Preparation and application of mouse monoclonal antibody for resisting bovine akabane virus

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-bovine akabane virus murine monoclonal antibody and application thereof. The invention firstly provides an anti-bovine akabane virus murine monoclonal antibody, the monoclonal antibody has the characteristics of good purity and good specificity, is only specifically bound with N protein of bovine akabane virus, has a titer greater than 1: 32,000, has an IgG1 type heavy chain and a kappa type light chain, and provides a reliable research tool for early differential diagnosis of bovine epidemic diseases; secondly, the monoclonal antibody is applied to AKAV detection, an AKAV blocking ELISA method is constructed, and the method has good sensitivity and specificity, can be applied to WB and IFA detection of akabane viruses at the same time and has wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the preparation and application of a murine monoclonal antibody against bovine Akabane virus. Background Technology

[0002] Akabane disease (also known as Akabane disease) is a vector-borne infectious disease caused by Akabane disease virus (AKAV). While pregnant animals may not show obvious symptoms themselves after infection, the infection induces epidemic abortions, premature births, or stillbirths. The offspring often exhibit typical malformations, directly causing fetal loss and leading to decreased conception rates and prolonged reproductive cycles in the mothers, severely impacting herd renewal efficiency and causing immediate economic losses. Currently, there is no effective treatment for this disease. Once an outbreak occurs, it can spread through vector insects, causing regional epidemics, making prevention and control extremely difficult. This leads to a vicious cycle of "infection-loss-reinfection" in livestock farms, threatening the entire livestock industry chain, including breeding stock trading, dairy production, and related industries.

[0003] Over the past five years, the epidemic trend of Akabane disease has shown a shift from local outbreaks to regional spread. After 2024, with the expansion of the vector distribution range, the epidemic gradually spread, exhibiting obvious seasonal characteristics, with summer and autumn being the peak seasons. The prevention and control situation is severe, and it is urgent to strengthen epidemic monitoring, promote vaccination, and control vectors in order to curb the further spread of the epidemic.

[0004] Monoclonal antibodies are prepared by fusing B lymphocytes with myeloma cells using hybridoma technology, screening for single hybridoma cell clones that stably secrete specific antibodies, and exhibit high homogeneity and specificity. Blocking ELISA is an enzyme-linked immunosorbent assay (ELISA) based on the principle of competitive inhibition. Monoclonal antibodies are used in blocking ELISA; the antibody in the sample competes with the enzyme-labeled monoclonal antibody for binding to a single antigen. The presence of specific antibodies in the sample is detected through competitive inhibition, thus determining the infection status. It has high specificity and sensitivity, effectively reducing false positives. Furthermore, the low batch-to-batch variability of monoclonal antibodies ensures detection stability and repeatability, improving result reliability. The short production cycle and low cost make it suitable for standardized and large-scale testing, and it is an important tool for the early diagnosis and epidemiological surveillance of Akabane disease. Summary of the Invention

[0005] This invention targets bovine Akabane virus (BAKV) and has screened a murine monoclonal antibody against the BAKV N protein, exhibiting advantages such as BAKV specificity, strong antigen binding ability, and good antigen site blocking effect. Furthermore, an AKAV blocking ELISA method has been established using this monoclonal antibody. By periodically detecting specific antibodies in serum, positive individuals can be identified in the early stages of infection, allowing for real-time monitoring of the epidemic situation and providing a scientific basis for timely isolation, immunization, and treatment.

[0006] Specifically, it includes the following:

[0007] In a first aspect, the present invention provides a monoclonal antibody against bovine Akabane virus, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain; the variable region CDR of the antibody heavy chain comprises amino acid sequences as shown in SEQ ID No. 1 CDR1, SEQ ID No. 2 CDR2 and SEQ ID No. 3 CDR3; the variable region CDR of the antibody light chain comprises amino acid sequences as shown in SEQ ID No. 4 CDR1, SEQ ID No. 5 CDR2 and SEQ ID No. 6 CDR3.

[0008] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID No. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No. 8.

[0009] In a second aspect, the present invention provides a nucleic acid that encodes the monoclonal antibody described in the first aspect above.

[0010] Thirdly, the present invention provides an expression vector containing the nucleic acid described in the second aspect above.

[0011] Fourthly, the present invention provides a host cell containing the expression vector described in the third aspect above, or having the nucleic acid described in the second aspect above integrated into its genome.

[0012] Fifthly, the present invention provides the application of the monoclonal antibody described in the first aspect above in the preparation of reagents for detecting serum antibodies against Akabane virus.

[0013] Preferably, the reagent includes test strips or kits.

[0014] In a sixth aspect, the present invention provides a detection kit for detecting serum antibodies against Akabane virus, the detection kit comprising the monoclonal antibody described in the first aspect above.

[0015] Preferably, the detection kit further includes a coated antigen, an enzyme-labeled plate, a blocking solution, a diluent, a washing solution, a chromogenic agent, and a stop solution.

[0016] In a seventh aspect, the present invention provides the application of the detection kit described in the sixth aspect above in the detection of Akabane virus serum antibodies for non-disease diagnosis purposes.

[0017] The beneficial effects of this invention are as follows:

[0018] (1) This invention provides a monoclonal antibody against bovine Akabane virus. The monoclonal antibody has good purity and specificity, and no cross-reaction with Brucella, O-type foot-and-mouth disease virus and A-type foot-and-mouth disease virus. It has IgG1 heavy chain and κ light chain, providing a reliable research tool for the early identification and diagnosis of swine diseases.

[0019] (2) The present invention provides the variable region sequence of the monoclonal antibody, which can be used for recombination and modification by conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells, and also facilitating antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity.

[0020] (3) The monoclonal antibody was applied to the detection of AKAV virus serum antibodies. The results showed that the AKAV blocking ELISA antibody detection method had specificity and sensitivity, and there was no cross-reaction in the detection of serum from animals infected with Brucella and Foot-and-Mouth Disease Virus. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 Results of titer assay for the prepared monoclonal antibody;

[0023] Figure 2 Purification results of the prepared monoclonal antibody;

[0024] Figure 3 Western blot (WB) reactivity verification of the prepared monoclonal antibody;

[0025] Figure 4 Verification of the IFA reactivity of the prepared monoclonal antibody;

[0026] Figure 5 The results of the subtype identification of the prepared monoclonal antibody;

[0027] Figure 6 Specificity validation of the AKAV blocking ELISA method;

[0028] Figure 7 Sensitivity validation of the AKAV blocking ELISA method. Detailed Implementation

[0029] The present invention will be described in detail below through specific embodiments. However, the scope of protection of the present invention is not limited to the following embodiments. Any technical solution that can be conceived by those skilled in the art based on the present invention and in combination with common knowledge in the art shall fall within the scope of protection of the present invention. In addition, for those embodiments where specific technical operation steps or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in general literature in the art or in accordance with the product instructions. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0030] Light chains are classified into κ chains or λ chains based on their constant regions. The subunit structures and three-dimensional conformations of different types of immunoglobulins are well known to those skilled in the art. In this invention, VH represents the variable region of the heavy chain, and VL represents the variable region of the light chain, which is divided into κ and λ types.

[0031] BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences; HRP Conjugation Kit - Lighting - The kits were purchased from Abcam, Freund's complete and incomplete adjuvants from Sigma, and the mouse monoclonal antibody subtype identification kit from Proteintech. Trizol, DNA fragment recovery kit, and plasmid extraction kit were OMEGA products. Molecular biology reagents were from Sigma. Other biochemical reagents were domestically produced analytical grade.

[0032] Sera containing type O FMDV and type A FMDV were obtained from the National Foot-and-Mouth Disease Reference Laboratory. Those skilled in the art who require their use may apply for their assistance. Sera containing AKAV, LSDV, and Brucella were obtained from the State Key Laboratory of Animal Disease Pathogen Biology. The recombinant AKAV-N protein was prepared using conventional methods. Those skilled in the art who require the above serum samples may apply for their assistance.

[0033] Example 1: Preparation of mouse-derived monoclonal antibody against bovine Akabane virus

[0034] 1. Immunizing mice

[0035] AKAVN protein (GenBank: AAQ92318.1) was expressed, diluted to 200 μg / mL with PBS, and thoroughly emulsified with an equal volume of Freund's complete adjuvant to form a stable water-in-oil emulsion. Six 6-week-old female BALB / c mice were primary immunized by subcutaneous injection of 200 μL of the emulsified antigen at multiple sites on the back. On days 15 and 30 post-primary immunization, booster immunizations were performed using the same dose of antigen emulsified with Freund's incomplete adjuvant to continuously stimulate the immune response. One week after the third immunization, serum was collected via tail vein, and antibody titers were detected using an indirect ELISA method to screen for OD. 450nm High-response mice with a value greater than 2.0. To enhance the activity of splenic B lymphocytes, the selected mice were given a final booster immunization three days before cell fusion by intraperitoneal injection of 0.5 mL of adjuvant-free pure antigen.

[0036] 2. Cell fusion

[0037] Positive serum was prepared from blood collected from BALB / c mice 3 days after booster immunization. Mice were euthanized, and spleens were aseptically harvested after disinfection, washed, and fat removed. The spleens were minced and ground in incomplete RPMI-1640, and spleen cells were collected. Spleen cells were mixed with Sp2 / 0 cells at a ratio of 1:5 to 1:10, centrifuged, and then PEG 2000 was added to promote fusion. The mixture was then diluted with incomplete RPMI-1640. The fused cells were resuspended in HAT selective medium, seeded in 96-well plates, and cultured at 37°C with 5% CO2.

[0038] 3. Screening and subcloning of positive hybridoma cells

[0039] The medium for fusion cells is changed halfway every 2-4 days (discarding 100 μL and adding 100 μL of fresh HAT medium). Observe after 7-8 days and label the surviving wells. When hybridoma cells have grown to more than 1 / 10 of the bottom of the well, collect the supernatant and use ELISA to screen positive wells. Transfer cells from positive wells to 24-well plates, count them after confluence, dilute to 10 cells / mL, and seed into 96-well plates containing feeder cells. Repeat the screening. Simultaneously, cryopreserve some cells. Perform subcloning three times consecutively until highly reactive monoclonal hybridoma cells are selected. Select monoclonal hybridoma cells with high titers and good reactivity, propagate them, and cryopreserve them.

[0040] 4. Preparation and identification of monoclonal antibodies

[0041] BALB / c female mice aged 10–12 weeks were pre-stimulated by intraperitoneal injection of 0.5 mL Freund's incomplete adjuvant. Seven days later, the selected monoclonal hybridoma cell line was immunized into the mice at a dose of 3 × 10⁻⁶. 6Cells / mouse. 7–10 days after immunization, when the mice’s abdomens were noticeably enlarged and fluctuant to the touch, ascites was collected. The ascites was purified and desalted using the lipoic acid-saturated ammonium sulfate method to prepare a monoclonal antibody against bovine Akabane virus N protein, named 3A10.

[0042] Monoclonal antibody ELISA reactivity assessment: AKAVN protein was coated into ELISA plates at 100 ng / well and incubated overnight at 4°C. After washing with PBST, commercial blocking buffer was added and the plates were blocked overnight. The previously prepared monoclonal antibody 3A10 was serially diluted and added to the coated plates. The plates were incubated at 37°C for 30 minutes, followed by 4 washes with PBST. HRP-labeled goat anti-mouse IgG (1:15000 dilution) was added, and the plates were incubated at 37°C for 30 minutes, followed by 4 washes with PBST. TMB was allowed to develop color for 15 minutes, and then stop buffer was added. OD values ​​were read using a microplate reader. 450nm Value. Result as follows Figure 1 As shown, the titer of the prepared monoclonal antibody 3A10 is greater than 1:32000.

[0043] Purity identification of monoclonal antibody purified products: The purity of the purified products was identified by SDS-PAGE electrophoresis and then staining.

[0044] The results are as follows Figure 2 As shown, the product exhibits pure bands at 25kDa and 55kDa, consistent with the size of the light and heavy chains of mouse IgG antibodies, indicating accurate purification results and good product purity.

[0045] Western blot reactivity of monoclonal antibody: After SDS-PAGE electrophoresis of AKAVN protein, it was transferred onto a PVDF membrane and subjected to Western blot reaction with the monoclonal antibody 3A10 prepared above to verify reactivity.

[0046] The results are as follows Figure 3 As shown, the monoclonal antibody 3A10 described in this application can specifically bind to the AKAVN protein and exhibits good reactivity.

[0047] Monoclonal antibody IFA reactivity verification: The pcDNA3.1 AKAVN plasmid expressing AKAVN was constructed and transiently transfected into BHK cells cultured in cell dishes. After 48 hours of culture, the cells were washed three times with sterile PBS and fixed by incubation at 4°C for 20 minutes with paraformaldehyde. After fixation, the cell dishes were washed three times with sterile PBS, blocked for 1 hour at room temperature with 5% BSAPBS solution, washed three times with sterile PBS, and incubated overnight at 4°C with a 1 μg / mL dose of monoclonal antibody 3A10. The cell dishes were washed three times with sterile PBS, and incubated for 1 hour at room temperature with a 1 μg / mL dose of goat anti-mouse IgG-FITC antibody. The cell dishes were washed three times with sterile PBS. DAPI staining solution diluted 1:2000 was added, and the cells were incubated for 10 minutes at room temperature. The cell dishes were washed three times with sterile PBS. The results were observed and recorded using an EVOSM5000 cell imaging system.

[0048] Cell imaging results as follows Figure 4 As shown, the monoclonal antibody 3A10 reacts with AKAVN protein with low negative background, extremely weak cross-reactivity, and high specificity; at the same time, the cell fluorescence is strong, indicating good affinity.

[0049] Monoclonal antibody subtype identification: The subtype of the monoclonal antibody 3A10 described in this application was determined using a commercially available monoclonal antibody subtype identification kit.

[0050] The results are as follows Figure 5 As shown, the monoclonal antibody 3A10 described in this application has an IgG1 type heavy chain and a κ type light chain.

[0051] Example 2: Amplification of the variable region sequence of a monoclonal antibody

[0052] 1. Extraction of total mRNA from positive hybridoma cells

[0053] Resuscitate the cryopreserved monoclonal hybridoma cells from Example 1, culture for 2-3 generations, and prepare a cell suspension. Add 1 mL of TRizol, mix well, and incubate at 4°C for 5 minutes. Add 250 μL of chloroform, mix well, incubate at 4°C for 10 minutes, and centrifuge at 12000 rpm for 15 minutes. Take 450 μL of the supernatant, add an equal volume of isopropanol, incubate at -20°C for 30 minutes, and centrifuge as above. Discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, and centrifuge for 5 minutes. Dry the precipitate, dissolve it in 25 μL of RNase-free water, and obtain total mRNA.

[0054] 2. cDNA double-strand synthesis and purification

[0055] Add the components shown in Table 1 to a 0.2 mL PCR amplification tube in sequence; after mixing, place the PCR amplification tube in a PCR instrument and amplify using the reaction program shown in Table 2. The obtained product is the whole genome cDNA, which should be stored at -20℃ for later use.

[0056] Table 1 Reverse transcription reagent system

[0057]

[0058] Table 2 Reverse Transcription Procedure

[0059]

[0060] 3. Determination of the variable region sequence of monoclonal antibodies

[0061] 3.1 Primer Synthesis

[0062] Primers were designed based on the reference (von Boehmer L, Liu C, Ackerman S, Gitlin AD, Wang Q, Gazumyan A, Nu ssenzweig MC. Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc. 2016 Oct;11(10):1908-1923. doi:10.1038 / nprot.2016.102. Epub 2016Sep 15.PMID:27658009.), and were synthesized by Beijing Qingke Biotechnology Co., Ltd. After preparing the primer premixes according to the literature, a PCR amplification program was designed and PCR amplification reaction was carried out.

[0063] 3.1.1 First-round amplification primers

[0064] (1) Primers for amplifying the VH sequence:

[0065] VH-I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 9);

[0066] VH-II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 10);

[0067] VH-III: 5'-TGGCAGCARCAGCTACAGG-3' (SEQ ID No. 11);

[0068] VH-IV: 5'-CTGCCTGGTGACATTCCCA-3' (SEQ ID No. 12);

[0069] VH-V: 5'-CCAAGCTGTGTCCTGTC-3' (SEQ ID No. 13);

[0070] VH-VI: 5'-TTTTAAAAGGTGTCCAGKGT-3' (SEQ ID No.14);

[0071] VH-VII: 5'-CCTGTCAGTAACTRCAGGTGTCC-3' (SEQ ID No.15);

[0072] VH-VIII: 5'-TTTTAAAAGGGGTCCAGTGT-3' (SEQ ID No.16);

[0073] VH-IX: 5'-CGTTCCTGGTATCCTGTCT-3' (SEQ ID No.17);

[0074] VH-X: 5'-ATGAAGTTGTGGYTRAACTGG-3' (SEQ ID No.18);

[0075] VH-XI: 5'-TGTTGGGGCTKAAGTGGG-3' (SEQ ID No.19);

[0076] VH-XII: 5'-AGAAGGTGTGCACACCGCTGGAC-3' (SEQ ID No.20).

[0077] (2) Primers for amplifying VL sequences

[0078] VL-I: 5'-RGTGCAGATTTTCAGCTTCCTGCT-3' (SEQ ID No.21);

[0079] VL-II: 5'-TGGACATGAGGGCYCCTGCTCAGT-3' (SEQ ID No.22);

[0080] VL-III: 5'-CTSTGGTTGTCTGGTGTTGAYGGA-3' (SEQ ID No.23);

[0081] VL-IV: 5'-GTTGCTGCTGCTGTGGCTTACA-3' (SEQ ID No.24);

[0082] VL-V: 5'-GTATCTGGTACCTGTGG-3' (SEQ ID No.25);

[0083] VL-VI: 5'-TGCCTGTTAGGCTGTTGGTGCT-3' (SEQ ID No.26);

[0084] VL-VII: 5'-GCTCAGTTCCTTGGTCTCCTGTTGC-3' (SEQ ID No. 27);

[0085] VL-VIII: 5'-TGGGTGCTGCTGCTCTGGGT-3' (SEQ ID No. 28);

[0086] VL-IX: 5'-CAGTTCCTGTTTCTGTTARTGCTCTGG-3' (SEQ ID No. 29);

[0087] VL-X: 5'-TGCTCTGGTTATATGGTGCTGATGGG-3' (SEQ ID No. 30);

[0088] VL-XI: 5'-ACTGAGGCACCTCCAGATGTT-3' (SEQ ID No. 31).

[0089] 3.1.2 Primers for the second round of amplification

[0090] Ⅰ: 5'-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3' (SEQ ID No. 32);

[0091] Ⅱ: 5'-GCTCAGGGAARTAGCCCTTGAC-3' (SEQ ID No. 33);

[0092] Ⅲ: 5'-GAYATTGTGMTSACCMCARWCTMCA-3' (SEQ ID No. 34);

[0093] Ⅳ: 5'-TGGGAAGATGGATACAGTT-3' (SEQ ID No. 35);

[0094] V: 5'-CAGGCTGTTGTGACTCAG-3' (SEQ ID No. 36);

[0095] VI: 5'-CAACTTGTGCTCACTCAG-3' (SEQ ID No. 37);

[0096] VII: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 38).

[0097] 3.2 First Round of Amplification

[0098] Add the components listed in Tables 3 and 4 to a 0.2 mL PCR amplification tube in sequence, and perform PCR amplification using the reaction program in Table 5. Store the amplification products at -20℃.

[0099] Table 3 Antibody VH gene PCR amplification system

[0100]

[0101] Table 4. Antibody VH gene PCR amplification system

[0102]

[0103] Table 5 Antibody VH / VL Amplification Program

[0104]

[0105] 3.3 Second round of expansion

[0106] The VH / VL products from the first round of amplification were amplified again using the amplification system shown in Table 6, following the reaction procedures shown in Table 7. The amplified products were stored at -20℃.

[0107] Table 6 Antibody Second-Round PCR Amplification System

[0108]

[0109] Table 7. Antibody Second-Round PCR Amplification Procedure

[0110]

[0111] 3.3 Comparison of gene sequences

[0112] After amplification, the target fragment was ligated into the pMD-19T vector to construct a sequencing plasmid. This plasmid was then transformed into DH5α competent cells to screen for positive plasmids and expanded. The plasmids were then extracted using an Omega plasmid miniprep kit and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained variable region sequence was compared with the mouse antibody heavy chain variable region sequence, Lambda light chain sequence, and Kappa light chain sequence published on the NCBI website using the NCBI and IMGT gene banks.

[0113] The amplified sequences are the complementarity determining regions (CDRs) of the heavy and light chain variable regions of the monoclonal antibody, as shown in Table 8; the amino acid sequences of the light and heavy chain variable regions are shown in Table 9.

[0114] Table 8. Complementation-determining region sequence of antibody variable region

[0115]

[0116] Table 9. Amino acid sequence of antibody variable region

[0117]

[0118] Example 3: Application of monoclonal antibodies in AKAV antibody detection

[0119] 1. Establishment and validation of the AKAV blocking ELISA method

[0120] According to HRP Conjugation Kit-Lighting- The kit procedure involved HRP labeling the purified monoclonal antibody 3A10. The enzyme-labeled monoclonal antibody was fixed at a dilution ratio of 1:15000. The optimal operating conditions for the ELISA method were determined using checkerboard titration: 100 ng / well of P54 antigen and a serum sample dilution ratio of 1:5. Under the ELISA operating conditions, the enzyme-labeled monoclonal antibody 3A10 was serially diluted, and the optimal dilution ratio was determined to be 1:30000. After optimizing the conditions, the OD of 93 AKAV antibody-negative serum samples was measured using this ELISA method. 450nm The critical value for this method was determined to be 0.40 using ROC analysis with Prism software.

[0121] Specificity validation of AKAV blocking ELISA method: Using optimized working conditions of the ELISA method, bovine serum positive for AKAV, Brucella, LSDV, and A / O type FMDV was detected.

[0122] Test results as follows Figure 6 As shown, the ELISA method established in this application can only detect AKAV positive antibodies. The detection method has no cross-reactivity with other viral serum antibodies, and the ELISA method has good specificity.

[0123] Sensitivity validation of the AKAV blocking ELISA method: AKAV-positive bovine serum was started with a 1:5 dilution ratio and detected using optimized working conditions of the ELISA method.

[0124] Test results as follows Figure 7 As shown, this ELISA method can detect bovine Akabane disease-positive serum at a dilution ratio of up to 1:160, demonstrating good sensitivity.

[0125] In summary, this invention provides a monoclonal antibody against bovine Akabane virus (BAV). This monoclonal antibody exhibits high purity, good specificity, high reactivity, and specific binding only to the N protein of BAV, with a titer greater than 1:32000. It possesses an IgG1 heavy chain and a κ light chain, demonstrates good reactivity in Western blotting (WB) and in vitro anabolism (IFA), and has a wide range of applications, making it a reliable tool for Akabane disease research. The monoclonal antibody sequence provided by this invention can be used for recombination and modification using conventional genetic engineering or protein engineering methods, avoiding antibody loss during long-term cryopreservation of hybridoma cells. It also facilitates antibody optimization at the gene and protein levels, thereby improving antibody specificity and affinity. Applying this monoclonal antibody to AKAV antibody detection showed that the AKAV antibody blocking ELISA method can be used for the diagnosis of Akabane disease infection. This provides a reliable tool for the differential diagnosis and development of related detection methods for BAV.

[0126] The embodiments described above are only some embodiments of the present invention and are not intended to limit the scope of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the patent claims of the present invention should be included within the scope of the patent claims of the present invention.

Claims

1. A monoclonal antibody against bovine Akabane virus, characterized in that, The monoclonal antibody comprises an antibody heavy chain and an antibody light chain; the variable region CDR of the antibody heavy chain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively; the variable region CDR of the antibody light chain comprises CDR1, CDR2, and CDR3 as shown in SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively.

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. 7, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID No.

8.

3. A nucleic acid, characterized in that, The nucleic acid encodes the monoclonal antibody of claim 1 or 2.

4. An expression carrier, characterized in that, The expression vector contains the nucleic acid as described in claim 3.

5. A host cell, characterized in that, The host cell contains the expression vector of claim 4, or the nucleic acid of claim 3 is integrated into its genome.

6. The use of the monoclonal antibody as described in claim 1 or 2 in the preparation of reagents for detecting serum antibodies against Akabane virus.

7. The application as described in claim 6, characterized in that, The reagents include test strips or kits.

8. A detection kit for detecting serum antibodies against Akabane virus, characterized in that, The detection kit includes the monoclonal antibody as described in claim 1 or 2.

9. The detection kit as described in claim 8, characterized in that, The test kit also includes coated antigen, ELISA plate, blocking solution, diluent, washing solution, chromogenic agent, and stop solution.

10. The application of the test kit as described in claim 8 or 9 in the detection of Akabane virus serum antibodies for non-disease diagnosis purposes.

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