Hybridoma cell strain secreting anti-eel herpesvirus monoclonal antibody, anti-eel herpesvirus monoclonal antibody and application of anti-eel herpesvirus monoclonal antibody

Abstract

The invention belongs to the technical field of monoclonal antibodies and biological detection, and particularly relates to a hybridoma cell strain secreting an anti-AngHV monoclonal antibody, the anti-AngHV monoclonal antibody and application of the anti-AngHV monoclonal antibody. The anti-eel herpesvirus monoclonal antibody provided by the invention is prepared by taking purified eel herpesvirus particles as antigens, and can specifically recognize eel herpesvirus; a double-antibody sandwich ELISA detection kit is developed on the basis of the anti-eel herpesvirus monoclonal antibody, the kit is prepared by taking the anti-eel herpesvirus monoclonal antibody as a capture antibody and taking an eel herpesvirus polyclonal antibody as a detection antibody, and the kit is suitable for detecting the eel herpesvirus. The method has good specificity and sensitivity, is simple, convenient and rapid, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of monoclonal antibodies and biological detection, and specifically relates to a hybridoma cell line secreting anti-eel herpes virus monoclonal antibodies, anti-eel herpes virus monoclonal antibodies and applications thereof. Background Art

[0002] Eels are known as "soft gold in water" and have important medicinal effects and nutritional value. "Debonding septicemia syndrome" is the main epidemic in eel farming. It breaks out all year round during the large-scale farming of eels, causing huge economic losses to farmers. It has been confirmed that Anguillid herpesvirus (AngHV) is the causative pathogen of eel "debonding septicemia syndrome", which can cause clinical symptoms such as skin mucus shedding and bleeding, fin bleeding, and bloody ascites in the abdominal cavity. It has the characteristics of high incidence, short course of disease, fast transmission rate and high cumulative mortality, which seriously threatens the sustainable development of the eel farming industry.

[0003] AngHV is one of the most destructive viruses and is ubiquitous in eel farming. Since AngHV was first isolated from Japanese eels (Anguilla japonica) farmed in Japan, it has been isolated in different parts of the world, including the main farmed species European eels (Anguilla anguilla) and American eels (Anguilla rostrata). Serological and molecular studies have confirmed that these virus strains are the same virus. AngHV is an enveloped linear double-stranded DNA virus belonging to the family Heteroherpesviridae of the order Herpesvirales. Proteomic analysis has identified that the AngHV virus particle structure contains 7 nucleocapsid proteins, 11 envelope proteins, and 22 envelope proteins. Among them, the ORF95 gene encodes the envelope structural protein of the AngHV virus particle, with a conserved sequence and high gene abundance, making it suitable as a target site for virus detection; while ORF36 is the capsid protein of the AngHV virus particle, with 13 potential antigenic epitopes and good immunogenicity. The expression of these structural proteins can be used as an important tool for establishing detection methods.

[0004] Clinical studies have found that AngHV has a high infection prevalence in young eels and is latent. It can be transmitted through water and easily activated and erupted by exogenous or endogenous stimuli, with a mortality rate of up to 30%. However, to date, there is still a lack of vaccines or antiviral drugs that can effectively prevent and control AngHV. Therefore, early diagnosis of eel infection with AngHV is crucial to prevent the spread of "disintegration sepsis syndrome".

[0005] So far, the established AngHV detection methods include in situ hybridization, polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), recombinase-assisted amplification combined with lateral flow test strips (RAA-LFD), loop-induced isothermal amplification (LAMP), and real-time quantitative loop-induced isothermal amplification (qLAMP).

[0006] However, existing technologies for detecting AngHV all rely on molecular diagnostic methods, which are cumbersome and time-consuming, have high requirements on the professional knowledge of the testing personnel, and often require expensive instruments, making them difficult to promote and apply. Summary of the invention

[0007] The purpose of the present invention is to provide a hybridoma cell line secreting anti-eel herpes virus monoclonal antibody, anti-eel herpes virus monoclonal antibody and application thereof. The anti-eel herpes virus monoclonal antibody has the function of specifically recognizing AngHV, can be used for the preparation of eel herpes virus detection products, and the operation is simple and fast.

[0008] The invention provides a hybridoma cell strain AngHV-10G8 which secretes an anti-eel herpes virus monoclonal antibody, and the deposit number is CCTCC NO: C2024410.

[0009] The present invention also provides an anti-eel herpes virus monoclonal antibody 10G8-9F4, wherein the anti-eel herpes virus monoclonal antibody 10G8-9F4 is obtained by secretion of the hybridoma cell line AngHV-10G8 described in the above technical solution.

[0010] The present invention also provides the use of the hybridoma cell line AngHV-10G8 described in the above technical solution or the anti-eel herpes virus monoclonal antibody 10G8-9F4 described in the above technical solution in the preparation of products for detecting eel herpes virus and / or in molecular biology research on eel herpes virus.

[0011] Preferably, the product is prepared based on immunoassay technology.

[0012] Preferably, the product comprises a reagent and / or a kit.

[0013] Preferably, the kit comprises a double antibody sandwich ELISA detection kit.

[0014] The present invention also provides a double antibody sandwich ELISA detection kit for detecting eel herpes virus, the double antibody sandwich ELISA detection kit comprises a capture antibody and a detection antibody, the capture antibody is an anti-eel herpes virus monoclonal antibody, and the anti-eel herpes virus monoclonal antibody is the anti-eel herpes virus monoclonal antibody 10G8-9F4 described in the above technical solution;

[0015] The detection antibody is an anti-eel herpes virus polyclonal antibody.

[0016] Preferably, the anguilla herpesvirus polyclonal antibody is obtained by immunizing animals with the envelope structural protein ORF95 of the anguilla herpesvirus as an antigen.

[0017] The present invention also provides the use of the double-antibody sandwich ELISA detection kit described in the above technical solution in the preparation of a product for detecting eel herpes virus.

[0018] Preferably, the sample to be tested includes one or more of blood, body fluids, tissues and cells.

[0019] Beneficial effects:

[0020] The present invention provides a hybridoma cell line AngHV-10G8 that secretes an anti-eel herpes virus monoclonal antibody, and the deposit number is CCTCC NO: C2024410. On this basis, an anti-eel herpes virus monoclonal antibody 10G8-9F4 secreted by the above hybridoma cell line AngHV-10G8 is provided. The present invention uses purified AngHV as an immunogen, and the obtained monoclonal antibody has high specificity and affinity, can specifically recognize AngHV, and can be used for the detection of eel herpes virus, drug preparation and related research.

[0021] Based on the anti-eel herpes virus monoclonal antibody, the present invention also provides a double antibody sandwich ELISA detection kit for detecting eel herpes virus, wherein the double antibody sandwich ELISA detection kit is prepared with anti-eel herpes virus monoclonal antibody as capture antibody and anti-eel herpes virus polyclonal antibody as detection antibody, and the double antibody sandwich ELISA detection kit has good specificity and sensitivity, has no cross reaction with other common aquatic viruses, and can be used for the detection of eel herpes virus. At the same time, the double antibody sandwich ELISA detection kit of the present invention does not require expensive instruments, and only needs to collect a small amount of samples including blood, body fluids, tissues or cells for detection, which has the advantages of simplicity, accuracy and speed, and is more convenient for promotion.

[0022] Biological deposit information

[0023] The hybridoma cell line AngHV-10G8 was deposited in the China Center for Type Culture Collection (CCTCC) on December 24, 2024, address: Wuhan University, Wuhan, China, with the deposit number CCTCCNO: C2024410 and the postal code 430072. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0025] Figure 1 The cross-reaction results of the monoclonal antibody against eel herpes virus detected by indirect ELISA in Example 1;

[0026] Figure 2 The specific results of the anti-eel herpes virus monoclonal antibody identified by Western-blot in Example 1; wherein, M: Marker; 1: EO cell supernatant infected with AngHV; 2: EO cell supernatant not infected with AngHV;

[0027] Figure 3 The specificity of the monoclonal antibody against eel herpes virus was detected by indirect immunofluorescence method in Example 1;

[0028] Figure 4 It is the specificity, tissue detection rate and sensitivity identification results of the eel herpes virus double antibody sandwich ELISA detection kit in Example 2; wherein, A is the specificity analysis result of the eel herpes virus double antibody sandwich ELISA detection kit, B is the tissue detection rate analysis result of the eel herpes virus double antibody sandwich ELISA detection kit, wherein 1 to 9 correspond to the heart, liver, spleen, kidney, gill, intestine, muscle, mucus and fin ray, respectively, and C is the detection limit analysis result of the eel herpes virus double antibody sandwich ELISA detection kit. DETAILED DESCRIPTION

[0029] The invention provides a hybridoma cell strain AngHV-10G8 which secretes an anti-eel herpes virus monoclonal antibody. The deposit number of the hybridoma cell strain AngHV-10G8 is CCTCCNO: C2024410.

[0030] The present invention uses purified AngHV virus particles as antigens to immunize mice, obtains the hybridoma cell line AngHV-10G8 that secretes anti-eel herpes virus monoclonal antibodies through cell fusion and indirect ELISA screening, prepares and collects mouse ascites, and obtains anti-eel herpes virus monoclonal antibodies 10G8-9F4 after purification. Furthermore, the anti-eel herpes virus monoclonal antibodies 10G8-9F4 also fall within the protection scope of the present invention.

[0031] Through verification, it was found that the anti-eel herpes virus monoclonal antibody 10G8-9F4 described in the present invention has the function of specifically recognizing AngHV and can be widely used in the detection, prevention and treatment of eel herpes virus and related research.

[0032] The present invention also provides the use of the hybridoma cell line described in the above technical solution or the anti-eel herpes virus monoclonal antibody described in the above technical solution in the preparation of products for detecting eel herpes virus and / or molecular biology research on eel herpes virus. As an embodiment, the product of the present invention is a product prepared based on immunoassay technology. Such as based on immunofluorescence technology, blotting detection technology, ELISA detection technology, flow sorting technology or IHC detection technology; as another embodiment, the product can be a reagent and / or a kit; as another embodiment, the kit can be an ELISA detection kit; as another embodiment, the ELISA detection kit can be a double antibody sandwich ELISA detection kit.

[0033] The present invention also provides a double antibody sandwich ELISA detection kit for detecting eel herpes virus, the double antibody sandwich ELISA detection kit comprises a capture antibody and a detection antibody, the capture antibody is an anti-eel herpes virus monoclonal antibody, the anti-eel herpes virus monoclonal antibody is the anti-eel herpes virus monoclonal antibody 10G8-9F4 described in the above technical solution; the detection antibody is an anti-eel herpes virus polyclonal antibody.

[0034] As an embodiment, the anti-eel herpes virus polyclonal antibody is obtained by immunizing animals with the envelope structural protein ORF95 of the eel herpes virus as an immunogen. The present invention does not specifically limit the preparation process of the anti-eel herpes virus polyclonal antibody, and the conventional polyclonal antibody preparation method in the art can be used. For example, in a specific embodiment, the present invention obtains the AngHV-ORF95 recombinant expression protein by cloning and expressing, and immunizes New Zealand rabbits with the AngHV-ORF95 recombinant expression protein as an immunogen, thereby obtaining an anti-eel herpes virus polyclonal antibody with high immunogenicity and specificity.

[0035] As an embodiment, the double antibody sandwich ELISA detection kit of the present invention further includes one or more of an enzyme-labeled plate, an enzyme-labeled secondary antibody, a washing solution, a chromogenic substrate, and a stop solution. As an embodiment, the enzyme-labeled secondary antibody of the present invention is a goat anti-rabbit secondary antibody labeled with HRP; as an embodiment, the washing solution is a PBST buffer; as an embodiment, the chromogenic substrate is an OPD substrate solution; as an embodiment, the stop solution is H2SO4 with a concentration of 2M.

[0036] The present invention also provides the use of the double antibody sandwich ELISA detection kit described in the above technical solution in the preparation of a product for detecting eel herpes virus. As an embodiment, the sample to be detected includes one or more of blood, body fluid, tissue and cell.

[0037] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] Preparation of monoclonal antibodies against eel herpesvirus

[0040] (1) AngHV virus purification

[0041] Eel ovary cell line (EO cells) was cultured in L15 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) to a monolayer, and AngHV-NA16108 (this strain is disclosed in the following literature [1] Yang Jinxian, Chen Qiang, Li Yingying, et al. Biological and physicochemical properties of eel herpes virus [J]. Journal of Fisheries of China, 2020(009):044.]) (MOI = 0.1) was inoculated for virus propagation; when 80% of the cells produced cytopathic When the cell membrane is exposed to the CPE, the supernatant and cells are collected and centrifuged at 15,000×g for 60 min; the supernatant is ultracentrifuged at 200,000×g for 2 h; the supernatant is removed and the precipitate is resuspended with sterile PBS; the precipitate is layered according to a 20%-60% discontinuous sucrose gradient and ultracentrifuged at 200,000×g for 2 h; the interlayer bands are collected, diluted with PBS, and centrifuged at 200,000×g for 1 h; the residual sucrose is removed and the virus particles are resuspended with PBS.

[0042] (2) Mouse immunization

[0043] Four 6-week-old female BALB / c mice were immunized with the AngHV purified in step (1) for a total of three immunizations, with multiple subcutaneous injections. The antigen immunization dose for each mouse was 25 μg each time. The first immunization was emulsified with an equal amount of Freund's complete adjuvant, and the second immunization was emulsified with an equal amount of Freund's incomplete adjuvant. Blood was collected from the tail vein 10 days after the second immunization, and the antibody titer was measured by indirect ELISA. The results are shown in Table 1; the titers of the four mice were compared, and the mouse with a higher antibody titer was selected for cell fusion (mouse number: 2#); three days before fusion, the antigen was used for direct booster immunization once, and the dose was the same as before.

[0044] Table 1 Determination of mouse serum titer after the second immunization

[0045] Mouse number 100 1K 3K 9K 27K 81K 243K Negative control Potency 1# 2.533 2.376 2.522 2.500 2.378 2.055 1.817 0.271 >1：243000 2# 2.757 2.463 2.621 2.333 2.468 2.243 2.046 0.166 >1：243000 3# 2.277 1.674 2.295 1.942 1.572 0.927 0.514 0.293 1：81000 4# 2.569 2.205 2.482 2.236 2.417 1.906 1.829 0.123 >1：243000

[0046] (3) Establishment of monoclonal antibody hybridoma cell lines

[0047] The mouse with the highest titer and enhanced immunization was selected, and the eyeball was removed to collect blood, and the serum was separated as the positive control serum for detection; the mouse was killed by cervical dislocation, and the spleen was aseptically removed and placed in a sterile plate containing basal culture medium for cleaning, and the connective tissue on the membrane was peeled off; the spleen was transferred to the filter membrane in another plate containing basal culture medium, and the spleen was pierced with the curved needles on two syringes, and then one of the curved needles was used to fix the filter membrane, and the other curved needle was used to squeeze the filter membrane, so that the spleen cells were completely released into the basal culture medium in the plate; the cells were blown with a sterile dropper to make a single cell suspension, and the spleen cell suspension was harvested; the spleen cell suspension in the plate was transferred to a centrifuge tube, centrifuged, the supernatant was discarded, and the cell culture medium was centrifuged and washed once; the separated spleen cells were fused with the myeloma cells that were resuscitated in advance with PEG, centrifuged, the supernatant was discarded, and the cell culture medium was centrifuged and washed once, and the cell culture medium with HAT was added to blow to make a single cell suspension, and the cell culture plate was plated.

[0048] On the 7th day after cell fusion, visible clones grow at the bottom of the culture plate wells. First, all cell wells are initially screened by the ELISA indirect method, and the cell wells showing strong positive results are recorded; each cell well that is initially positive should be expanded and cloned in a 24-well cell culture plate in a timely manner; the cell suspension is continuously diluted by the limiting dilution method until each well contains only a single cell, and then transferred to a 96-well plate for culture. After several cloning operations, when the positive rate of all cloned cell wells is 100%, it can be determined that the hybridoma cell lines NA-1, NA-2 and NA-3 that secrete monoclonal antibodies have been obtained; the obtained positive hybridoma cells NA-1, NA-2 and NA-3 are expanded and cultured in cell culture bottles, and then frozen and ascites is prepared.

[0049] (4) Large-scale preparation and purification of monoclonal antibodies

[0050] Six-week-old healthy female mice were sensitized by intraperitoneal injection of 0.5 mL of liquid paraffin. One to two weeks later, each mouse was intraperitoneally injected with 2 × 10 6hybridoma cells, observe the mouse status for 7-10 days, collect ascites after the mouse abdominal cavity is obviously swollen, and the supernatant is ascites, which is divided for use; the ascites is centrifuged at 4000rpm for 15min at room temperature, take the supernatant, add saturated ammonium sulfate drop by drop slowly to half saturation under stirring at 4℃, continue stirring for 30min, centrifuge at 13000rpm for 30min (4℃), and discard the supernatant; the precipitate is dissolved in an appropriate amount of PBS (0.01M, pH 7.4); saturated ammonium sulfate is slowly added drop by drop to 33% under stirring at 4℃, continue stirring for 30min, centrifuge at 13000rpm for 30min (4℃), and discard the supernatant; the precipitate is dissolved in an appropriate amount of PBS (0.01M, pH 7.4), and dialyzed at 4℃ overnight; after ammonium sulfate precipitation, continue to purify using Protein G column, first pass 5mL ultrapure water through the column, and then balance the purification column with 5mL0.4M PB buffer (pH 7.0); continue 10mL The purification column was equilibrated with 0.4 M PB buffer (pH 7.0); 5 mL 0.1 M glycine-HCl buffer (pH 2.7) was used to elute the antibody on the binding site, and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine to keep the pH at a neutral value suitable for antibody storage, thereby obtaining monoclonal antibodies 3G10-5G1, 9E8-7E9 and 10G8-9F4, respectively.

[0051] (5) Characterization of monoclonal antibodies

[0052] The monoclonal antibodies 3G10-5G1, 9E8-7E9 and 10G8-9F4 in step (4) above were identified by indirect ELISA, specifically: a 96-well plate was coated with AngHV (1 μg / mL) and incubated at 4°C overnight; the next day, the wells were washed three times with PBST and blocked with 2% bovine serum albumin (BSA) at 37°C for 1 h; the monoclonal antibodies 3G10-5G1, 9E8-7E9 and 10G8-9F4 were serially diluted with PBS (1:1000; 1:2000; 1:4000; 1:8000; ... : 16000; 1:32000; 1:64000; 1:128000; 1:256000; 1:512000; 1:1024000) as primary antibody, incubated at 37°C for 1h; washed wells three times with PBST, anti-mouse IgG diluted 1:3000 with PBST as secondary antibody, 100μL per well, incubated at 37°C for 1h; washed wells three times with PBST, washed the plate thoroughly to remove unbound conjugates, and then added 100μL of OPD substrate solution to each well, incubated at 37°C for 15min in the dark, and detected by xMark TMThe absorbance was read at 595 nm by a microplate spectrophotometer; a ratio of the absorbance between the test group and the control group (P / N value) > 2.1 was considered positive. The results are shown in Table 2, and the titers of the three monoclonal antibodies were all higher than 1:128,000.

[0053] Table 2 OD differences between three monoclonal antibodies and negative control 595 Ratio (P / N value)

[0054]

[0055] In order to test the specificity of the above monoclonal antibodies, several common aquatic viruses were collected: eel herpesvirus AngHV, adenovirus AEAdoV, koi herpesvirus KHV, largemouth bass iridovirus LMB V, grass carp reovirus GCRV, infectious pancreatic necrosis virus IPNV and large yellow croaker iridovirus LYC IV. Indirect ELISA analysis was performed according to the above operation. The results are shown in Figure 1 shown.

[0056] Depend on Figure 1 It can be concluded that the three monoclonal antibodies were positive only to AngHV (P / N value>2.1), among which 10G8-9F4 had the most obvious reaction, indicating that the three monoclonal antibodies had no cross-reaction with other aquatic viruses.

[0057] The monoclonal antibodies were identified by Western blot. TM The supernatant of EO cells infected with AngHV was separated by precast gel and transferred to polyvinylidene fluoride (PVDF) membrane; after blocking in PBST containing 5% skim milk for 2 h, the membrane was incubated with primary antibodies (monoclonal antibodies 3G10-5G1, 9E8-7E9 and 10G8-9F4 were diluted 1000 times) at 37°C overnight; after washing three times with PBST, the membrane was 800CW goat anti-mouse IgG (1:5000) was incubated at 37°C for 1 h; Li-Cor was used for imaging, and the data were analyzed using ImageJ software. Figure 2 shown.

[0058] Depend on Figure 2 It can be concluded that all three monoclonal antibodies showed specific reaction bands, among which 3G10-5G1 and 9E8-7E9 recognized a single band with a molecular weight of approximately 110 kDa, while 10G8-9F4 recognized a single band of approximately 35 kDa, and its reaction intensity was stronger than that of other monoclonal antibodies, while no band appeared in the negative control group (lane 2).

[0059] The above monoclonal antibodies were identified by indirect immunofluorescence assay (IFA). Round coverslips were placed in 24-well plates and seeded with EO cells; when confluent monolayers were formed, AngHV (10 4 TCID 50 / mL) at 27°C for 1 h, then the excess virus solution was removed and replaced with fresh L15 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin); 48 h later, the cells were washed with PBS, fixed with pre-cooled paraformaldehyde (4%) for 10 min at room temperature, permeabilized with TritonX-100 (0.1%) for 10 min, then washed with goat serum (5%) and blocked for 2 h; after washing, the cells were incubated with monoclonal antibodies 3G10-5G1, 9E8-7E9 or 10G8-9F4 (diluted 1000 times with PBS) at 4°C overnight, and mouse negative serum was used as a negative control; after washing three times with PBS, the cells were 594 donkey anti-mouse IgG was stained at 37°C for 1 h; then DAPI was stained at 37°C in the dark; BZ-X800LE was used for observation, and ImageJ software was used for data analysis. The results are shown in the figure. Figure 3 As shown, Figure 3 The scale bar is 10 μm.

[0060] Depend on Figure 3 It can be concluded that clear red fluorescence signals can be seen in cells treated with the three monoclonal antibodies, with 10G8-9F4 having the strongest signal, followed by 3G10-5G1, and 9E8-7E9 having relatively weak signals, while no red fluorescence signal was shown in the negative control, indicating that all developed monoclonal antibodies can specifically recognize AngHV virus particles, among which 10G8-9F4 has higher specificity and affinity.

[0061] Example 2

[0062] Preparation of double antibody sandwich ELISA kit for detection of eel herpes virus

[0063] (1) Capture Antibody

[0064] According to the identification results of the monoclonal antibodies in Example 1, 10G8-9F4 showed high affinity and high specificity for AngHV and was the most preferred capture antibody in the double antibody sandwich ELISA assay.

[0065] (2) Detection of antibodies

[0066] ORF95 is one of the main envelope proteins of AngHV. It has a conservative sequence and high gene abundance, making it suitable as a target site for virus detection. In the preliminary laboratory research, the AngHV-ORF95 recombinant expression protein was obtained by cloning and expression, and it was used as an immunogen to immunize New Zealand rabbits to obtain a polyclonal antibody 9NA with high immunogenicity and specificity. The polyclonal antibody 9NA was referenced to the literature [Chen Xi, Yang Jinxian, Chen Hua, et al. Identification, expression and characteristic analysis of the immunogenic protein ORF36 of eel herpes virus [J]. Journal of Agricultural Biotechnology, 2023. DOI: 10.3969 / j.issn.1674-7968.2023.08.014.】was prepared, and the ORF95 recombinant expression protein was prepared according to the literature [Li Youjuan, Ge Junqing, Song Tieying, Lin Tianlong, Cloning of ORF95 gene of eel herpes virus and its expression in Escherichia coli [J]. Journal of Fujian Agricultural Sciences, 2012.], and it was used as an immunogen to immunize New Zealand rabbits, and a polyclonal antibody 9NA with high immunogenicity and specificity was obtained. The ORF95 primer sequence was: upstream primer 95F: 5'-gaattcATGAAGACTTTTATTGCG-3' (SEQ ID NO: 1); downstream primer 95R: 5'-ctcgagTCAACGAGTCGAAAAGA-3' (SEQ ID NO: 2); at the same time, the polyclonal antibody has the ability to recognize multiple epitopes, which can improve the sensitivity of the detection method, and is the best choice as the detection antibody in the double antibody sandwich ELISA detection.

[0067] (3) Preparation method

[0068] 100 μL of capture antibody (most preferably 50 ng / well) was added to each 96-well microplate for coating and incubated at 37°C for 5 h; after washing 3 times with PBST, 200 μL of 2% BSA was added to each well and blocked overnight at 4°C; after washing 3 times with PBST, AngHV-infected samples were added to each well and incubated at 37°C for 1 h; after washing 3 times with PBST, 100 μL of detection antibody (most preferably 1:500 dilution) was added to each well and incubated at 37°C for 1 h; after washing 3 times with PBST, 100 μL of enzyme-labeled secondary antibody (goat anti-rabbit, most preferably 1:20,000 dilution) was added to each well and incubated at 37°C for 1 h; after washing 3 times with PBST, 100 μL of OPD substrate solution was added to each well and incubated at 37°C for 15 min; after adding stop buffer (2MH2SO4), the absorbance at 495 nm was measured; the OD of the detection sample and the negative control sample was compared. 495 A ratio (P / N value) greater than 2.1 was considered positive.

[0069] (4) Identification of double antibody sandwich ELISA (DAS-ELISA) kit

[0070] In order to detect the specificity of the double antibody sandwich ELISA detection kit, eel herpesvirus AngHV, American eel adenomas virus AEAdoV, koi herpesvirus KHV, largemouth bass iridovirus LMBV, grass carp reovirus GCRV, infectious pancreatic necrosis virus IPNV, and large yellow croaker iridovirus LYCIV were used for cross-reaction test. Among them, large yellow croaker iridovirus LYCIV was reported in the literature [XH Chen, KB Lin and XW Lang. Outbreak sofaniridovirus disease in mariculture d large yellow croaker, Larimichthys crocea (Richardson), in China [J]. Journal of fFishDiseases, 2003.】, and the remaining viruses are disclosed in the literature [Kong Wendi, Chen Xi, Yang Jinxian, et al. Establishment and application of conventional PCR and SYBRGreenI real-time fluorescence quantitative PCR detection methods for American eel adenoma virus (AEAdoV) [J]. Journal of Fisheries of China, 2024, 48(4): 049429. DOI: 10.11964 / jfc.20230714084.].

[0071] The steps of the cross-reaction test are as follows: 96-well plates were coated with virus solutions of eel herpesvirus AngHV, American eel adenomatous virus AEAdoV, koi herpesvirus KHV, largemouth bass iridovirus LMBV, grass carp reovirus GCRV, infectious pancreatic necrosis virus IPNV and large yellow croaker iridovirus LYCIV, respectively, and indirect ELISA was performed using monoclonal antibody 10G8-9F4 as the primary antibody and goat anti-mouse as the secondary antibody. P / N>2.1 was considered positive. Figure 4 As shown in Figure A, the detection kit has good specificity and does not cross-react with other aquatic viruses.

[0072] In order to analyze the detection rate of the above-mentioned double antibody sandwich ELISA test kit for different tissues of eels, eel tissues infected with AngHV were collected for detection, including heart, liver, spleen, kidney, gill, intestine, muscle, mucus and fin rays. The specific detection steps are as follows: 96-well plates were coated with capture monoclonal antibody 10G8-9F4 (500ng / well) and incubated at 37°C for 5 hours. After blocking with 2% BSA at 4°C overnight, homogenates of different tissues were added to the wells and incubated at 37°C for 1 hour. Wash three times with PBST. Then, ORF95 polyclonal antibody 9NA (1:500) was added to each well and incubated at 37°C for 1 hour. After washing, HRP-labeled goat anti-mouse polyclonal antibody (1:20000) was added to each well and incubated at 37°C for 1 hour. Subsequently, the plate was washed, 100 μL TMB was added, and the reaction was incubated at 37°C for 15 min. The reaction was stopped by adding the stop buffer, and the absorbance at 495 nm was measured. A P / N > 2.1 was considered positive. Figure 4 All tissues of eels infected with AngHV were positive (P / N>2.1), with the strongest reaction in the intestine.

[0073] In order to test the sensitivity of the double antibody sandwich ELISA detection kit, the intestine of eel infected with Ang HV was used as the test sample in this example, and the intestinal tissue supernatant with known AngHV viral load was diluted to different copy numbers (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copy / μL), and tested according to the gradient. The results are as follows Figure 4 As shown in C, compared with qPCR, the qPCR detection method was carried out according to the literature [Li Yingying, Yang Jinxian, Chen Xi, et al. Establishment and application of SYBRGreenⅠ real-time fluorescence quantitative PCR detection method for eel herpes virus [J]. Journal of Fisheries of China, 2021, 45(5): 9. DOI: 10.11964 / jfc.20200712326.], and the detection limit of the above double antibody sandwich ELISA detection kit was 10 4 Virus copy number.

[0074] Example 3

[0075] Clinical application of double antibody sandwich ELISA kit for detection of eel herpes virus

[0076] In order to determine the clinical application effect of the above-mentioned double antibody sandwich ELISA detection kit, this example collected 9 intestinal clinical samples from different eel farms in Fujian and surrounding areas, and detected the presence of AngHV according to the above operation, with PBS as a blank control; at the same time, the qPCR detection method was used to verify the same samples, and the consistency rate of DAS-ELISA and qPCR was compared. The results are shown in Table 3.

[0077] It can be seen that a total of 8 positive samples were detected using the above-mentioned double antibody sandwich ELISA detection kit, while 9 positive samples were detected by qPCR, indicating that compared with qPCR, the above-mentioned double antibody sandwich ELISA detection kit has a compliance rate of 88.89% (8 / 9), has high sensitivity, and can be used for clinical detection.

[0078] Table 3 Comparison of the results of qPCR and DAS-ELISA in the detection of eel clinical samples

[0079] sample 1 2 3 4 5 6 7 8 9 PBS qPCR test results + + + + + + + + + - P / N value detection by DAS-ELISA <2.1 3.3 2.8 3.6 3.0 4.9 4.7 5.1 4.7 <2.1

[0080] In summary, the present invention provides three monoclonal antibodies against eel herpes virus, and their specificity and affinity are confirmed by WB and IFA experiments, which provide important tools for further studying the biological characteristics of AngHV and developing effective AngHV vaccines or antiviral drugs. At the same time, the present invention also provides a double antibody sandwich ELISA (DAS-ELISA) detection kit for eel herpes virus, which has good specificity and sensitivity, is suitable for the detection of AngHV in eel tissues and body fluids, is simple to operate, and has low cost, and can provide technical support for the clinical diagnosis and epidemiology of AngHV.

[0081] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A hybridoma cell line AngHV-10G8 secreting anti-eel herpes virus monoclonal antibody, with a deposit number of CCTCC NO: C2024410.

2. An anti-eel herpes virus monoclonal antibody 10G8-9F4, characterized in that: The anti-eel herpes virus monoclonal antibody 10G8-9F4 is obtained by secretion of the hybridoma cell line AngHV-10G8 described in claim 1.

3. Use of the hybridoma cell line AngHV-10G8 according to claim 1 or the anti-eel herpes virus monoclonal antibody 10G8-9F4 according to claim 2 in the preparation of products for detecting eel herpes virus and / or in molecular biology research on eel herpes virus.

4. The use according to claim 3, characterized in that: The product is prepared based on immunoassay technology.

5. The use according to claim 3 or 4, characterized in that: The products include reagents and / or kits.

6. The use according to claim 5, characterized in that: The kit comprises a double antibody sandwich ELISA detection kit.

7. A double antibody sandwich ELISA detection kit for detecting eel herpes virus, the double antibody sandwich ELISA detection kit comprising a capture antibody and a detection antibody, characterized in that: The capture antibody is an anti-eel herpes virus monoclonal antibody, and the anti-eel herpes virus monoclonal antibody is the anti-eel herpes virus monoclonal antibody 10G8-9F4 according to claim 2; The detection antibody is an anti-eel herpes virus polyclonal antibody.

8. The double antibody sandwich ELISA detection kit according to claim 7, characterized in that: The anti-eel herpesvirus polyclonal antibody is obtained by immunizing animals with the envelope structural protein ORF95 of the eel herpesvirus as an immunogen.

9. Use of the double-antibody sandwich ELISA detection kit according to claim 7 or 8 in the preparation of a product for detecting eel herpes virus.

10. The use according to claim 9, characterized in that: The sample to be tested includes one or more of blood, body fluid, tissue and cells.

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