep disease virus. BACKGROUND

[0002] Nairobi sheep disease virus (NSDV) is an important tick-borne virus belonging to the Orthonairovirus genus of the Nairoviridae family, which can cause Nairobi sheep disease (NSD) and lead to more than 90% mortality of sheep and goats. The virus mainly infects small ruminants such as sheep and goats, causing serious economic losses to the livestock industry. NSDV infection in sheep can cause the diseased sheep to appear depression, high fever, progressive weight loss, severe diarrhea (some cases show characteristic bloody watery stool) and respiratory symptoms. The acute course and high mortality of the disease make it one of the important diseases affecting the health of ruminants. However, there is currently a lack of effective commercial prevention vaccines and specific treatment drugs for NSD, and this prevention and control situation highlights the urgency of carrying out related research.

[0003] Nairobi sheep disease virus (NSDV) is a hemorrhagic fever virus transmitted by ticks, which can cause high mortality in sheep and goats. The virus was first reported in an epidemic in Kenya in 1917, when it caused 90% mortality in sheep populations. It is listed as a notifiable animal disease by the World Organization for Animal Health (WOAH). NSDV and Crimean-Congo hemorrhagic fever virus (CCHFV) belong to the same Nairobi virus genus, and the polymerase and glycoprotein encoded by the genome have similar immune escape mechanisms, suggesting potential zoonotic risk. When susceptible animals are introduced from non-epidemic areas to epidemic areas, they often trigger large-scale epidemics.

[0004] Nairobi sheep disease (NSD) is a tick-borne disease, which is mainly transmitted by Dermacentor appendiculatus in East Africa and Haemaphysalis intermedia in Asia, and has a wide range of public hazards. After the virus invades the host body through tick bites, it targets vascular endothelial cells to trigger systemic inflammatory response, and the clinical manifestations are hemorrhagic gastroenteritis, fever and abortion. Traditionally, the virus mainly prevails in East Africa and South Asia. In recent years, NSDV has been detected in tick serum collected in Liaoning Province, Jilin Province, Heilongjiang Province and Hubei Province of China, which indicates that climate change may promote the spread of tick species to temperate zones. Studies have shown that the widespread transmission of NSDV in China may pose a serious potential risk, and this virus not only poses a major threat to the breeding production of China's animal husbandry, but also may have a profound negative impact on the economic value of the related industry chain. Therefore, it is urgent to detect the virus. SUMMARY

[0005] Therefore, the present application aims to solve the problem of accurately detecting whether the sheep is infected with Nairobi sheep disease virus.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical scheme: a monoclonal antibody of Nairobi sheep disease virus (NSDV) is applied to prepare a kit for detecting the Nairobi sheep disease virus, wherein the kit comprises a monoclonal antibody of the Nairobi sheep disease virus, the amino acid sequence of CDR-H1 of the heavy chain variable region of the monoclonal antibody of the Nairobi sheep disease virus is shown in SEQ ID NO. 3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO. 4; the amino acid sequence of CDR-H3 is shown in SEQ ID NO. 5; the amino acid sequence of CDR-L1 of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO. 6; the amino acid sequence of CDR-L2 is LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO. 8.

[0007] The present application provides a kit for detecting Nairobi sheep disease virus, wherein the kit comprises a monoclonal antibody of the Nairobi sheep disease virus, the amino acid sequence of CDR-H1 of the heavy chain variable region of the monoclonal antibody of the Nairobi sheep disease virus is shown in SEQ ID NO. 3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO. 4; the amino acid sequence of CDR-H3 is shown in SEQ ID NO. 5; The amino acid sequence of the CDR-L1 of the light chain variable region of the Nairobi sheep disease virus monoclonal antibody is shown in SEQ ID NO. 6; the amino acid sequence of CDR-L2 is LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO. 7.

[0008] With further limitation, the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO. 8; and the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO. 9.

[0009] With further limitation, the genetic sequence encoding the amino acid sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO. 10; and the genetic sequence encoding the amino acid sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO. 11.

[0010] With further limitation, the kit further comprises a goat anti-mouse HRP-IgG.

[0011] With further limitation, the kit further comprises a positive control and a negative control, wherein the positive control is NSDV positive serum and the negative control is NSDV negative serum.

[0012] With further limitation, the kit further comprises a blocking solution, wherein the blocking solution is skimmed milk.

[0013] With further limitation, the kit further comprises TMB color developing solution and H2SO4 stop solution.

[0014] With further limitation, the dilution ratio of the Nairobi sheep disease virus monoclonal antibody is 1:320, and the dilution ratio of the goat anti-mouse HRP-IgG is 1:8000.

[0015] The present application provides a method for non-diagnostic and detection using the above-mentioned kit, characterized in that the method is: Step 1: Antigen coating: dilute NSDV N protein at a ratio of 1:800 using CBS coating solution, add 100 μL / well to an enzyme-labeled plate, wash the plate with PBST, and block with 5% skimmed milk; Step 2: Add 50 μL of the sheep serum sample to be detected, then add an equal volume of the Nairobi sheep disease virus monoclonal antibody of claim 2; discard the liquid in the well, wash the plate with 200 μL of PBST, and block with 5% skimmed milk; wash the plate with PBST; Step 3: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody, incubate at 37℃ for 1 h; discard the liquid in the well, and wash the plate with PBST; Step 4: Add 100 μL of TMB color developing solution, incubate at room temperature for 15 min in the dark; Step 5: color termination: 100 μL / well of 2 M H2SO4 termination solution; Step 6: enzyme reader reading: OD450nm value is read by an enzyme reader at a wavelength of 450 nm; and PI value is calculated; when PI≥35.06%, it is determined to be positive.

[0016] Compared with the prior art, the beneficial effects of the present application are: a monoclonal antibody is obtained, which is named 6D12. The Western blot and IFA experimental results show that the monoclonal antibody can specifically bind to NSDV, and a high-titer and high-specificity monoclonal antibody against NSDV nucleocapsid protein is successfully prepared. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 PCR amplification result of N gene; Note: M: Marker, 1: negative control, 2: PCR product; Figure 2 Bacterial liquid PCR identification result; Note: M: Marker, 1: positive control, 2: negative control, 3, 4: different strains selected; Figure 3 Restriction enzyme identification result of recombinant plasmid pCold-Gst-NP; Note: M: Marker, 1: restriction enzyme vector; Figure 4 SDS-PAGE identification result of protein expression result; Note: M: Marker 1: before induction, 2: after induction, 3: ultrasonic supernatant, 4: ultrasonic precipitate; Figure 5 SDS-PAGE identification result of protein purification result; Note: M: Marker 1: before column, 2: after column, 3: washing, 4: eluate; Figure 6 Western blot identification result of NSDV N protein; Note: M: Marker 1: negative control, 2: target protein; Figure 7 SDS-PAGE identification result of 6D12 monoclonal antibody after mouse ascites purification; Note: M: protein Marker, 1: unpurified monoclonal antibody, 2: purified monoclonal antibody; Figure 8 IFA identification result (150 x) of monoclonal antibody; A: after inoculation of NSDV, IFA test is carried out with 12D6 as a primary antibody; B: negative control; Figure 9Figure of Western blot identification result of anti-NSDV 6D12 monoclonal antibody; Note: M: Protein Marker 1: pCold-Gst empty vector 2: Target protein; Figure 10 Figure of competitive detection result of monoclonal antibody. DETAILED DESCRIPTION

[0018] Example 1. Preparation of monoclonal antibody 1. N protein primer design: By comparing and analyzing the full genome sequence of Nairobi sheep disease virus (NSDV) included in the NCBI database, a pair of primers for amplifying N gene were designed. The primer pair can amplify a 1449 bp long N gene fragment. The design and synthesis of the primers were completed by Rui Boxingke Biotechnology Co., Ltd. (located in Harbin), and the specific primer sequence information is as follows: NSDV-F: (XhoI) CTCGAG ATGCAGAATCAGATT (SEQ ID NO. 1); NSDV-R: (ECORI) GAATTC TTAAATAATCTGAATGCTGGTCGC (SEQ ID NO. 2).

[0019] 2. Construction and identification of recombinant plasmid: PCR amplification of NSDV N gene: The cDNA of NSDV strain was used as a template for PCR amplification, and the reaction program was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, 35 cycles; final extension at 72°C for 5 min; the PCR system was as follows: NSDV-F, 1 μL; NSDV-R, 1 μL; template, 2 μL; 2x Rapid TaqMaster Mix, 12.5 μL; RNase-free Water to complete 20 μL; the PCR amplification product was subjected to 1% agarose gel electrophoresis, and the size of the target band was observed to be normal. The gel was labeled and reserved.

[0020] Extraction of nucleic acid of NSD strain for RT-PCR. The N gene of NSDV was amplified by specific primers, as shown in Figure 1 the agarose gel electrophoresis experiment result showed that the size of the amplification product was about 1500 bp, which was consistent with the expected size, indicating that the N gene amplification was successful.

[0021] 3. Gene and vector connection and transformation: The target gene fragment and linearized vector were gel purified, respectively, and the In-Fusion high-efficiency ligase system was used for directional ligation to obtain the pCold-Gst-NSDV-N recombinant plasmid. The specific reaction system is as follows: 5x In-Fusion HD Enzyme Premix, 2 μL; pClod-GST, 1 μL; NSDV-N, 1 μL; RNase-free Water, complete to 10 μL, and the reaction condition is set to 50°C constant temperature reaction for 15 min. After the completion of the ligation reaction, 5 μL of the ligation product was immediately transformed into DH5α chemically competent cells.

[0022] 4. pCold-Gst-NSDV-N recombinant plasmid identification: After 1% agarose gel electrophoresis analysis, the amplified product of one colony selected after PCR showed a specific amplification band at about 1500 bp position, as shown in Figure 2 , which is consistent with the expected size of N gene fragment. The length of N gene is 1457 bp, and the preliminary determination is positive clone. Restriction enzyme double digestion of recombinant plasmid: the recombinant plasmid pCold-Gst-NSDV-N was treated with Kpn I and EcoR I restriction enzymes, and the nucleic acid electrophoresis detection showed two specific bands with molecular weights of about 5050 bp and 1457 bp, which were consistent with the expected size. The results show that the N gene of NSDV has been accurately inserted between the Kpn I and EcoR I enzyme cutting sites of the pCold-Gst prokaryotic expression vector Figure 3 ).

[0023] 5. NSDV N protein induced expression: the pCold-Gst-NSDV-N recombinant plasmid was introduced into BL21 competent cells by heat shock transformation method, and the uninduced control group sample. The pre-configured IPTG solution (100 mm storage solution) was added to the remaining bacterial liquid, so that the final concentration reached 1 mm. The induced culture was lysed by ultrasonic cell disrupter. The supernatant of the bacterial liquid was purified by column, and protein electrophoresis was performed on 12% protein gel. After completing SDS-PAGE electrophoresis, the protein was efficiently transferred from the gel to the PVDF membrane, and after blocking, the membrane was immersed in NSDV positive serum primary antibody diluted by 5% skimmed milk powder at 1:10. After incubation of the primary antibody, the secondary antibody was incubated, and then the signal was collected by chemiluminescence imaging system to obtain clear protein detection results.

[0024] Results: SDS-PAGE experiment was performed on the pre-induction, post-induction, bacterial supernatant and bacterial precipitate, which proved that the target protein was successfully produced, and mainly existed in the form of solubility in the lysate supernatant Figure 4 ).

[0025] NSDV N protein purification SDS-PAGE electrophoresis was used to identify the purified NSDV N protein. The results are shown in Figure 5 Figure 2, the purified product showed a single clear band at about 79 kDa, which was consistent with the expected size, and the band was not significantly contaminated with impurities, indicating that the protein purification was good and met the requirements of subsequent experiments.

[0026] NSDV N protein identification: The results of Western blot are shown in Figure 6 Figure 3, after SDS-PAGE, the protein was transferred to a PVDF membrane, and NSDV positive serum was used as a primary antibody. A clear band appeared at 79 kDa, which was consistent with the expected size, confirming that the recombinant N protein had natural antigen epitopes and reacted specifically with N protein.

[0027] 6. Preparation of NSDV monoclonal antibody (1) Animal immunization: NSDV-N protein was used as an immunogen to immunize 6-week-old male mice 3 times with an interval of 14 days between each immunization. The immunization method was subcutaneous multiple point injection. For the first immunization, 100 μg of NSDV N protein was emulsified with Freund's complete adjuvant at a ratio of 1:1; for the 2nd and 3rd immunizations, 100 μg of NSDV N protein was emulsified with Freund's incomplete adjuvant at a ratio of 1:1. Seven days after the 3rd immunization, mouse serum was collected and serum titer was detected by indirect ELISA. The mouse with the highest serum titer was selected for booster immunization, and the booster immunization method was intraperitoneal injection of NSDV N protein. Three days after booster immunization, cell fusion experiment was performed.

[0028] (2) Preparation of positive and negative serum: cheek blood was collected from unimmunized healthy mice and mice immunized with NSDV N protein, and centrifuged at 4000 x g at 4°C for 10 min. After centrifugation, the upper serum was collected to obtain NSDV negative serum and NSDV positive serum.

[0029] (3) Serum titer determination: After 3 immunizations, cheek blood was collected from mice to collect serum, and serum titer was determined by indirect ELISA method. NSDV N protein was coated on the enzyme-labeled plate at a concentration of 1:800, and the enzyme-labeled plate was incubated at 4°C overnight to ensure that the protein was fully adsorbed on the bottom of the plate. Discard the coating solution, block with 5% skim milk at 37°C for 1 hour, wash the plate 3 times with PBST on a shaker for 5 minutes. Dilute the serum 8 times, 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:640000, dilute by ratio, then add 100 μL of diluted solution to each well of the enzyme-labeled plate, incubate at 37°C for 1 hour, discard the serum, wash the plate 3 times with PBST on a shaker for 5 minutes. Add 100 μL of HRP goat anti-mouse IgG secondary antibody (1:5000 dilution) and incubate at 37°C for 1 hour, discard the secondary antibody, wash the plate 3 times with PBST on a shaker for 5 minutes. Finally, add 100 μL of TMB color developing solution, incubate at room temperature for 15 minutes, then immediately add 100 μL of 2 M H2SO4 to stop the reaction, and immediately measure the OD 450nm .

[0030] After the third immunization, blood was collected from 4 immunized mice and the serum was separated. The serum was diluted by ratio from 1:500 to 1:640000, and the serum titer was determined by indirect ELISA method. The positive value determination standard is 2 times the OD 450 of the negative serum. According to the results, the serum titers of the 4 mice all reached the positive value. Since the serum titer of No. 4 mouse was the highest, it was given a booster immunization, and 3 days later, cell fusion experiment was performed.

[0031] Table 1 Mouse serum titer determination

[0032] (4) Preparation of SP2 / 0 cells: 5 days before cell fusion, SP2 / 0 myeloma cells were recovered in advance, and the culture medium was changed to 20% FBS 1640 medium for culturing SP2 / 0 myeloma cells. When the cell density reached 80-90% confluence, the cells were passaged at a ratio of 1:3.

[0033] (5) Preparation of spleen cells: BALB / c mice with serum titer detection up to standard were selected, and the spleen was transferred to a new culture dish containing 10 mL of serum-free 1640 medium. Spleen cells were flushed out by hydraulic impact method. Cell counting was performed using a hemocytometer under an inverted microscope, and the proportion of living cells was required to be ≥90%, and the cell density was adjusted to 1×10 7Reserve.

[0034] (6) Cell fusion: the spleen cells and SP2 / 0 cells were mixed in a new 50 mL centrifuge tube at an optimal ratio of 1:8, gently inverted and mixed for 10 times to ensure sufficient cell contact. 1 mL of preheated 45% PEG fusion agent (containing 5% DMSO) was added and incubated in a 37°C water bath for 2 min. The fusion reaction was terminated using the gradient dilution method.

[0035] (7) After 5 days of fusion, observe whether the cells have successfully fused under an inverted microscope. When the cells in the plate grow to about 1 / 3, remove the supernatant and use the indirect ELISA method. According to the indirect ELISA detection results, 1) antigen coating: coat the NSDVN protein on the enzyme-labeled plate at a ratio of 1:800, 100 μL / well, 4°C overnight to ensure that the protein is fully adsorbed on the plate bottom. 2) Blocking: block with 5% skim milk at 37°C for 1 h to block non-specific binding sites. 3) Primary antibody incubation: wash with PBST for 3 times, 5 min each time, add 100 μL of monoclonal antibody supernatant to each well, incubate at 37°C for 1 h, 4) secondary antibody incubation: wash with PBST for 3 times, 5 min each time, then dilute HRP goat anti-mouse IgG1:5000 and incubate at 37°C for 1 h, 5) color development: wash with PBST for 3 times, 5 min each time, add 100 μL of Biyun Tian TMB color developing solution to each well, incubate at room temperature for 15 min in the dark, 6) color development termination and reading: finally, add 100 μL of 2 M H2SO4 to the enzyme-labeled plate to terminate the reaction, and immediately measure the OD450nm of each well. Perform preliminary screening according to the indirect ELISA detection results, select strong positive wells with high antibody secretion levels, and mark them for subsequent subcloning experiments. Use cell counting method for subcloning, resuspend the original well cells in 100 μL of HT medium, mix 20 μL of medium with 20 μL of trypan blue at a ratio of 1:1, and perform cell counting. Calculate and plate 200 μL containing 300 cells for cell culture. Select strong positive wells with high antibody secretion levels and mark them for subsequent subcloning experiments. Use cell counting method for subcloning, resuspend the original well cells in 100 μL of HT medium, mix 20 μL of medium with 20 μL of trypan blue at a ratio of 1:1, and perform cell counting. Calculate and plate 200 μL containing 300 cells for cell culture.

[0036] After each subcloning, select a single cell for detection, detect the cell supernatant, and detect it by indirect ELISA method. When the positive rate reaches 100%, use indirect immunofluorescence experiment for the second screening, use indirect ELISA and IFA experiment for positive well screening, and finally screen out the strain monoclonal antibody, named 6D12 (8) Ascites preparation: 7 days before the ascites preparation experiment, 12-week-old BALB / c female mice were injected with 1 mL of sterile liquid paraffin into the abdominal cavity, and 7 days later, the hybridoma cells were blown off with serum-free 1640 medium, and 10 5 cells were taken for intraperitoneal injection by cell counting, and the mice were observed daily for changes in the abdominal cavity. After intraperitoneal injection for one week, the mice had a significant bulge in the abdomen and a fluctuating feeling, and ascites was taken using an ascites needle, and the collected ascites was centrifuged at 2000 r / min at 4°C for 30 min, and the supernatant was collected and stored at -20°C.

[0037] (9) Ascites purification: The pretreated ascites sample was purified by using Protein G Resin.

[0038] Example 2. 1. Monoclonal antibody titer determination: The purified ascites was diluted by 1 : 200 to 1 : 25600, and the ascites titer was detected by indirect ELISA method, and negative control was prepared, and HRP goat anti-mouse IgG secondary antibody was diluted by 1 : 5000, 100 μL was added to each well, and incubated at 37°C for 1 h, 100 μL TMB color developing liquid was added, and color development was carried out at room temperature for 15 min; immediately add 100 μL 2 M H2SO4 to terminate the reaction. The OD 450nm values of each dilution were read by a microplate reader.

[0039] 2. Monoclonal antibody subclass identification: The prepared ascites was used as a primary antibody, and the operation was carried out according to the operation instruction of the Biyun Tian kit: the corresponding Ag specifically combined with the monoclonal antibody was coated with CBS buffer in the enzyme-labeled plate, 100 ng was coated in each well, and incubated at 4°C on a shaking bed for 12 h, and the coating solution was discarded, and PBST was washed once for 5 min. Add 200 μL ELISA blocking solution to each well, and incubate at 37°C for 1 h, and wash with PBST for three times, each for 5 min; dilute the prepared ascites with PBS by 1 : 1000, add 100 μL to each well, and incubate at 37°C for 1 h, and wash with PBST for three times, each for 5 min; add 8 kinds of enzyme labels, 2 wells for each kind, 100 μL for each well, and incubate at 37°C for 1 h, and wash with PBST for five times, each for 5 min; add TMB color developing liquid and incubate at 37°C for 30 min, and immediately add 100 μL 2 M H2SO4 to terminate the reaction. The OD 450nm values were read by a microplate reader, and the results were judged.

[0040] 3. Indirect immunofluorescence identification: trypsinized BSR cells were plated in 6-well plates, when the cells reached about 90%, 1 ug / mL NSDV in 2% FBS medium was used for incubation for 2 h, after 2 h, the medium was discarded, 100 μL of 2% FBS medium was added for continuous culture, and the medium was discarded after 72 h. Cell fixation: 500 μL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 20 min, and the fixing solution was discarded. Membrane permeation treatment: 0.02% trion X-100 was used for membrane permeation at room temperature for 20 min, blocking treatment: 1% BSA was used for blocking at room temperature for 1 h, and PBS was used for washing 3 times; primary antibody incubation: 100 μL of monoclonal antibody was added to each well, and incubation was carried out at 4°C overnight, and PBS was used for washing 3 times the next day; secondary antibody incubation: 100 μL of 488 goat anti-mouse secondary antibody was added to each well, diluted with PBS at 1:1000, incubated at room temperature for 1 h, and washed with PBS 3 times in the dark; DAPI staining: DAPI was diluted at 1:1000, incubated at room temperature for 10 min, and washed with PBS 3 times; and the experimental results were observed by indirect immunofluorescence instrument.

[0041] 4. Single antibody reaction identification, sample preparation: 100 μL of prepared NSDV N protein was added to an EP tube containing 25 μL of 5x loading buffer and mixed, and then the protein was denatured by boiling in a metal bath at 100°C for 10 min. The prepared protein sample was subjected to Western-blot experiment. The specific operation is as follows: the prepared 12% SDS-PAGE gel was placed in the electrophoresis tank, and SDS electrophoresis solution was added, and the prepared protein mark and protein sample were spotted into the gel well at a loading amount of 20 μL per well, and electrophoresis experiment was carried out, and the program was 80V for 30 min and 120V for 1 h. The membrane transfer was soaked in the membrane transfer solution with sponge and filter paper for standby; the PVDF membrane was cut to the appropriate size and soaked in anhydrous methanol for 1-2 min to activate the PVDF membrane. The protein gel was taken out and the excess gel was cut off. The membrane transfer was carried out using a rapid wet transfer instrument, and after membrane transfer, 5% skim milk was used for room temperature blocking for 1 h, and then PBST was used for rapid shaking washing 3 times, 5 min each time. The monoclonal antibody was diluted at 1:1000 with 5% skim milk as the primary antibody, and incubated at 4°C overnight, and the next day, PBST was used for rapid shaking washing 3 times on a shaking table, 5 min each time. The secondary antibody was diluted at 1:5000 with HRP goat anti-mouse IgG secondary antibody, and incubated at room temperature for 1 h, and then PBST was used for rapid shaking washing 3 times on a shaking table, 5 min each time. The western-blot instrument was used for gel exposure experiment.

[0042] Results: The hybridoma single antibody cells were injected into the abdominal cavity of mice, and then the ascites induced was purified by Protein G affinity chromatography. The purified product was analyzed by SDS-PAGE electrophoresis, and the results are shown in Figure 7 Figure 1. After purification, specific bands appeared at the heavy chain (50 kDa) and light chain (20 kDa), respectively, and no obvious impurity bands were observed, indicating good purification effect.

[0043] Monoclonal antibody titer determination The purified monoclonal antibody was subjected to monoclonal antibody titer detection, and the detection results showed that the ascites produced by the 1 strain of hybridoma cells had a titer of more than 1:12800 (the OD value of the negative control was 0.275, and the determination standard was that 2 times of the negative control was determined as positive). 450nm

[0044] Table 2 Monoclonal antibody serum titer determination

[0045] Monoclonal antibody subclass identification The monoclonal antibody secreted by the hybridoma cell strain 6D12 was subjected to subtype analysis using the IgG class / subclass identification kit of Biyun Tian, and the results are as follows: the heavy chain of the 6D12 monoclonal antibody was IgG I subtype, and the light chain type was IgG kappa.

[0046] Monoclonal antibody IFA identification: After inoculating BSR cells with NSDV, the purified monoclonal antibody was identified by IFA, and mouse negative serum was used as negative control. The monoclonal antibody had specific green fluorescence, and the negative control had no fluorescence, indicating that the antibody could specifically bind to NSDV. The results are shown in Figure 8 Figure 2.

[0047] Western blot results showed that the prepared 6D12 monoclonal antibody was used as a primary antibody for incubation, and the NSDV N protein sample showed a target band at 79 kDa as Figure 9 shown in Figure 3, while no band was found in the pCold-Gst empty vector, verifying the specificity of the monoclonal antibody, indicating that the 2 strains of monoclonal antibodies could recognize NSDV N protein and had good specificity.

[0048] Example 3.612D Monoclonal Antibody Sequencing 1. Amino acid sequence: (1) The amino acid sequence of the CDR-H1 of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 3: GYTFTRYW (2) The amino acid sequence of CDR-H2 is shown in SEQ ID NO. 4: INPTNGHI; ​(3) the amino acid sequence of CDR-H3 is as shown in SEQ ID NO. 5: SANIY; (4) the amino acid sequence of CDR-L1 of the light chain variable region of the monoclonal antibody is as shown in SEQ ID NO. 6: QSLLYSNGKTY; (5) the amino acid sequence of CDR-L2 is as shown below: LVS; (6) the amino acid sequence of CDR-L3 is as shown in SEQ ID NO. 7: VQGTHFPQT; (7) the amino acid sequence of the heavy chain of the monoclonal antibody is as shown in SEQ ID NO. 8: QVQLQQPGAELVKPGAAVKLSCKASGYTFTRYWMHWVKQRPGQGLEWIGEINPTNGHINYNEKFKSKAILTVDKSSSTAYMQLSSLTSEDSAVYYCSANIYWGQGTLVTVSA; the amino acid sequence of the light chain of the monoclonal antibody is as shown in SEQ ID NO. 9: DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK.

[0049] 2. nucleic acid sequences: (1) heavy chain encoding gene: SEQ ID NO. 10 CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTGCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGGTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTACCAACGGTCATATTAATTACAATGAGAAGTTCAAGAGCAAGGCCATCCTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAGCCAATATTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA; (2) light chain encoding gene: SEQ ID NO. 11 GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTCCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0050] Example 4. Preliminary establishment of competitive ELISA method I. Antigen coating: NSDV N protein was diluted according to 1:800 with CBS coating solution, mixed well, and 100 μL / well was added to the enzyme-labeled plate and incubated at 4°C overnight (12-16 h). Washing the plate: discard the liquid in the wells, add 200 μL PBST per well, shake quickly on the shaker for 5 min, wash the plate 3 times, and pat the enzyme-labeled plate dry after washing. Blocking: block with 5% skim milk, 200 μL / well, cover with plastic film, and block at 37°C for 1 h. Washing the plate: discard the liquid in the wells, add 200 μL PBST per well, shake quickly on the shaker for 5 min, wash the plate 3 times, and pat the enzyme-labeled plate dry after washing. Incubate the primary antibody: add 50 μL NSDV positive serum to each well, then add an equal volume of monoclonal antibody supernatant, mix gently, cover with plastic film, and incubate at 37°C for 1 h, while preparing negative serum controls. Washing the plate: discard the liquid in the wells, add 200 μL PBST per well, shake quickly on the shaker for 5 min, wash the plate 3 times, and pat the enzyme-labeled plate dry after washing. Incubate the secondary antibody: use HRP-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution), add 100 μL per well. Incubate at 37°C for 1 h. Washing the plate: discard the liquid in the wells, add 200 μL PBST per well, shake quickly on the shaker for 5 min, wash the plate 3 times, and pat the enzyme-labeled plate dry after washing. Color development: add 100 μL of Bicun TMB color developing solution to each well, incubate at room temperature in the dark for 15 min, stop color development: 100 μL / well, 2 M H2SO4 stop solution. Enzyme reader: read OD at 450 nm wavelength with an enzyme reader 450nm value. Calculate P / N value, calculate PI value, (inhibition rate (PI) = [(negative control well OD 450nm-Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Select the group with the smallest P / N value and the largest PI value as the optimal condition (P represents the sample well, and N represents the negative control well).

[0051] II. Optimization of Competitive ELISA Detection Methods 1. Competitive Detection: Five different NSDV positive and NSDV negative sera were selected for competitive ELISA experiments. The steps for the competitive ELISA experiment are as described in Example 4. The primary antibody was incubated with 6D12 monoclonal antibody, the P / N value was calculated, the PI value was calculated, and the monoclonal antibody with the largest difference in PI value between the positive and negative sera was selected.

[0052] 2. Determination of antigen coating conditions and optimal serum dilution: The optimal antigen coating conditions and optimal serum dilution were determined using the checkerboard method. The procedure was as follows: NSDV N protein was diluted with coating buffer at ratios of 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000 to coat ELISA plates and incubate overnight at 4°C. The next day, NSDV-infected positive and negative sera were diluted at ratios of 1:1, 1:2, 1:4, 1:8, and 1:16 to perform competitive ELISA experiments. The coating conditions were determined based on the P / N and PI values, and the group with the lowest P / N and highest PI value was selected.

[0053] 3. Determination of the optimal blocking solution: NSDV N protein was subjected to a competitive ELISA experiment according to the determined coating conditions and optimal serum dilution. Blocking solutions were prepared using ELISA stabilizer I, ELISA stabilizer II, 5% skim milk, and 5% whey protein, respectively, and incubated at 37°C for 1 h. The optimal blocking solution was determined based on the P / N and PI values, and the solution with the lowest P / N and highest PI value was selected.

[0054] 4. Determination of optimal blocking time: Determine the antigen coating conditions, optimal serum dilution factor, and blocking solution. Change the blocking time to 5% skim milk and block at 37℃ for 0.5 h, 1 h, 1.5 h, and 2 h respectively, while keeping other conditions unchanged. Determine the optimal blocking time based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0055] 5. Determination of optimal blocking concentration: Determine the antigen coating conditions, optimal serum dilution factor, blocking solution, and blocking time. Change the blocking conditions and use 1% skim milk, 3% skim milk, 5% skim milk, and 10% skim milk for blocking at 37℃. Keep other conditions unchanged. Determine the optimal blocking solution concentration based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0056] 6. Determination of the optimal working concentration of monoclonal antibody: Determine the antigen coating conditions, optimal serum dilution factor, blocking solution, and optimal blocking time. Change the monoclonal antibody dilution factor by diluting the monoclonal antibody with PBS at 1:50, 1:100, 1:150, and 1:200 and then perform the assay. Keep other conditions unchanged. Determine the optimal working concentration of the monoclonal antibody based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0057] 7. Determination of Optimal Monoclonal Antibody Incubation Time: With the antigen coating conditions, optimal serum dilution, blocking buffer conditions, and monoclonal antibody concentration unchanged, the monoclonal antibody incubation time was varied. Primary antibody incubation was performed at 37℃ for 30 min, 60 min, 90 min, and 120 min, respectively. The optimal incubation time was determined based on the P / N and PI values, and the group with the lowest P / N and highest PI value was selected.

[0058] 8. Determination of enzyme-labeled secondary antibody dilution: Determine the antigen coating conditions, optimal serum dilution factor, blocking buffer, optimal blocking time, monoclonal antibody concentration, and optimal monoclonal antibody incubation time. Adjust the dilution factor of goat anti-mouse HRP-IgG enzyme-labeled secondary antibody by diluting it with PBS at 1:2000, 1:4000, 1:8000, and 1:16000, and then perform the assay. Determine the enzyme-labeled secondary antibody dilution based on P / N and PI, and select the group with the lowest P / N and highest PI value.

[0059] 9. Determination of enzyme-labeled secondary antibody incubation time: Determine the antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, optimal monoclonal antibody conditions, and enzyme-labeled secondary antibody dilution. Change the enzyme-labeled secondary antibody incubation time. Incubate goat anti-mouse HRP-IgG enzyme-labeled secondary antibody at 37℃ for 30 min, 45 min, 60 min, and 75 min. Determine the enzyme-labeled secondary antibody incubation time based on P / N and PI, and select the group with the lowest P / N and the highest PI value.

[0060] 10. Determination of Chromogenic Conditions: Determine the antigen coating conditions, optimal serum dilution, blocking buffer, optimal blocking time, monoclonal antibody concentration, optimal monoclonal antibody incubation time, enzyme-labeled secondary antibody dilution, and enzyme-labeled secondary antibody incubation time. Incubate the TMB chromogenic solution at room temperature and 37°C for 5 min, 10 min, 12 min, 15 min, and 20 min, respectively. Select the group with the lowest P / N ratio and the highest PI value.

[0061] 11. Criteria for Determining Positive and Negative Results of the Competitive ELISA Method: Forty-one sheep negative serum samples stored in the laboratory were tested using the optimized competitive ELISA method described above. A positive control was also included. The absorbance (OD) values ​​of the samples at 450 nm were recorded. 450nm According to the formula for calculating the blocking rate (PI): (PI) = [(OD of negative control wells)] 450nm -Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Calculate the mean (xˉ) and standard deviation (SD) of the blocking rate for negative samples. The positive threshold is set as the mean blocking rate of negative serum plus 3 times the standard deviation, and the negative threshold is set as the mean blocking rate of negative serum plus 2 times the standard deviation. When the blocking rate of the sample is ≥ the positive threshold, it is judged as positive; if the blocking rate is ≤ the negative threshold, it is judged as negative. For samples with blocking rates between the two, repeated testing is required. If the repeated test results are still within this range, it is finally judged as negative.

[0062] 12. Sensitivity Evaluation of Competitive ELISA Method: A random sample of NSDV-positive serum was diluted 1:1, 1:2, 1:4, 1:8, and 1:16 for neutralization and competitive ELISA experiments. The neutralization experiment was performed as follows: Diluted sheep serum was inactivated in a water bath at 56°C for 30 min. A sterile 96-well cell culture plate was prepared, with 50 μL of serum-free DMEM medium added to each well. In the first row of wells, 50 μL of serum was added to each well, with four replicates per serum sample. 50 μL of the mixture was drawn from the first row and diluted downwards row by row. The last row was mixed thoroughly and 50 μL of liquid was discarded. NSDV virus strains with known titers were diluted with serum-free DMEM medium to a concentration of 1:1, 1:2, 1:4, 1:8, and 1:16. Working concentration of virus particles / mL. Gently vortex to mix, incubate at 37°C for 5%. Incubate for 2 hours in an incubator to promote virus-antibody neutralization. Digest healthy BSR cells at appropriate density, centrifuge them, resuspend them in 1 mL of 2% FBS DMEM medium, and perform cell counting. Dilute to 4.5 × 10⁻⁶. 5Cells were seeded at a density of 100 μL per well in a 96-well plate. IFA assay was performed after 4 days. A competitive ELISA method was used for detection, and the concordance rate between the two methods for clinical samples was calculated.

[0063] 13. Specificity evaluation of the competitive ELISA method: Positive sera for foot-and-mouth disease, small ruminant virus, sheep pox virus, sheep oral thrush virus, and akaban virus stored in the laboratory were serially diluted 2-fold and used as primary antibodies for incubation in a competitive ELISA experiment. NSDV positive serum was used as a positive control, and negative serum as a negative control. Specificity was evaluated based on OD... 450nm Values ​​determine the specificity of the results.

[0064] 14. Repeatability evaluation of the competitive ELISA detection method: Five NSDV positive serum samples and five negative serum samples were selected and subjected to competitive ELISA detection using ELISA plates coated from the same batch under standard experimental conditions. The intra-assay repeatability of the method was evaluated by calculating the coefficient of variation of the serum inhibition rate (CV = S / X × 100%, where S is the standard deviation and X is the arithmetic mean).

[0065] Five NSDV-positive serum samples and five NSDV-negative serum samples were collected and tested using different batches of ELISA plates under the same experimental conditions. The coefficient of variation (CV = S / X × 100%) of the serum inhibition rate was calculated to evaluate the reproducibility of this method among different batches of ELISA plates.

[0066] 15. Clinical application of competitive ELISA method: 179 local sheep serum samples were tested using competitive ELISA and neutralization test, and the concordance rate of the two methods in detecting clinical samples was calculated.

[0067] Results: The results of the competitive testing are as follows Figure 10 As shown: The ability of monoclonal antibodies to distinguish between positive and negative sera was determined by cELISA. The shape of each symbol indicates the identity inhibition of the serum against the corresponding monoclonal antibody. Five positive NSDV sera and five negative sera were tested, and the mean PI for positive and negative sera for each monoclonal antibody was recorded. According to the competitive detection results, the 6D12 monoclonal antibody performed very well. Determination of antigen coating conditions and optimal serum dilution: Using the checkerboard method, under otherwise identical conditions, the optimal antigen coating concentration and optimal serum dilution were determined. N protein coating buffer was diluted to 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, and 1:1000. Positive and negative controls were diluted to 1:1, 1:2, 1:4, 1:8, and 1:16, respectively. Checkerboard titration was performed using positive and negative controls at different protein concentrations and dilutions, and OD was measured using a competitive ELISA method. 450nm The optimal conditions were determined by the PI value, and the results are shown in Table 3. When the antigen coating concentration was 300 μg / mL and the serum dilution ratio was 1:2, the PI value was the highest and the blocking rate was the highest.

[0068] Table 3 Screening results based on antigen coating concentration and serum dilution factor

[0069] Determination of the optimal blocking solution: Based on the optimized antigen coating conditions and optimal serum dilution factor, competitive ELISA experiments were conducted using different blocking solutions, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 4. The optimal blocking solution is 5% skim milk.

[0070] Table 4 Determination of the Optimal Sealing Fluid

[0071] Determination of optimal blocking time: Based on the optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution type, competitive ELISA experiments were conducted using different blocking times, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 5. The optimal closure time is 1.5 h.

[0072] Table 5 Determination of Optimal Closure Time

[0073] Determination of optimal blocking concentration: Based on the optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution and blocking time, competitive ELISA experiments were conducted using different blocking solution concentrations, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 6. The optimal blocking solution concentration is 5% skim milk blocking.

[0074] Table 6 Determination of Optimal Blocking Concentration

[0075] Determination of the optimal working concentration of monoclonal antibodies: Based on the optimized antigen coating conditions, optimal serum dilution factor, and optimal blocking solution conditions, competitive ELISA experiments were conducted using different monoclonal antibody dilution factors, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 7. The optimal dilution factor for the monoclonal antibody is 1:320.

[0076] Table 7 Optimal dilution factor for antibodies

[0077] Determination of optimal antibody incubation time: Based on optimized antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, and antibody dilution, competitive ELISA experiments were conducted using different antibody incubation times, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 8. The optimal antibody action time is 1.5 h.

[0078] Table 8 Optimal Antibody Inaction Time

[0079] The determination of enzyme-labeled secondary antibody dilution was based on optimized antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, and optimal antibody conditions. Different secondary antibody dilutions were used in competitive ELISA experiments to measure OD. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 9. The optimal dilution factor for the second antibody is 1:8000.

[0080] Table 9 Optimal Dilution Factors for Secondary Antibodies

[0081] The determination of enzyme-labeled secondary antibody incubation time was based on optimized antigen coating conditions, optimal serum dilution, optimal blocking buffer conditions, and optimal antibody conditions. Competitive ELISA experiments were conducted using different incubation times for the secondary antibody, and OD was measured. 450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 10: the optimal incubation time for the two antibodies is 1 hour.

[0082] Table 10 Optimal Action Time of Secondary Antibody

[0083] Color development conditions determined: Based on the above conditions, the development times for the color development solution were 5 min, 10 min, 12 min, 15 min, and 20 min, respectively. OD was measured after the color development was completed.450nm The PI value was calculated, and the highest PI value was selected as the optimal condition. The results are shown in Table 11. The optimal color development condition is to develop the color at room temperature for 15 minutes.

[0084] Table 11 Determination of Optimal Color Development Time

[0085] The determination of the critical value: According to the formula for calculating the blocking rate (PI): (PI) = [(OD of negative control wells)] 450nm -Sample well OD 450nm ) / Negative control well OD 450nm (×100%). Calculate the OD of 41 sheep negative serum samples. 450nm The results showed that the average blocking rate of negative serum was 21.275%, with a standard deviation of 4.59%. The cutoff values ​​for positive and negative sera were determined as follows: Positive threshold = Average blocking rate of negative serum + 3 times the standard deviation of the negative serum blocking rate = 21.275% + 3 * 4.59% = 35.06%; Negative threshold = Average blocking rate of negative serum + 2 times the standard deviation of the negative serum blocking rate = 21.275% + 2 * 4.59% = 30.47%. When the PI ≥ 35.06%, it is considered positive; when the PI ≤ 30.47%, it is considered negative; and when the PI is between 30.47% and 35.06%, it is considered suspicious and requires repeat testing. If it is suspicious again, it is considered negative.

[0086] Table 12 Results of competitive ELISA detection of negative serum samples

[0087] The sensitivity evaluation of the competitive ELISA method was based on the results of the competitive ELISA and neutralization assays (Table 13). The competitive ELISA result was determined by the PI value; a PI value > 35.06% was considered positive. The neutralization assay criteria were: a neutralizing antibody titer ≥ 4 was considered positive, and < 4 was considered negative. The results of the competitive ELISA and neutralization assays were consistent; positive serum could be diluted to 1:4, demonstrating the good sensitivity of this method.

[0088] Table 13 Sensitivity test results

[0089] Specificity evaluation of the competitive ELISA method: Positive sera for peste des petits ruminants virus (PPR), foot-and-mouth disease virus (FMD), sheep pox virus (EPV), sheep stomatitis virus (SPV), and akaban virus (AKAV), as well as NSDV positive sera, were subjected to a competitive ELISA experiment, and the specificity was determined by measuring OD. 450nmThe specificity of the competing ELISA was assessed, and the results are shown in Table 14. Only NSDV-positive serum showed a blocking rate of ≥35.06% (68.50%), and the result was considered positive. The blocking rates of other pathogen-positive serum samples were all ≤30.47%, and the results were considered negative. The experimental data indicate that this method can effectively distinguish NSDV from other related viral antibodies and has high detection specificity.

[0090] Table 14 Specificity Test Results

[0091] Repeatability evaluation of the competitive ELISA detection method: Five NSDV positive serum samples were randomly selected for competitive ELISA testing. Inter-batch and intra-batch replicates were performed, and the competitive inhibition rate, standard deviation, and coefficient of variation were measured. According to the results in Table 15, the coefficients of variation for different batches were all low (maximum 6.01%). A coefficient of variation ≤10% is generally considered to indicate good repeatability, suggesting that the test results have good reproducibility.

[0092] Table 15 Repeatability Test Results

[0093] 179 local sheep serum samples were tested using a competitive ELISA method and a neutralization assay. Using the established competitive ELISA method, 35 samples were positive (19.5%). Using the neutralization assay, 42 samples were positive (23.4%). The positive concordance rate was 83%, and the negative concordance rate was 95.8%. Kappa test results showed a Kappa value of 0.78 > 0.610, indicating a high degree of consistency between the two methods. The calculated concordance rate was 93%, which is relatively high.

[0094] Table 16 Comparison of detection results between competitive ELISA method and neutralization method

Claims

1. The application of a monoclonal antibody against Nairobi sheep disease virus (NSDV) in the preparation of a kit for detecting Nairobi sheep disease virus, characterized in that, The kit includes a Nairobi sheep disease virus monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region CDR-H1 of the Nairobi sheep disease virus monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

5. The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of CDR-L2 is: LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

7.

2. A kit for detecting Nairobi sheep disease virus, characterized in that, The kit includes a Nairobi sheep disease virus monoclonal antibody, the amino acid sequence of the heavy chain variable region CDR-H1 of the Nairobi sheep disease virus monoclonal antibody is shown in SEQ ID NO.3; the amino acid sequence of CDR-H2 is shown in SEQ ID NO.4; and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

5. The amino acid sequence of the light chain variable region CDR-L1 of the monoclonal antibody is shown in SEQ ID NO.6; the amino acid sequence of CDR-L2 is LVS; and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

7.

3. The reagent kit according to claim 2, characterized in that, The heavy chain amino acid sequence of the Nairobi sheep disease virus monoclonal antibody is shown in SEQ ID NO.8; the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.

9.

4. The reagent kit according to claim 3, characterized in that, The gene sequence encoding the heavy chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.10; the gene sequence encoding the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO.

11.

5. The reagent kit according to claim 2, characterized in that, It also includes goat anti-mouse HRP-IgG.

6. The reagent kit according to claim 2, characterized in that, It also includes positive and negative controls. The positive control is NSDV positive serum, and the negative control is NSDV negative serum.

7. The reagent kit according to claim 2, characterized in that, It also includes a sealing solution, which is skim milk.

8. The reagent kit according to claim 2, characterized in that, It also includes TMB colorimetric solution and H2SO4 stop solution.

9. The reagent kit according to claim 2, characterized in that, The dilution factor for Nairobi sheep disease virus monoclonal antibody was 1:320, and the dilution factor for goat anti-mouse HRP-IgG was 1:8000.

10. A method for non-diagnostic and diagnostic testing using the kit according to any one of claims 2-9, characterized in that, The method is as follows: Step 1: Antigen coating: Dilute NSDV N protein 1:800 with CBS coating buffer, add 100 μL / well to the microplate, wash with PBST, and block with 5% skim milk; Step 2: Add 50 μL of sheep serum sample to be tested, followed by an equal volume of the Nairobi sheep disease virus monoclonal antibody as described in claim 2; discard the liquid in the wells, wash the plate with 200 μL of PBST, and block with 5% skim milk; wash the plate with PBST. Step 3: Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37°C for 1 h; discard the liquid in the wells and wash the plate with PBST; Step 4: Add 100 μL TMB colorimetric solution and incubate at room temperature in the dark for 15 min; Step 5: Stop color development: 100 μL / well of 2 M H2SO4 stop solution; Step 6: Microplate reader reading: Read the OD450nm value at a wavelength of 450 nm using a microplate reader; and calculate the PI value. When PI ≥ 35.06%, it is considered positive.

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