Infectious pancreatic necrosis virus monoclonal antibody and application thereof

Abstract

The invention discloses an infectious pancreatic necrosis virus monoclonal antibody and application thereof. The invention provides hybridoma cell strains IPNV-vp2-178-2A7 (CCTCC (China Center For Type Culture Collection) NO: C2024368) and IPNV-vp2-380-1G12 (CCTCC NO: C2024369) as well as two monoclonal antibodies secreted by the two hybridoma cell strains. According to the invention, two monoclonal antibodies are utilized to establish an antigen capture ELISA detection method aiming at the VP2 protein of the IPNV virus, and the effect of the antigen capture ELISA detection method is evaluated to find that the antigen capture ELISA detection method can specifically, rapidly and sensitively detect the infectious pancreatic necrosis virus.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly relates to a monoclonal antibody against infectious pancreatic necrosis virus and its application. Background Art

[0002] Infectious pancreatic necrosis (IPN) is an acute and highly contagious disease that mainly affects salmonids. It is caused by infectious pancreatic necrosis virus (IPNV), and the mortality rate is generally 10%-90%. IPNV is divided into 7 genotypes (genome types I-VII). In China, the main prevalent genome types are I and V, and it is listed as a class III animal disease in China.

[0003] The VP2 protein of IPNV has multiple important biological significances. On the one hand, it can bind to viral RNA and participate in various viral biological activities. On the other hand, its high immunogenicity can enable the body to produce a large number of antibodies shortly after being infected with the virus. Therefore, the VP2 protein of IPNV can be used as an ideal target antigen for the early detection and diagnosis of large-scale outbreaks of salmonids. Summary of the Invention

[0004] The purpose of the present invention is to provide a monoclonal antibody against infectious pancreatic necrosis virus and its application.

[0005] In the first aspect, the present invention claims to protect a hybridoma cell line or a set of hybridoma cell lines.

[0006] The hybridoma cell line claimed to be protected by the present invention is the hybridoma cell line IPNV-vp2-178-2A7 or the hybridoma cell line IPNV-vp2-380-1G12.

[0007] The set of hybridoma cell lines claimed to be protected by the present invention is composed of the hybridoma cell line IPNV-vp2-178-2A7 and the hybridoma cell line IPNV-vp2-380-1G12.

[0008] The preservation number of the hybridoma cell line IPNV-vp2-178-2A7 at the China Center for Type Culture Collection is CCTCC NO: C2024368;

[0009] The preservation number of the hybridoma cell line IPNV-vp2-380-1G12 at the China Center for Type Culture Collection is CCTCC NO: C2024369.

[0010] In the second aspect, the present invention claims to protect a monoclonal antibody or a set of monoclonal antibodies.

[0011] The monoclonal antibodies claimed in the present invention are monoclonal antibody VP2-178 or monoclonal antibody VP2-380.

[0012] The set of monoclonal antibodies claimed in the present invention is composed of the monoclonal antibody VP2-178 and the monoclonal antibody VP2-380.

[0013] The monoclonal antibody VP2-178 is secreted by the hybridoma cell line IPNV-vp2-178-2A7 described in the first aspect above;

[0014] The monoclonal antibody VP2-380 is secreted by the hybridoma cell line IPNV-vp2-380-1G12 described in the first aspect above.

[0015] In the third aspect, the present invention claims an enzyme-linked immunosorbent assay kit for detecting infectious pancreatic necrosis virus (IPNV).

[0016] The enzyme-linked immunosorbent assay kit for detecting infectious pancreatic necrosis virus (IPNV) claimed in the present invention comprises a capture antibody and a detection antibody; the capture antibody is the monoclonal antibody VP2-178 described in the second aspect above, and the detection antibody is the monoclonal antibody VP2-380 described in the second aspect above.

[0017] Furthermore, the enzyme-linked immunosorbent assay kit may also contain other commonly used reagents and / or consumables for ELISA detection, such as 96-well enzyme-linked immunosorbent assay plates, IPNV VP2 standard antigen, coating buffer, washing solution, blocking solution, chromogenic solution and / or termination solution, etc.

[0018] In the enzyme-linked immunosorbent assay kit, the capture antibody (i.e., the monoclonal antibody VP2-178) can be pre-coated on a solid phase carrier such as a 96-well enzyme-linked immunosorbent assay plate; the detection antibody can be the monoclonal antibody VP2-380 labeled with a labeling agent such as HRP or biotin.

[0019] In the fourth aspect, the present invention claims the application of the hybridoma cell line or set of hybridoma cell lines described in the first aspect above, or the monoclonal antibody or set of monoclonal antibodies described in the second aspect above, or the enzyme-linked immunosorbent assay kit described in the third aspect above in any of the following:

[0020] (A1) Detecting whether an infectious pancreatic necrosis virus is contained in a test sample;

[0021] (A2) Preparing a product for detecting whether an infectious pancreatic necrosis virus is contained in a test sample;

[0022] (A3) Monitoring or warning of infectious pancreatic necrosis;

[0023] (A4) Preparation of products for monitoring or warning of infectious pancreatic necrosis

[0024] In a fifth aspect, the present invention claims protection for a non-disease diagnosis method for detecting infectious pancreatic necrosis virus.

[0025] The non-disease diagnosis method for detecting infectious pancreatic necrosis virus claimed by the present invention is the ELISA double antibody sandwich method; wherein, the monoclonal antibody VP2-178 described in the second aspect above is used as the capture antibody, and the monoclonal antibody VP2-380 described in the second aspect above is used as the detection antibody.

[0026] Relatively preferably, in the ELISA double antibody sandwich method, the working concentration of the monoclonal antibody VP2-178 as the capture antibody is 0.5 μg / well, and / or, the dilution of the monoclonal antibody VP2-380 as the detection antibody is 1:2000, and / or, the reaction conditions are: the monoclonal antibody VP2-178 (capture antibody) is added to the enzyme-linked immunosorbent assay (ELISA) plate at 100 μl / well and incubated overnight at 4 °C, blocked with 5% non-fat milk powder for 2 h, the antigen to be detected is reacted at 37 °C for 1.5 h, the monoclonal antibody VP2-380 (detection antibody) is reacted at 37 °C at 100 μl / well for 1.5 h, after adding the TMB reaction substrate, the reaction is carried out for 15 min and then the reaction is terminated by adding the termination solution.

[0027] In a sixth aspect, the present invention claims protection for any one of the following substances:

[0028] (B1) An antibody against IPNV VP2, which is antibody A (corresponding to the monoclonal antibody VP2-178) or antibody B (corresponding to the monoclonal antibody VP2-380);

[0029] The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of antibody A are shown as positions 44-58, 74-80, and 113-121 of SEQ ID No. 3 in sequence; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of antibody A are shown as positions 50-54, 69-85, and 118-127 of SEQ ID No. 4 in sequence;

[0030] The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of antibody B are shown as positions 44-58, 74-80, and 113-121 of SEQ ID No. 5 in sequence; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of antibody B are shown as positions 50-54, 69-85, and 118-127 of SEQ ID No. 6 in sequence.

[0031] (B2)A kit of antibodies consisting of the antibody A and the antibody B described in (B1).

[0032] (B3)An enzyme-linked immunosorbent assay (ELISA) kit for detecting infectious pancreatic necrosis virus (IPNV), comprising a capture antibody and a detection antibody; the capture antibody is the antibody A described in (B1), and the detection antibody is the antibody B described in (B1).

[0033] Further, in (B1), the amino acid sequence of the light chain variable region of the antibody A is shown as SEQ ID No. 3; the amino acid sequence of the heavy chain variable region of the antibody A is shown as SEQ ID No. 4.

[0034] Further, in (B1), the amino acid sequence of the light chain variable region of the antibody B is shown as SEQ ID No. 5; the amino acid sequence of the heavy chain variable region of the antibody B is shown as SEQ ID No. 6.

[0035] Further, in (B3), the ELISA kit may further contain other commonly used reagents and / or consumables for ELISA detection, such as 96-well enzyme-linked immunosorbent assay plates, IPNV VP2 standard antigen, coating buffer, washing solution, blocking solution, chromogenic solution, and / or termination solution, etc.

[0036] In the ELISA kit, the capture antibody (i.e., the antibody A, corresponding to the monoclonal antibody VP2-178) can be pre-coated on a solid phase carrier such as a 96-well enzyme-linked immunosorbent assay plate; the detection antibody can be the antibody B (corresponding to the monoclonal antibody VP2-380) labeled with HRP or a labeling agent such as biotin.

[0037] In a seventh aspect, the present invention claims the use of the substance described in the sixth aspect above in any of the following:

[0038] (A1)Detecting whether an infectious pancreatic necrosis virus is contained in a test sample;

[0039] (A2)Preparing a product for detecting whether an infectious pancreatic necrosis virus is contained in a test sample;

[0040] (A3)Monitoring or warning of infectious pancreatic necrosis;

[0041] (A4)Preparing a product for monitoring or warning of infectious pancreatic necrosis.

[0042] In an eighth aspect, the present invention claims a non-disease diagnosis method for detecting infectious pancreatic necrosis virus.

[0043] The non-disease diagnosis method for detecting infectious pancreatic necrosis virus claimed by the present invention can be the double antibody sandwich ELISA method; wherein, the antibody A (corresponding monoclonal antibody VP2-178) described in the sixth aspect above is used as the capture antibody, and the antibody B (corresponding monoclonal antibody VP2-380) described in the sixth aspect above is used as the detection antibody.

[0044] In a ninth aspect, the present invention claims to protect the IPNV VP2 antigenic epitope peptide.

[0045] The amino acid sequence of the IPNV VP2 antigenic epitope peptide claimed by the present invention is SEQ ID No.1 or SEQ ID No.2.

[0046] In a tenth aspect, the present invention claims to protect the nucleic acid molecule encoding the IPNV VP2 antigenic epitope peptide described in the ninth aspect above.

[0047] In an eleventh aspect, the present invention claims to protect an expression cassette or a recombinant vector or a recombinant bacterium containing the nucleic acid molecule described in the tenth aspect above.

[0048] In a twelfth aspect, the present invention claims to protect the IPNV VP2 artificial antigen.

[0049] The IPNV VP2 artificial antigen claimed by the present invention is obtained by conjugating the IPNV VP2 antigenic epitope peptide described in the ninth aspect above with a carrier protein.

[0050] In an embodiment of the present invention, the carrier protein is specifically keyhole limpet hemocyanin (KLH).

[0051] In a thirteenth aspect, the present invention claims the application of the IPNV VP2 antigenic epitope peptide described in the ninth aspect above or the IPNV VP2 artificial antigen described in the twelfth aspect above as an immunogen in the preparation of an IPNV VP2 antibody.

[0052] In each of the above related aspects, in an embodiment of the present invention, the infectious pancreatic necrosis virus (IPNV) is type I and / or type V IPNV.

[0053] The present invention screens the antigenic epitopes of VP2, screens out two short peptides, VP2-178 and VP2-380 (the amino acid sequences are SEQ ID No.1 and SEQ ID No.2 respectively), uses them to prepare monoclonal antibodies respectively, and establishes an antigen capture ELISA detection method for the VP2 protein of the IPNV virus with the two monoclonal antibodies, and evaluates its effect, and finds that it can specifically, rapidly and sensitively detect the infectious pancreatic necrosis virus (IPNV).

[0054] Depository Instructions

[0055] Name of the deposited culture: Hybridoma cell line IPNV-vp2-178-2A7

[0056] Depository institution: China Center for Type Culture Collection

[0057] Abbreviation of the depository institution: CCTCC

[0058] Address: Wuhan University, Wuhan, China

[0059] Date of deposit: October 30, 2024

[0060] Deposit number in the depository center: CCTCC NO: C2024368

[0061] Name of the deposited culture: Hybridoma cell line IPNV-vp2-380-1G12

[0062] Depository institution: China Center for Type Culture Collection

[0063] Abbreviation of the depository institution: CCTCC

[0064] Address: Wuhan University, Wuhan, China

[0065] Date of deposit: October 30, 2024

[0066] Deposit number in the depository center: CCTCC NO: C2024369 Description of the drawings

[0067] Figure 1 Predicting antigenic sites of IPNV VP2 on the IEDB website

[0068] Figure 2 Predicting antigenic sites of IPNV VP2 on the Bepipred website

[0069] Figure 3 Diagram of antigenic sites located on the protein surface. A: Positions 17 - 30 of the IPNV-VP2 protein sequence, in red; B: Positions 139 - 152 of the IPNV-VP2 protein sequence, in purple; C: Positions 178 - 191 of the IPNV-VP2 protein sequence, in blue; D: Positions 197 - 210 of the IPNV-VP2 protein sequence, in light blue; E: Positions 380 - 393 of the IPNV-VP2 protein sequence, in orange; F: Positions 399 - 412 of the IPNV-VP2 protein sequence, in yellow.

[0070] Figure 4 For the relative positions of the two antigenic epitopes VP2-178 and VP2-380.

[0071] Figure 5 SDS-PAGE detection of monoclonal antibody VP2-178 before and after purification.

[0072] Figure 6 SDS-PAGE detection of monoclonal antibody VP2-380 before and after purification.

[0073] Figure 7 Western blot for detecting the specificity of monoclonal antibodies VP2-178 (A) and VP2-380 (B).

[0074] Figure 8 Indirect immunofluorescence for detecting the specificity of monoclonal antibody VP2-178.

[0075] Figure 9 Indirect immunofluorescence for detecting the specificity of monoclonal antibody VP2-380.

[0076] Figure 10 Determination of capture antibody and detection antibody. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. 178 / 380 indicates that monoclonal antibody VP2-178 is the capture antibody and HRP-labeled monoclonal antibody VP2-380 is the detection antibody; 380 / 178 indicates that monoclonal antibody VP2-380 is the capture antibody and HRP-labeled monoclonal antibody VP2-178 is the detection antibody.

[0077] Figure 11 Determination of the optimal working concentration of capture antibody VP2-178. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV.

[0078] Figure 12 Determination of the optimal working concentration of detection antibody VP2-380. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV.

[0079] Figure 13 Determination of the optimal coating time. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value of type I IPNV detection; N represents the OD450 value of negative sample (PBS) detection; P / N represents the OD450 value of type I IPNV detection / OD450 value of negative sample detection. The left vertical axis is the OD450 value, and the right vertical axis is the P / N value.

[0080] Figure 14Determination of the best blocking solution. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value for the detection of type I IPNV; N represents the OD450 value for the detection of the negative sample (PBS); P / N represents the OD450 value for the detection of type I IPNV / the OD450 value for the detection of the negative sample. The left vertical axis represents the OD450 value, and the right vertical axis represents the P / N value.

[0081] Figure 15 Determination of the best blocking time. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value for the detection of type I IPNV; N represents the OD450 value for the detection of the negative sample (PBS); P / N represents the OD450 value for the detection of type I IPNV / the OD450 value for the detection of the negative sample. The left vertical axis represents the OD450 value, and the right vertical axis represents the P / N value.

[0082] Figure 16 Determination of the best antigen reaction time. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value for the detection of type I IPNV; N represents the OD450 value for the detection of the negative sample (PBS); P / N represents the OD450 value for the detection of type I IPNV / the OD450 value for the detection of the negative sample. The left vertical axis represents the OD450 value, and the right vertical axis represents the P / N value.

[0083] Figure 17 Determination of the best action time of the detection antibody VP2 - 380. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value for the detection of type I IPNV; N represents the OD450 value for the detection of the negative sample (PBS); P / N represents the OD450 value for the detection of type I IPNV / the OD450 value for the detection of the negative sample. The left vertical axis represents the OD450 value, and the right vertical axis represents the P / N value.

[0084] Figure 18 Determination of the best TMB chromogenic time. A shows the detection results for type I IPNV; B shows the detection results for type V IPNV. In the figure, P represents the OD450 value for the detection of type I IPNV; N represents the OD450 value for the detection of the negative sample (PBS); P / N represents the OD450 value for the detection of type I IPNV / the OD450 value for the detection of the negative sample. The left vertical axis represents the OD450 value, and the right vertical axis represents the P / N value.

[0085] Figure 19 Determination of the positive and negative critical values of the antigen capture ELISA method. Among them, A is the receiver operating characteristic curve (ROC curve); B is the interactive dot plot.

[0086] Figure 20 This is the specificity test result of the optimized antigen capture ELISA method of the present invention. In the figure, the negative sample is PBS.

[0087] Figure 21 This is the broad-spectrum test result of the optimized antigen capture ELISA method of the present invention. In the figure, the negative sample is PBS. Specific Embodiments

[0088] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0089] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0090] Example 1: Preparation and Application of Monoclonal Antibodies Against Infectious Pancreatic Necrosis Virus

[0091] I. Experimental Methods

[0092] 1. Selection of Antigen Epitopes

[0093] Screen antigen epitopes for the VP2 protein of IPNV virus. Download the common IPNV VP2 protein sequences of type I and type V from the NCBI website, perform protein sequence alignment through the Snapgene software, and select the fragment sequences with high conservation. With the help of software such as DNAMAN, websites such as IEDB and Bepipred, screen out the peptide segments with high antigenicity. Download the constructed IPNV VP2 structure from the Uniprot and PDB websites, visualize the VP2 protein through the PyMOL software, and screen out the peptide segments located on the protein surface. After determining the peptide segments, hand them over to Genescript for synthesis. Since the short peptides are short, in order to enhance their immunogenicity, synthesize naked peptides and polypeptides conjugated with KLH respectively. Among them, the polypeptides conjugated with KLH are used to immunize mice, and the naked peptides are used as antigens for screening positive hybridomas and antibodies.

[0094] 2. Preparation, Purification and Identification of Monoclonal Antibodies

[0095] The peptide-KLH conjugate protein at 1 mg / ml was used as an antigen and mixed with Quick Antibody 3W adjuvant (purchased from Bioron Biotech Co., Ltd.) at a ratio of 1:1, and then injected into the hind leg muscles of 6-8-week-old BALB / c mice, with 100 μl injected into each mouse. Booster immunization was carried out 14 days later, and non-immunized mice were used as negative controls. Seven days after booster immunization, blood was collected from the tail tips of the mice, incubated overnight at 4 °C, centrifuged to collect the supernatant, and ELISA titer detection was performed. When the titer reached 1:6400, cell fusion was carried out. Through subcloning and screening of hybridoma cells, the 3 wells with the highest OD values were selected for the next subcloning each time. Finally, two cell lines, IPNV-vp2-178-2A7 and IPNV-vp2-380-1G12, which could stably secrete monoclonal antibodies, were obtained. 6-8-week-old BALB / c mice were intraperitoneally injected with Freund's incomplete adjuvant, and the expanded hybridoma cells were injected 7 days later. The ascites of the mice were harvested 7-10 days later. The antibody was purified using a Protein A antibody purification column, and after measuring the antibody concentration, it was stored at -80 °C; the antibody was labeled with HRP using an HRP conjugation kit from Abcam. The antibody secreted by IPNV-vp2-178-2A7 was named VP2-178, and the antibody secreted by IPNV-vp2-380-1G12 was named VP2-380.

[0096] Identification of the antibody by Western blotting: EPC cells (ATCC CRL-2872) were seeded in a six-well plate. After 12 h, suspensions of infectious hematopoietic necrosis virus (IHNV, GenBank: MT242597), spring viremia of carp virus (SVCV, GenBank: MT675953), and viral hemorrhagic septicemia virus (VHSV, described in the article "Xu Liming, Liu Miao, et al. Prokaryotic expression and application of the antigenic epitope enrichment region of infectious hematopoietic necrosis virus. Freshwater Fisheries, 2015, 45(2): 49-55", which can be obtained from the applicant by the public and can only be used for repeating the experiments of this invention and not for other purposes) were added respectively; CHSE-214 cells (described in the article "Xu Liming, Liu Miao, et al. Prokaryotic expression and application of the antigenic epitope enrichment region of infectious hematopoietic necrosis virus. Freshwater Fisheries, 2015, 45(2): 49-55", which can be obtained from the applicant by the public and can only be used for repeating the experiments of this invention and not for other purposes) were seeded in a six-well plate. After 12 h, a suspension of infectious pancreatic necrosis virus (IPNV, GenBank: KX234591) was added. The cells were cultured at 15 °C for 48 h, and then the cells were collected for Western blotting detection. Using the purified VP2 monoclonal antibody as the primary antibody and the HRP-labeled goat anti-mouse IgM antibody as the secondary antibody, after incubation at 37 °C for 1 h, development and analysis were carried out using ECL luminescent solution in an imager.

[0097] Indirect immunofluorescence identification of antibodies: Infectious pancreatic necrosis virus (IPNV, GenBank: KX234591) was inoculated into CHSE-214 cells, and infectious hematopoietic necrosis virus (IHNV, GenBank: MT242597), spring viremia of carp virus (SVCV, GenBank: MT675953), and viral hemorrhagic septicemia virus (VHSV, described in the article "Xu Liming, Liu Miao, et al. Prokaryotic expression and application of antigenic epitope enrichment region of infectious hematopoietic necrosis virus. Freshwater Fisheries, 2015, 45(2): 49-55", which can be obtained from the applicant by the public and can only be used for repeating the experiments of the present invention and shall not be used for other purposes) were inoculated into EPC cells. After culturing at 15 °C for 48 h, the cells were washed twice with sterile PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min, and the cells were perforated with 0.5% Triton-X 100 for 20 min. The cells were blocked with 5% skim milk powder at 37 °C for 1 h. Using the prepared VP2 monoclonal antibody as the primary antibody and Alexa Fluor 488-labeled goat anti-mouse IgM as the secondary antibody, after incubating at 37 °C for 1 h, the cells were washed three times with PBS, 1 μg / mL DAPI was added, and the reaction was carried out at 37 °C for 5 min. After washing three times with PBS, the results were observed using a fluorescence microscope.

[0098] 3. Establishment of antigen capture ELISA method

[0099] Test procedure:

[0100] (1) Coating capture antibody: Dilute the capture antibody with coating buffer (pH 9.6 carbonate buffer), add 100 μL to each well for coating.

[0101] (2) Blocking: After washing away the unbound capture antibody, add the blocking solution (100 μL / well), block at 37 °C to reduce non-specific binding.

[0102] (3) Sample addition and incubation: Add the diluted test sample (100 μL / well), incubate at 37 °C to allow the antigen to bind to the capture antibody.

[0103] (4) Washing: Wash 3-5 times with PBST (PBS containing 0.05% Tween-20), 5 minutes each time, to remove unbound substances.

[0104] (5) Adding detection antibody: Add the HRP-labeled detection antibody (100 μL / well), incubate at 37 °C to form a "capture antibody - antigen - enzyme-labeled antibody" complex.

[0105] (6) Color development and termination: After washing the plate, add the TMB reaction substrate, develop color in the dark, and then add the termination solution (2M H2SO4) to terminate the reaction.

[0106] (7) Detection and calculation: Read OD450 with an enzyme-linked immunosorbent assay (ELISA) reader.

[0107] By optimizing the reaction conditions, the optimal capture antibody and detection antibody were determined, and their optimal working concentrations were determined as follows: VP2-178 antibody and VP2-380 antibody were used as the capture antibody and detection antibody respectively. The capture antibody was serially diluted 4-fold (1000 - 256000) to screen out the optimal capture antibody and detection antibody. The concentrations of the screened capture antibody (1 μg / well, 0.5 μg / well, 0.1 μg / well, 0.05 μg / well, 0.01 μg / well) and detection antibody (stock concentration 1 mg / ml, dilution ratios 1:1000, 1:2000, 1:4000, 1:8000) were optimized. At the same time, the optimal coating time (overnight at 4 °C, overnight at 4 °C + 1 h at 37 °C, 1 h at 37 °C, 2 h at 37 °C), optimal blocking solution (3% skim milk powder, 5% skim milk powder, 3% fetal bovine serum (FBS), 5% FBS, 3% bovine serum albumin (BSA), 5% BSA), optimal blocking time (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h), optimal antigen reaction time (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h), optimal detection antibody incubation time (0.5 h, 1 h, 1.5 h, 2 h, 2.5 h), and optimal TMB chromogenic time (10 min, 15 min, 20 min, 30 min) in the ELISA detection were determined. The optimal working conditions were determined according to the OD450 values under different conditions, where the larger the OD450 value, the better the detection effect.

[0108] 4. Determination of the cut-off value for the antigen capture ELISA method

[0109] Fifteen IPNV-negative and 33 IPNV-positive samples previously determined by the RT-qPCR detection method (the optimal method screened in the article "Zhang Wen et al. Establishment and application of a real-time fluorescence quantitative RT-PCR detection method for infectious pancreatic necrosis virus. China Animal Health Inspection, Vol. 40, No. 4, 2023", including the optimal primers, probes, and detection conditions) were selected for detection. Interactive dot plot analysis was performed using MedCalcv20.0.10 to determine the cut-off value of the antigen capture ELISA method.

[0110] 5. Evaluation of the antigen capture ELISA detection method

[0111] (1) Specificity detection

[0112] The antigen capture ELISA method optimized by step 3 was used to detect the virus dilutions of infectious pancreatic necrosis virus (IPNV, GenBank: KX234591), infectious hematopoietic necrosis virus (IHNV, GenBank: MT242597), spring viremia of carp virus (SVCV, GenBank: MT675953), and viral hemorrhagic septicemia virus (VHSV, described in the article "Xu Liming, Liu Miao, et al. Prokaryotic expression and application of the antigenic epitope enrichment region of infectious hematopoietic necrosis virus. Freshwater Fisheries, 2015, 45(2): 49-55", which is available to the public from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes). The specific method is as follows: ① Coat the ELISA plate with 0.5 μg / well of VP2-178 antibody, wash the ELISA plate 3 times with PBST after coating overnight at 4°C; ② Block the ELISA plate with 5% skim milk powder at 37°C for 2 h, and then wash the ELISA plate 3 times with PBST; ③ Add the virus solutions of IPNV, IHNV, SVCV, and VHSV respectively, incubate at 37°C for 1.5 h, and then wash the ELISA plate 3 times with PBST; ④ Add the HRP-labeled VP2-380 antibody, incubate at 37°C for 1.5 h, and then wash the ELISA plate 3 times with PBST; ⑤ Add the TMB chromogenic solution, incubate at room temperature for 15 min, and then read the absorbance value of OD450 using an ELISA reader. The specificity of this method was evaluated by comparing the OD450nm value with the cut-off value.

[0113] (2) Broad-spectrum detection

[0114] The original solutions of IPNV type I viruses (GenBank: KX234591, KX355401, MW662100, MW662098, MW662095) and IPNV type V viruses (GenBank: MW662108, MW662092, MW662091) (each of the above IPNVs is described in the article "Kaiyue Duan, Jingzhuang Zhao, Guangming Ren, et la. Molecular Evolution of Infectious Pancreatic Necrosis Virus in China. Viruses, 2021, 13, 488. https: / / doi.org / 10.3390 / v13030488", which is available to the public from the applicant and can only be used for repeating the experiments of the present invention and not for other purposes) were diluted 1000-fold, and the titer was detected by the antigen capture ELISA method optimized by step 3 to verify its broad-spectrum property.

[0115] (3) Sensitivity detection

[0116] First, the original solution of IPNV virus (GenBank: KX234591) was serially diluted 10-fold to determine the detection limit range of this detection method. Then, the IPNV virus solution was serially diluted 2-fold to accurately detect and determine the detection limit.

[0117] (4) Repeatability test

[0118] Using the ELISA plates coated with the optimized method in step 3 from the same batch, 4 IPNV virus samples (GenBank: KX234591, KX355401, MW662108, MW662092) were detected. Four replicates were set in each group to measure the OD values and calculate the within-batch coefficient of variation. Using the ELISA plates coated with different batches, 4 virus samples were detected. Four replicates were set in each group to measure the OD values and calculate the between-batch coefficient of variation. The repeatability was determined.

[0119] 6. Clinical application

[0120] Forty clinical tissue samples infected or not infected with IPNV were detected by antigen capture ELISA method. At the same time, the optimal method (including the optimal primers and probes and optimal detection conditions) screened from the article "Zhang Wen et al. Establishment and application of a real-time fluorescence quantitative RT-PCR detection method for infectious pancreatic necrosis virus. China Animal Quarantine, Vol. 40, No. 4, 2023" was used as the control method to detect the tissue samples. According to the detection results, the coincidence rate between the two was calculated.

[0121] II. Results and analysis

[0122] 1. Antigenic epitope

[0123] Through software such as DNAMAN, websites such as IEDB and Bepipred, the antigenicity and hydrophilicity of the IPNV-VP2 protein sequence were analyzed. It was found that the peptide segments at positions 178-191aa, 197-210aa, 17-30aa, 32-45aa, 399-412aa and 380-393aa of the IPNV-VP2 protein sequence all had strong hydrophilicity, and the peptide segments at positions 17-30aa, 165-220aa, 370-421aa had strong antigenicity, and the peptide segment length was greater than 14. It was comprehensively found that 178-191aa, 197-210aa, 17-30aa, 399-412aa, 380-393aa of the VP2 protein had strong antigenicity. As shown in Table 1, Figure 1 and Figure 2 shown.

[0124] Table 1. Prediction of antigenic sites of IPNV VP2

[0125] Serial number Start site Epitope Length Antigenicity / Hydrophilicity 1 178 CPQGPQSMNGARMR 14 2.15 / 0.50 2 197 CPRRYEIDLPSERLP 14 2.04 / 0.58 3 17 PASIPDDITERHILC 14 1.94 / 0.27 4 32 QETSSYNLEVSESGC 14 1.90 / 0.26 5 399 CLSHREELDIRTVWR 14 1.76 / 0.58 6 427 CTDFTSDLPTSKAWG 14 1.76 / 0.09 7 279 CENHRGASAKFTQSI 14 1.65 / 0.18 8 380 CMVTKYGKYDPEGLN 14 1.51 / 0.29

[0126] 2. Short peptides on the protein surface

[0127] The structure of VP2 protein was queried on the Uniprot website and visualized in the PyMOL software. Antigenic sites on the protein surface were screened out from it. Among them, positions 17 - 30, 139 - 152, and 399 - 412 of the IPNV - VP2 protein sequence were partially blocked, and positions 178 - 191, 197 - 210, and 380 - 393 were on the protein surface. As Figure 3 shown

[0128] To establish an ELISA detection method, two different antigenic epitopes need to be selected on each protein respectively and used as capture antibody and labeled antibody. Considering that the short peptides may affect each other if they are too close, two short peptides, VP2 - 178 and VP2 - 380, were selected as antigenic epitopes (see Table 2), Figure 4 indicating their relative positions

[0129] Table 2. Final antigenic epitope sequences of VP2

[0130] Name Sequence VP2-178 sequence PQGPQSMNGARMRC (SEQ ID No.1) VP2-380 sequence MVTKYGKYDPEGLNC (SEQ ID No.2)

[0131] 3. Solubility analysis

[0132] The solubility of the peptide segments was evaluated through the official website of https: / / www.genscript.com, and it was found that all the above - mentioned sites had strong solubility. Since the short peptides were short, to enhance their immunogenicity, naked peptides and peptides conjugated with KLH were synthesized respectively. Among them, the peptides conjugated with KLH were used to immunize mice, and the naked peptides were used as antigens for screening positive hybridomas and antibodies. They were synthesized by a biological company and dissolved in appropriate solvents, as shown in Table 3

[0133] Table 3. Solubility of different antigenic epitopes in different solvents

[0134] Antigen site Water 1×PBS DMSO VP2-178 √ × √ VP2-380 √ √ √ VP2-178 conjugated to KLH √ √ × VP2-380 conjugated to KLH √ × ×

[0135] Note: √ indicates soluble, × indicates insoluble

[0136] 4. Preparation and identification of monoclonal antibodies

[0137] The antigen - KLH conjugated protein was injected into the hind leg muscles of mice once every 2 weeks. Blood was collected from the mice 7 days after injection for ELISA titer detection. After the titer reached 1:6400, cell fusion was carried out with SP2 / 0. Through multiple sub - cloning cultures, hybridoma cells IPNV - vp2 - 178 - 2A7 and IPNV - vp2 - 380 - 1G12 that could stably secrete antibodies were obtained

[0138] The hybridoma cell line IPNV-vp2-178-2A7 is a hybridoma cell line prepared by immunizing mice with the epitope peptide VP2-178. This hybridoma cell line was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2024, and the deposit number at the deposit center is CCTCC NO: C2024368.

[0139] The hybridoma cell line IPNV-vp2-380-1G12 is a hybridoma cell line prepared by immunizing mice with the epitope peptide VP2-380. This hybridoma cell line was deposited at the China Center for Type Culture Collection (CCTCC) on October 30, 2024, and the deposit number at the deposit center is CCTCC NO: C2024369.

[0140] Ascites of mice was obtained by intraperitoneal injection of hybridoma cells into mice, and antibodies were purified from the ascites. The specific operation method was carried out according to the instruction manual of the Protein A antibody purification column (P2015) of Beyotime Biotechnology Co., Ltd. Liquids at each stage were collected for gel electrophoresis. Before purification, the ascites was named: before purification, the purified flow-through was named: flow-through, the first wash was named: wash 1, the tenth wash was named: wash 10, the glycine eluate was named: elution, the protein solution after solution replacement without adding DTT was named: desalting without DTT, and the protein solution after solution replacement with DTT added was named: desalting with DTT. Among them, the monoclonal antibody prepared using the hybridoma cell line IPNV-vp2-178-2A7 was named VP-178; the monoclonal antibody prepared using the hybridoma cell line IPNV-vp2-380-1G12 was named VP-380. SDS-PAGE detection was performed on samples before and after purification and at each sampling point during the purification process. The results showed that the purification results were good, and the antibody concentrations of both reached 1 mg / ml, and they could be labeled with HRP. As Figure 5 and Figure 6 shown.

[0141] The subtypes of the monoclonal antibodies VP-178 and VP-380 were identified respectively using the SBA Clonotyping System-HRP kit (Southern Biotech, catalog number 5300-05), and the results are shown in Table 4.

[0142] Table 4. Results of antibody subtype identification (OD405nm values)

[0143] Antibody name Subtype result IgG1 IgG2a IgG2b IgG2c IgG3 IgA IgM K λ vp2-178 IgM.K 0.087 0.100 0.118 0.110 0.130 0.099 2.370 2.349 0.091 vp2-380 IgM.K 0.097 0.116 0.094 0.095 0.100 0.088 2.319 2.229 0.079

[0144] The variable region sequences of the monoclonal antibodies VP-178 and VP-380 were detected respectively, and the results are as follows:

[0145] The amino acid sequence of the light chain variable region of monoclonal antibody VP-178 is shown in SEQ ID No.3, wherein LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No.3 at positions 44-58, 74-80 and 113-121, respectively; the amino acid sequence of the heavy chain variable region of monoclonal antibody VP-178 is shown in SEQ ID No.4, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No.4 at positions 50-54, 69-85 and 118-127, respectively.

[0146] The amino acid sequence of the light chain variable region of monoclonal antibody VP-380 is shown in SEQ ID No.5, wherein the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No.5 at positions 44-58, 74-80 and 113-121, respectively; the amino acid sequence of the heavy chain variable region of monoclonal antibody VP-380 is shown in SEQ ID No.6, wherein the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No.6 at positions 50-54, 69-85 and 118-127, respectively.

[0147] The specificity of two monoclonal antibodies, VP-178 and VP-380, was tested by Western blot and indirect immunofluorescence, and it was found that both reacted specifically with IPNV VP2 protein and had no cross-reaction with other viruses. Figure 7 , Figure 8 and Figure 9 shown.

[0148] 5. Establishment of antigen capture ELISA method

[0149] After experimental optimization, it was determined that the best capture antibody was monoclonal antibody VP2-178, the best working concentration was 0.5μg / well, the detection antibody was HRP-labeled monoclonal antibody VP2-380, and the best dilution was 1:2000. The best reaction conditions were: capture antibody VP2-178 was added to the ELISA plate at 4°C overnight at 100μl per well, blocked with 5% skim milk powder for 2h, and the antigen to be detected was reacted at 37°C for 1.5h. Detection antibody VP2-380 was reacted at 37°C for 1.5h at 100μl / well, and after adding TMB reaction substrate, the reaction was stopped by adding stop solution for 15min. The details are as follows:

[0150] (1) Determination of capture antibody and detection antibody

[0151] The capture antibody was serially diluted 4-fold (1000 - 256000), and the detection antibody was diluted 1:2000. The original virus stocks of IPNV type I (GenBank: KX234591) and IPNV type V (GenBank: MW662108) were initially diluted 1:1000. Two paired experiments were set up: monoclonal antibody VP2-178 as the capture antibody and HRP-labeled monoclonal antibody VP2-380 as the detection antibody; monoclonal antibody VP2-380 as the capture antibody and HRP-labeled monoclonal antibody VP2-178 as the detection antibody. The operation was carried out according to the test procedure in step 3. Among them, the dosage of the capture antibody was 0.1 μg / well, the coating time in ELISA was overnight at 4 °C, the blocking solution was 5% skim milk powder, the blocking time was 1 h, the antigen reaction time was 1 h, the action time of the detection antibody was 1 h, and the TMB color development time was 10 min.

[0152] According to the ELISA test results, monoclonal antibody VP2-178 was selected as the capture antibody and HRP-labeled monoclonal antibody VP2-380 as the detection antibody. As Figure 10 shown.

[0153] (2) Determination of the optimal working concentration of the capture antibody

[0154] The optimized concentrations of the capture antibody VP2-178 were set as follows: 1 μg / well, 0.5 μg / well, 0.1 μg / well, 0.05 μg / well, 0.01 μg / well. The antigens to be detected were the original virus stocks of IPNV type I (GenBank: KX234591) and IPNV type V (GenBank: MW662108) and they were diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the dilution factor of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4 °C, the blocking solution was 5% skim milk powder, the blocking time was 1 h, the antigen reaction time was 1 h, the action time of the detection antibody was 1 h, and the TMB color development time was 10 min.

[0155] According to the test results, the optimal concentration of the capture antibody VP2-178 was 0.5 μg / well. As Figure 11 shown.

[0156] (3) Determination of the optimal working concentration of the detection antibody

[0157] The optimized dilution factors of the HRP-labeled detection antibody VP2-380 are 1:1000, 1:2000, 1:4000, and 1:8000. The antigens to be detected are the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108), which are diluted 2-fold. The operation is carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 is 0.5 μg / well, the coating time in ELISA detection is overnight at 4°C, the blocking solution is 5% skim milk powder, the blocking time is 1 h, the antigen reaction time is 1 h, the action time of the detection antibody is 1 h, and the TMB color development time is 10 min.

[0158] According to the test results, the optimal dilution factor of the detection antibody VP2-380 is 1:2000. As Figure 12 shown.

[0159] (4) Determination of the optimal coating time

[0160] Under the conditions of the optimal capture antibody concentration and the optimal detection antibody concentration, the coating time is optimized, and 4 parallels are set as follows: overnight at 4°C; 1 h at 37°C; overnight at 4°C first and then 1 h at 37°C; 2 h at 37°C. Among them, the overnight time is specifically 12 h. The antigens are the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, which are diluted 2-fold. The operation is carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 is 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 is 1:2000, the blocking solution in ELISA detection is 5% skim milk powder, the blocking time is 1 h, the antigen reaction time is 1 h, the action time of the detection antibody is 1 h, and the TMB color development time is 10 min.

[0161] The results show that the optimal coating time is overnight at 4°C. As Figure 13 shown.

[0162] (5) Determination of the optimal blocking solution

[0163] Under the above optimal conditions, the blocking effects of 3% skim milk powder, 5% skim milk powder, 3% FBS, 5% FBS, 3% BSA, and 5% BSA were compared. Here, % represents g / 100 ml, and the solvent for each of the above blocking solutions was PBS. The antigens used were the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, which were serially diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 was 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4°C, the blocking time was 1 h, the antigen reaction time was 1 h, the action time of the detection antibody was 1 h, and the TMB color development time was 10 min.

[0164] The results showed that 5% skim milk powder had the best blocking effect. Therefore, 5% skim milk powder was selected as the blocking solution in subsequent experimental operations. As Figure 14 shown.

[0165] (6) Determination of the optimal blocking time

[0166] Under the above optimal conditions, the blocking effects at different blocking times (0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h) were compared. The antigens used were the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, which were serially diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 was 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4°C, the blocking solution was 5% skim milk powder, the antigen reaction time was 1 h, the action time of the detection antibody was 1 h, and the TMB color development time was 10 min.

[0167] The experimental results showed that the optimal blocking time was 2 h. As Figure 15 shown.

[0168] (7) Determination of the optimal antigen reaction time

[0169] Under the above optimal conditions, the effects of different antigen reaction times (0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h) on the detection results were compared. The antigens used were the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, and they were diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 was 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4°C, the blocking solution was 5% skim milk powder, the blocking time was 2 h, the action time of the detection antibody was 1 h, and the TMB color development time was 10 min.

[0170] The results showed that the optimal antigen reaction time was 1.5 h. As Figure 16 shown.

[0171] (8) Determination of the optimal action time of the detection antibody

[0172] Under the above optimal conditions, the effects of different action times (0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h) of the detection antibody on the detection results were compared. The antigens used were the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, and they were diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 was 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4°C, the blocking solution was 5% skim milk powder, the blocking time was 2 h, the antigen reaction time was 1.5 h, and the TMB color development time was 10 min.

[0173] The results showed that the optimal action time of the detection antibody was 1.5 h. As Figure 17 shown.

[0174] (9) Determination of the optimal TMB color development time

[0175] Under the above optimal conditions, the effects of different TMB chromogenic times (10 min, 15 min, 20 min, and 30 min) on the detection results were compared. The antigens used were the original virus solutions of type I IPNV (GenBank: KX234591) and type V IPNV (GenBank: MW662108) with a protein concentration of 2.5 mg / ml, and they were diluted 2-fold. The operation was carried out according to the test procedure in step 3. Among them, the coating amount of the capture antibody VP2-178 was 0.5 μg / well, the dilution ratio of the detection antibody VP2-380 was 1:2000, the coating time in ELISA was overnight at 4°C, the blocking solution was 5% skim milk powder, the blocking time was 2 h, the antigen reaction time was 1.5 h, and the action time of the detection antibody was 1.5 h.

[0176] The results showed that the optimal TMB chromogenic time was 15 min. As Figure 18 shown.

[0177] (10) Determination of the critical value

[0178] Using the established ELISA method, 15 IPNV-negative and 33 IPNV-positive tissue samples with clear backgrounds were detected. According to the OD450nm values and the results detected by the optimal method (including the optimal primer-probe and optimal detection conditions) screened in the article "Zhang Wen et al. Establishment and application of a real-time fluorescence quantitative RT-PCR detection method for infectious pancreatic necrosis virus. China Animal Quarantine, Vol. 40, No. 4, 2023", the critical value was calculated using MedCalcv20.0.10 software. The results showed that when the sensitivity was 97.9% and the specificity was 100%, the optimal cut-off value was 0.2137. Therefore, the critical value OD450nm of ELISA was determined to be 0.2137 (see Figure 19 ), and when OD450nm was greater than or equal to this value, it was considered positive, and when OD450nm was less than this value, it was considered negative.

[0179] 6. Specificity

[0180] The optimized antigen capture ELISA method in step 5 was used to detect IHNV, IPNV, SVCV, and VHSV respectively. The results showed that except for the OD450nm value of IPNV being greater than the critical value, the OD450nm values of the other viruses were all below the critical value, indicating that this method had good specificity. As Figure 20 shown.

[0181] 7. Broad-spectrum

[0182] The antigen capture ELISA method established in step 5 above was used to detect different strains of IPNV, and its broad-spectrum property was evaluated. The results showed that this method could effectively detect IPNV strains of type I and type V, and had the broad-spectrum detection ability for IPNV. As Figure 21 shown.

[0183] 8. Sensitivity

[0184] The sensitivity of the antigen capture ELISA method established in step 5 above was detected. The original virus solution of IPNV (GenBank: KX234591) was diluted serially at a 10-fold ratio. After measurement, its detection limit was around 10 2 TCID 50 . Serial 2-fold dilutions were made within this range, and finally the detection limit of this method was determined to be 62.5 TCID 50 / mL. The results showed that this ELISA detection method had high sensitivity. As shown in Table 5 and Table 6.

[0185] Table 5. Results of 10-fold dilution sensitivity test

[0186] Dilution factor <![CDATA[10 2 > <![CDATA[10 3 > <![CDATA[10 4 > <![CDATA[10 5 > <![CDATA[10 6 > <![CDATA[10 7 > <![CDATA[10 8 > <![CDATA[10 9 > <![CDATA[Virus titer (TCID 50 / mL)]]> <![CDATA[10 6 > <![CDATA[10 5 > <![CDATA[10 4 > <![CDATA[10 3 > <![CDATA[10 2 > 10 1 0.1 <![CDATA[OD 450nm > 2.356 2.263 1.258 0.645 0.267 0.120 0.079 0.071 Detection result + + + + + - - -

[0187] Table 6. Results of 2-fold dilution sensitivity test

[0188] Dilution factor <![CDATA[10 5 > <![CDATA[2×10 5 > <![CDATA[4×10 5 > <![CDATA[8×10 5 > <![CDATA[1.6×10 6 > <![CDATA[3.2×10 6 > <![CDATA[6.4×10 6 > <![CDATA[1.3×10 7 <!-- 12 -->]]> <![CDATA[Virus titer (TCID 50 / mL)]]> <![CDATA[10 3 > 500 250 125 62.5 31.25 15.6 7.8 <![CDATA[OD 450nm > 0.637 0.555 0.423 0.321 0.229 0.139 0.099 0.086 Detection result + + + + + - - -

[0189] 9. Repeatability

[0190] Using the same batch of pre-coated ELISA plates, 4 virus samples were detected. The results showed that the coefficient of variation for the detection of this batch of samples was 3.778 - 5.468%. Using pre-coated ELISA plates from different batches, 4 virus samples were detected. The results showed that the coefficient of variation for the detection of this batch of samples was 3.995 - 6.518%. The coefficients of variation of the two groups of experiments were both lower than 10%. Based on this result, it can be inferred that this detection method has good repeatability and stability. As shown in Table 7.

[0191] Table 7. Results of antigen capture ELISA repeatability test (n = 4)

[0192]

[0193] 10. Clinical application

[0194] In this study, 40 clinical samples were selected, including the mixed tissues of liver, spleen and head kidney of rainbow trout infected and not infected with IPNV. The tissues were ground with PBS solution containing 1% TritonX–100. After centrifugation at 12,000 r for 10 min, the supernatant was taken for ELISA detection (performed according to Step 1-3 with the optimal parameters). At the same time, the optimal method (including the optimal primer-probe and optimal detection conditions) screened from the article "Zhang Wen, et al. Establishment and application of a real-time fluorescence quantitative RT-PCR detection method for infectious pancreatic necrosis virus. China Animal Quarantine, Vol. 40, No. 4, 2023" was used as a control for detection. The detection results of the present invention were compared and analyzed with those of the control method, and the coincidence rate of the two results reached 100%. As shown in Table 8.

[0195] Table 8. Comparison of ELISA and control method detection results

[0196]

[0197] Note: + indicates that the detection result by the control method is positive, and - indicates that the detection result by the control method is negative.

[0198] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. A hybridoma cell line or a set of hybridoma cell lines, characterized in that: The hybridoma cell line is a hybridoma cell line IPNV-vp2-178-2A7 or a hybridoma cell line IPNV-vp2-380-1G12; The set of hybridoma cell lines consists of the hybridoma cell line IPNV-vp2-178-2A7 and the hybridoma cell line IPNV-vp2-380-1G12; The deposit number of the hybridoma cell line IPNV-vp2-178-2A7 in the China Center for Type Culture Collection is CCTCCNO: C2024368; The deposit number of the hybridoma cell line IPNV-vp2-380-1G12 in China Center for Type Culture Collection is CCTCC NO: C2024369.

2. A monoclonal antibody or a set of monoclonal antibodies, characterized in that: The monoclonal antibody is monoclonal antibody VP2-178 or monoclonal antibody VP2-380; The set of monoclonal antibodies consists of the monoclonal antibody VP2-178 and the monoclonal antibody VP2-380; The monoclonal antibody VP2-178 is secreted and produced by the hybridoma cell line IPNV-vp2-178-2A7 described in claim 1; The monoclonal antibody VP2-380 is secreted and produced by the hybridoma cell line IPNV-vp2-380-1G12 described in claim 1.

3. An enzyme-linked immunosorbent assay kit for detecting infectious pancreatic necrosis virus, comprising a capture antibody and a detection antibody; the capture antibody is the monoclonal antibody VP2-178 described in claim 2, and the detection antibody is the monoclonal antibody VP2-380 described in claim 2.

4. Use of the hybridoma cell line or the set of hybridoma cell lines according to claim 1, the monoclonal antibody or the set of monoclonal antibodies according to claim 2, or the enzyme-linked immunosorbent assay kit according to claim 3 in any of the following: (A1) detecting whether the sample to be tested contains infectious pancreatic necrosis virus; (A2) preparing a product for detecting whether a sample to be tested contains infectious pancreatic necrosis virus; (A3) Monitoring or early warning of infectious pancreatic necrosis; (A4) Prepare products for monitoring or early warning of infectious pancreatic necrosis disease.

5. A non-disease diagnostic method for detecting infectious pancreatic necrosis virus, characterized in that: The method is an ELISA double antibody sandwich method; wherein the monoclonal antibody VP2-178 described in claim 2 is used as a capture antibody, and the monoclonal antibody VP2-380 described in claim 2 is used as a detection antibody.

6. Any of the following substances: (B1) an anti-IPNV VP2 antibody, which is antibody A or antibody B; The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody A are shown in SEQ ID No. 3, positions 44-58, 74-80 and 113-121, respectively; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody A are shown in SEQ ID No. 4, positions 50-54, 69-85 and 118-127, respectively; The amino acid sequences of LCDR1, LCDR2 and LCDR3 in the light chain variable region of the antibody B are shown in SEQ ID No. 5, positions 44-58, 74-80 and 113-121, respectively; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of the antibody B are shown in SEQ ID No. 6, positions 50-54, 69-85 and 118-127, respectively; (B2) a set of antibodies consisting of the antibody A and the antibody B described in (B1); (B3) An enzyme-linked immunosorbent assay kit for detecting infectious pancreatic necrosis virus, comprising a capture antibody and a detection antibody; the capture antibody is antibody A described in (B1), and the detection antibody is antibody B described in (B1).

7. The substance according to claim 6, characterized in that: The amino acid sequence of the light chain variable region of the antibody A is shown in SEQ ID No.3; the amino acid sequence of the heavy chain variable region of the antibody A is shown in SEQ ID No.4; and / or The amino acid sequence of the light chain variable region of the antibody B is shown in SEQ ID No.5; the amino acid sequence of the heavy chain variable region of the antibody B is shown in SEQ ID No.

6.

8. Use of the substance according to claim 6 or 7 in any of the following: (A1) detecting whether the sample to be tested contains infectious pancreatic necrosis virus; (A2) preparing a product for detecting whether a sample to be tested contains infectious pancreatic necrosis virus; (A3) Monitoring or early warning of infectious pancreatic necrosis; (A4) Prepare products for monitoring or early warning of infectious pancreatic necrosis disease.

9. A non-disease diagnostic method for detecting infectious pancreatic necrosis virus, characterized in that: The method is an ELISA double antibody sandwich method; wherein the antibody A described in claim 6 or 7 is used as a capture antibody, and the antibody B described in claim 6 or 7 is used as a detection antibody.

10. IPNV VP2 antigen epitope peptide, whose amino acid sequence is SEQ ID No.1 or SEQ ID No.2; or A nucleic acid molecule encoding the IPNV VP2 antigenic epitope peptide; or An expression cassette or a recombinant vector or a recombinant bacterium containing the nucleic acid molecule; or The IPNV VP2 artificial antigen is obtained by coupling the IPNV VP2 antigen epitope peptide with a carrier protein; or The IPNV VP2 antigen epitope peptide or the IPNV VP2 artificial antigen is used as an immunogen in the preparation of IPNV VP2 antibodies.

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