A monoclonal antibody capable of recognizing novel duck reovirus σB protein, preparation method and application thereof

Abstract

The present invention discloses a monoclonal antibody capable of recognizing a novel duck reovirus NDRVσB protein, a preparation method and its application. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises V sequences as shown in SEQ ID NOs: 11, 12, and 13. H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes the amino acid sequences shown in SEQ ID NOs: 14, 15, and 16. L CDR1, V L CDR2 and V L A double-antibody sandwich ELISA method for quantifying σB protein was established based on the σB 6B9 mouse monoclonal antibody and the σB rabbit polyclonal antibody. This method offers advantages such as high sensitivity, a wide linear range, low cost, ease of use, and a short operation time. This invention provides a new approach for quality control and immune efficacy evaluation of NDRV subunit vaccines, and also lays the foundation for the upgrading of NDRV vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody capable of identifying a novel duck reovirus NDRV σB protein, a preparation method and an application thereof. Technical Background

[0002] In recent years, waterfowl reoviruses (WRVs) have become a serious threat to the sustainable development of waterfowl farming. Based on their host and genotype, waterfowl reoviruses are primarily classified into three categories: Muscovy duck reovirus (MDRV), goose reovirus (GRV), and novel duck reovirus (NDRV).

[0003] Novel duck reovirus (NDRV) disease is a newly emerging immunosuppressive disease that is prevalent in major waterfowl breeding areas, including Shandong, Hebei, Henan, Anhui, Jiangsu, Guangdong, Sichuan, Fujian, Zhejiang and other regions.

[0004] NDRV can be transmitted vertically and horizontally, causing mass mortality in newly hatched ducklings around one week of age. It can also cause spleen and liver enlargement, irregular bleeding, and necrosis in ducks of all ages, leading to reduced egg production in breeder ducks, weight loss in broiler ducks, and a decrease in the qualified rate of broiler ducks. Furthermore, NDRV's reservoir is expanding, affecting all duck species (such as Muscovy, Muscovy, Shelduck, and Pekin) and goslings, causing significant economic losses to the waterfowl industry.

[0005] There is currently no commercial vaccine against NDRV on the market.

[0006] NDRV vaccines in preclinical development primarily include whole-virus inactivated vaccines, attenuated live vaccines, and subunit vaccines. However, there are currently no methods for quantifying subunit vaccine antigens. While some studies have attempted to quantify NDRV σC antigens through His-tag protein purification, this purification process is complex, costly, and time-consuming, making it difficult to implement in large-scale vaccine production. Summary of the Invention

[0007] In response to at least one of the above technical issues, the present invention provides a monoclonal antibody capable of recognizing the novel duck reovirus (NDRV) σB protein, as well as a preparation method and application thereof. The present invention also provides a double-antibody sandwich ELISA method based on a mouse monoclonal antibody against σB and a rabbit polyclonal antibody against σB. This method can also be used for quality control of NDRV σB vaccines and determination of vaccine immunization doses.

[0008] The technical solutions provided by the present invention are as follows:

[0009] The present invention provides a monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3;

[0010] The V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 11;

[0011] The V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 12;

[0012] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 13;

[0013] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 14;

[0014] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 15;

[0015] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:16.

[0016] Furthermore, the heavy chain variable region further comprises: a heavy chain variable region framework region V having an amino acid sequence as shown in SEQ ID NO: 3, 4, 5, or 6. H FR1, V H FR2 and V H FR3 and V H FR4, and the light chain variable region framework region V having the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10, respectively. L FR1, V L FR2, V L FR3 and V L FR4.

[0017] In some embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 18; and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 20.

[0018] In some embodiments, the monoclonal antibody comprises all or a portion of an antibody heavy chain constant region and / or an antibody light chain constant region.

[0019] The present invention also provides a nucleic acid molecule, which encodes the monoclonal antibody.

[0020] Furthermore, the nucleic acid molecule has a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody as shown in SEQ ID NO: 17 and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody as shown in SEQ ID NO: 19.

[0021] The present invention also provides a method for preparing the monoclonal antibody, comprising:

[0022] The purified σB protein is used as an immunogen to immunize animals, and the monoclonal antibody is prepared by hybridoma technology.

[0023] Furthermore, the nucleotide sequence of the gene encoding the σB protein is shown in SEQ ID NO: 1.

[0024] Furthermore, the amino acid sequence of the σB protein is shown in SEQ ID NO: 2.

[0025] The present invention also provides use of the monoclonal antibody and the nucleic acid molecule in preparing a NDRV σB protein detection reagent.

[0026] The present invention also provides a double-antibody sandwich ELISA method for quantifying NDRV σB protein, using the monoclonal antibody as a capture antibody to determine the concentration of σB protein. In some embodiments, a rabbit-derived polyclonal antibody against NDRV σB protein is used as a detection antibody.

[0027] This method uses the above-mentioned monoclonal antibody as a capture antibody and can be used to determine the concentration of σB protein in vaccines or other biological preparations.

[0028] Furthermore, the dilution ratio of the monoclonal antibody capable of recognizing σB protein is 100 to 128,000 times, and the optimal dilution ratio is 1:2000.

[0029] The method uses a monoclonal antibody (6B9) capable of recognizing NDRV σB protein as a capture antibody and a rabbit-derived polyclonal antibody against NDRV σB protein as a detection antibody to quantify the captured σB antigen.

[0030] Furthermore, the present invention also provides a σB protein capture antibody, which is a mouse monoclonal antibody. The antibody uses the prokaryotically expressed σB protein as an immunogen and is prepared by monoclonal hybridoma technology.

[0031] Furthermore, the present invention also provides a σB protein detection antibody, which is a rabbit-derived polyclonal antibody. The antibody is obtained by immunizing New Zealand white rabbits with prokaryotically expressed σB protein as an immunogen, harvesting serum, and purifying the serum.

[0032] Furthermore, the present invention also provides an operating method for a quantitative double-antibody sandwich ELISA for NDRV σB protein, comprising: diluting the σB capture antibody 6B9 at a dilution of 1:2000, coating the ELISA plate, and coating overnight at 4°C; the blocking solution is PBST containing 5% skim milk, and the blocking is performed at 37°C for 1 h; the σB protein standard is preferably 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078125 μg / mL, 0.00390625 μg / mL, 0.001953125 μg / mL, and 0.001953125. These 12 concentrations are used for drawing a standard curve; the ELISA incubation conditions are 37°C for 1 h; the preferred dilution of the detection antibody is 1:800, and the incubation is 1 h at 37°C; the preferred dilution of the enzyme-labeled antibody (horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG) is 1:6000, and the incubation is 1 h at 37°C; the reaction substrate is tetramethylbenzidine (TMB), and the incubation is carried out at room temperature for 20 min. After that, 2M sulfuric acid solution is added to terminate the ELISA reaction; the OD value is read. 450 value.

[0033] Furthermore, to verify the effectiveness of the established double antibody sandwich ELISA, the present invention also provides a σB expression product obtained by infecting insect Sf9 cells with a recombinant baculovirus expressing the σB protein, which is used as a test sample for the double antibody sandwich ELISA. The σB gene sequence is shown in SEQ ID NO: 1.

[0034] Furthermore, the present invention also provides a method for quantifying σB protein in an expression product obtained by an insect cell-baculovirus expression system, which specifically comprises: diluting the capture antibody 6B9 at a ratio of 1:2000, coating an ELISA plate, and coating at 4°C overnight; blocking with PBST containing 5% skim milk, and incubating at 37°C for 1 h; drawing a standard curve with different concentrations of σB protein (2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078125 μg / mL, 0.00390625 μg / mL, 0.001953125 μg / mL, 0.001953125 μg / mL), and adding the baculovirus expression product at the same time. The incubation conditions are 37°C for 1 h. h; the detection antibody was diluted 1:800 and incubated at 37°C for 1 h; HRP-labeled goat anti-rabbit IgG was diluted 1:6000 and incubated at 37°C for 1 h; TMB substrate was added and incubated at room temperature for 20 min; 2 M sulfuric acid solution was added to terminate the reaction; the OD was read. 450 A standard curve was drawn based on the σB protein standard, and the content of σB protein in the expression product obtained based on the insect cell-baculovirus expression system was calculated according to the standard curve.

[0035] The main objectives of the present invention are achieved through the following technical solutions: prokaryotic expression and purification of NDRV σB protein; preparation and identification of mouse monoclonal antibodies and rabbit polyclonal antibodies specific for NDRV σB protein; determination of the optimal dilution factors of capture and detection antibodies; establishment of a double-antibody sandwich ELISA method and its application in determining the content of σB protein obtained based on an insect cell-baculovirus expression system.

[0036] Beneficial effects: The monoclonal antibody provided by the present invention has high specificity and can be directly detected against NDRV σB protein, which is convenient, fast and highly accurate.

[0037] The double-antibody sandwich ELISA method provided by this invention can accurately quantify the NDRV σB protein in vaccines and other similar biological products. This double-antibody sandwich ELISA method is highly sensitive, low-cost, easy to use, and time-efficient, and can be used for antigen quantification of NDRV σB subunit vaccines. This invention provides a new method for quality control and immune efficacy evaluation of NDRV subunit vaccines, and also lays the foundation for upgrading NDRV vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Schematic diagram of SDS-PAGE analysis of NDRV σB protein expression in the examples of the present invention. Lane 1: Marker. Lane 2: Supernatant protein, induced at 37°C with 1.0 mM IPTG for 4 hours. Lane 3: Inclusion body protein, induced at 37°C with 1.0 mM IPTG for 4 hours. Lane 4: Purified protein.

[0039] Figure 2 Schematic diagram of the ELISA reactivity characteristics identification results of the σB monoclonal antibody and the σB protein in the examples of the present invention.

[0040] Figure 3 Schematic diagram of the identification results of the purified σB rabbit polyclonal antibody in the examples of the present invention, wherein: A is the SDS-PAGE identification result of the purified σB rabbit polyclonal antibody, and B is the Western blotting identification result of the purified σB rabbit polyclonal antibody.

[0041] Figure 4 Schematic diagram of a standard curve generated using σB protein standards in an embodiment of the present invention.

[0042] Figure 5 Schematic diagram of the IFA identification results of the recombinant baculovirus (rBac-σB) expressing the NDRV σB protein in the examples of the present invention; wherein: A represents cells infected with rBac-σB, and B represents uninfected negative cells. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0044] Example 1: Prokaryotic expression and purification of NDRV σB protein

[0045] (1) Construction and identification of prokaryotic expression plasmid expressing σB

[0046] The consensus sequence of the NDRV σB gene was analyzed and codon-optimized using GenSmart™. The optimized sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The optimized σB gene sequence was synthesized into the PET-28a prokaryotic expression vector, designated PET28a-σB. PET28a-σB was transformed into large DH5a competent cells, and the plasmid was extracted and identified by PCR to obtain a positive PET28a-σB.

[0047] (2) Induced expression and detection of σB protein

[0048] The PET28a-σB recombinant plasmid was transformed into E. coli BL21 competent cells and plated on LB agar plates containing ampicillin resistance. Positive single clones were selected and expanded in 20 mL LB liquid medium containing ampicillin resistance at 37°C and 220 rpm. 600 When the value reaches 0.6-0.8, 1 mM IPTG is added to induce expression. Induce overnight at 37°C and collect the bacterial culture for SDS-PAGE analysis.

[0049] The collected bacterial suspension was centrifuged at 4°C and 12,000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 4 mL of PBS, sonicated, and centrifuged. The supernatant and pellet were collected separately. An empty vector containing pET-28a induced under the same conditions was used as a blank control. The sample was mixed with 25 μL of 5× SDS loading buffer and denatured in a 100°C metal bath for 10 minutes. The treated sample was subjected to SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane. Mouse anti-His monoclonal antibody was used as the primary antibody, and HRP-conjugated goat anti-mouse IgG was used as the secondary antibody. The color development reaction was performed using an ECL colorimetric kit.

[0050] (3) Purification and detection of σB protein

[0051] The σB protein was purified using a nickel column. SDS-PAGE showed that the purified σB protein had a single band and high purity, with a molecular weight of approximately 47 kDa ( Figure 1 ).

[0052] Example 2: Preparation and identification of NDRV σB protein-specific monoclonal antibodies

[0053] (1) Immunization of BALB / c mice with σB protein

[0054] Purified prokaryotic σB protein was mixed with an equal volume of adjuvant (Freund's complete or incomplete adjuvant) at a concentration of 1.52 mg / mL and thoroughly emulsified. Six-week-old BALB / c mice were immunized according to the immunization schedule shown in Table 1. Blood was collected 2, 4, and 6 weeks after the initial immunization to measure serum antibody levels against σB.

[0055] Table 1 Immunization schedule for BALB / c mice

[0056]

[0057] (2) Preparation and identification of σB monoclonal antibodies

[0058] 2.1 Cell fusion

[0059] Three days before cell fusion, perform pulse immunization with antigen. Select the immunized mouse with the highest antibody titer and transfer it to a biosafety cabinet. Secure it with a foam board. Remove the spleen and place it on a copper grid. Add HAT culture medium and gently grind it with a grinding rod. Wash it several times with culture medium and collect the cell fluid into a sterile 50 mL centrifuge tube.

[0060] Myeloma cells (SP2 / 0 cell line) were cultured to the logarithmic growth phase for fusion with spleen cells. Preheat the tube in a 40°C water bath. Slowly add the PEG solution, preheated to 37°C, dropwise to the fusion tube while swirling to mix thoroughly. Mixing should be completed within 45-60 seconds. Within 90 seconds, add 30 mL of culture medium, preheated to 37°C, to terminate the fusion.

[0061] Add approximately 5 mL of HAT medium to the tube to resuspend the cell pellet. Add additional HAT medium to a total volume of 80 mL. Add 100 μL of the cell suspension to the feeder cells prepared 1 day prior.

[0062] Place the cell plate in a 37°C, 6% CO2 incubator and culture for 5 days. Replace half of the medium in the wells with HAT medium containing 15% FBS. On the 10th day, replace all of the medium in the wells with HT medium containing 15% FBS. Aspirate the medium after it turns yellow.

[0063] 2.2 Screening and subcloning of positive hybridoma cells

[0064] Screening for positive hybridoma cells: Use immunized mouse serum as a positive control and non-immunized mouse serum as a negative control. Positive wells are identified when the ratio of the OD value of the cell supernatant to the OD value of the negative control is greater than 2.1.

[0065] Subcloning of hybridoma cells: Hybridoma cells are subcloned by limiting dilution. The resulting monoclonal hybridoma cells are plated in 96-well cell plates. The supernatant of each well is tested for reactivity against σB protein by ELISA. Wells with higher OD values ​​are selected for the next round of subcloning. Positive clones screened are expanded and cultured. Subcloning is repeated three times in succession, and the resulting monoclonal cells are expanded and cultured.

[0066] 2.3 Sequencing of hybridoma cells

[0067] Using PrimeScript TM The total RNA was reverse transcribed into cDNA using a reverse transcription primer using a synthesis kit; the V of σB was amplified using a kit from Biointron Biology Inc. H and VL Antibody fragment; the amplified fragment was cloned into the TA / Blunt-Zero cloning vector and sequenced for identification.

[0068] 2.4 Preparation of ascites

[0069] Multiparous BALB / c female mice were injected intraperitoneally with sterile liquid paraffin. Ten days later, hybridoma cells were injected intraperitoneally into the mice. Seven days later, ascites fluid was collected and assayed for reactivity with the σB protein by ELISA. The results showed that two σB monoclonal antibodies generated using hybridoma technology showed strong reactivity with the σB protein. 6B9 was selected for subsequent double-antibody sandwich ELISA.

[0070] 2.6 Reactivity of monoclonal antibodies with NDRV

[0071] To evaluate the reactivity between the prepared ascites and NDRV-infected cells, Western blot and indirect immunofluorescence (IFA) were used. The results showed that the four monoclonal antibodies targeting the NDRV σB protein had no Western blot or IFA characteristics.

[0072] 2.7 Affinity determination of monoclonal antibodies for σB and σC proteins

[0073] The affinity of mouse ascites to σB protein was detected by indirect ELISA. Ascites was diluted to 10 dilutions (1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240, 1:40960, and 1:81920) at 100 μL / well and the OD was measured. 450 It was found that all mAbs showed high affinity to σB protein ( Figure 2 ).

[0074] Example 3: Preparation and purification of rabbit polyclonal antibodies against σB protein

[0075] Preparation of rabbit polyclonal antibodies: Purified σB protein was mixed with an equal volume of adjuvant (Freund's complete or incomplete adjuvant) and thoroughly emulsified. New Zealand white rabbits were then immunized. After three immunizations, serum was collected and antibodies were purified using the Protein A + GAgarose method. Western blotting was performed to verify the antibodies. Results showed that rabbit polyclonal antibodies against the NDRV σB protein were successfully prepared and purified (Figure 3).

[0076] Example 4: Establishment of double antibody sandwich ELISA method

[0077] 1. Establishment of the double antibody sandwich ELISA method

[0078] A double antibody sandwich ELISA method was established using σB monoclonal antibody 6B9 as the capture antibody, σB rabbit polyclonal antibody as the detection antibody, and prokaryotic expressed σB protein as the standard. The optimal dilution of the capture antibody and detection antibody was determined using the checkerboard method:

[0079] 6B9 monoclonal antibody was diluted with carbonate buffered saline (PBST) at dilution multiples of 1: 250, 1: 500, 1: 1000, 1: 2000, 1: 4000, 1: 8000, 1: 16000, 1: 32000, 1: 64000, 1: 128000, and 1: 256000, and coated on the plate at 4°C overnight.

[0080] Wash three times with PBST buffer, 5 min each time, and pat dry for the final wash. Block with 5% skim milk, 100 μL / well, at 37°C for 1 h.

[0081] Wash three times with PBST buffer, 5 min each time, and pat dry for the last time.

[0082] Add 0.08 μg / mL σB protein (100 μL / well) and incubate at 37°C for 1 h. Add PBST to another plate as a negative control. Wash three times with PBST buffer, 5 min each time, and pat dry for the final wash.

[0083] σB rabbit polyclonal antibody was serially diluted with PBST at dilutions of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, with 100 μL / well incubated at 37°C for 1 h. The cells were washed three times with PBST buffer, 5 min each time, and patted dry for the final wash.

[0084] HRP-labeled goat anti-rabbit IgG antibody was diluted 1:5000 in PBST, 100 μL / well, and incubated at 37°C for 1 hour. Wash with PBST buffer three times, 5 minutes each time, and pat dry for the final wash.

[0085] Develop with TMB (100 μL / well) for 20 min at room temperature. Terminate the reaction with 2 M sulfuric acid (50 μL / well).

[0086] Determine OD 450The optimal dilution of the σB capture antibody was 1:2000, and the optimal dilution of the σB detection antibody was 1:800 (Tables 2 and 3).

[0087] Table 2 OD after different σB capture antibody and detection antibody pairing 450 value

[0088]

[0089] Table 3 P / N values ​​after different σB capture antibody and detection antibody pairing

[0090]

[0091] 2. Establishment of σB protein standard curve

[0092] Based on the above, 1:2000 was used as the dilution factor of the σB capture antibody, and 1:800 was used as the optimal dilution factor of the σB detection antibody for ELISA experiments. Standard curves were established using different concentrations of σB protein (2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL, 0.03125 μg / mL, 0.015625 μg / mL, 0.0078125μg / mL, 0.00390625μg / mL, 0.001953125μg / mL, and 0.001953125). It was found that the optimized ELISA experimental parameters were used to detect the σB standard, with good linear reactivity and a standard curve R of 0.01. 2 The detection limit was 12.5 ng (Figure 4).

[0093] Example 5: Preparation and quantitative analysis of σC protein expression products

[0094] To further validate the performance of the established double-antibody sandwich ELISA method in detecting σB protein in expression products, a recombinant baculovirus expressing NDRV σB protein was constructed, and the expression product expressing σB protein was prepared based on the insect cell-baculovirus expression system. The specific steps are as follows:

[0095] Based on the NDRV σB gene sequence published on GeneBank, after extensive sequence alignment analysis, the most conserved antigen sequence was obtained, and the NDRV σB gene was synthesized into the pvL1393 vector (designated pvL1393-σB). pvL1393-σB was co-transfected with the linearized Autographa californica polyhedrosis virus genome into sf9 cells to rescue the recombinant baculovirus rBac-σB. The expression of the NDRV σB protein was then identified using IFA, and the results showed that the recombinant baculovirus expressing the NDRV σB protein was successfully constructed ( Figure 5 ).

[0096] sf9 cells were inoculated with the recombinant baculovirus rBac-σB at an MOI of 0.1. After 96 hours of culture, the cells were harvested, lysed by sonication, centrifuged, and the supernatant collected. σB protein in the supernatant was quantified using the established double-antibody sandwich ELISA assay. The results showed that σB protein expression was detectable using the established double-antibody sandwich ELISA assay, with an expression level of 0.48 μg / mL.

Claims

1. A monoclonal antibody that recognizes the novel duck reovirus NDRV σB protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes V H CDR1, V H CDR2 and V H CDR3, the light chain variable region includes V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO: 11; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO: 12; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO: 13; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO: 14; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO: 15; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO:

16.

2. The monoclonal antibody according to claim 1, characterized in that The heavy chain variable region further comprises: The heavy chain variable region framework region V of the amino acid sequence shown in SEQ ID NO: 3, 4, 5, and 6, respectively H FR1, V H FR2 and V H FR3 and V H FR4, and the light chain variable region framework region V having the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10, respectively. L FR1, V L FR2, V L FR3 and V L FR4.

3. The monoclonal antibody according to claim 2, characterized in that The heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 18, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

20.

4. The monoclonal antibody according to any one of claims 1 to 3, characterized in that The monoclonal antibody includes all or part of the antibody heavy chain constant region and / or the antibody light chain constant region.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the monoclonal antibody according to any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that The nucleic acid molecule has a nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody as shown in SEQ ID NO: 17 and a nucleotide sequence encoding the light chain variable region of the monoclonal antibody as shown in SEQ ID NO:

19.

7. Use of the monoclonal antibody according to any one of claims 1 to 4 or the nucleic acid molecule according to claim 5 or 6 in the preparation of a reagent for detecting NDRVσB protein.

8. A double antibody sandwich ELISA method for quantification of NDRV σB protein for non-diagnostic purposes, characterized in that: The monoclonal antibody according to any one of claims 1 to 4 is used as a capture antibody to measure the concentration of σB protein.

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