Hybridoma cell line secreting anti-eel herpes virus monoclonal antibody, anti-eel herpes virus monoclonal antibody and use thereof

Abstract

The present invention belongs to the field of monoclonal antibodies and biological detection technology, and specifically relates to a hybridoma cell line that secretes anti-eel herpes virus (AngHV) monoclonal antibodies, anti-eel herpes virus monoclonal antibodies, and applications thereof. The anti-eel herpes virus monoclonal antibodies of the present invention are prepared using purified eel herpes virus particles as antigens and are capable of specifically identifying eel herpes virus. A double-antibody sandwich ELISA detection kit has been developed based on the anti-eel herpes virus monoclonal antibodies. The kit is prepared using anti-eel herpes virus monoclonal antibodies as capture antibodies and eel herpes virus polyclonal antibodies as detection antibodies. The kit is suitable for the detection of eel herpes virus, has good specificity and sensitivity, and the method is simple and rapid, with broad application prospects.

Description

Technical Field

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

[0002] Eels, known as "soft gold in water," possess significant medicinal and nutritional value. "Desmear septicemia syndrome" (DSS) is a major epidemic in eel farming, with frequent outbreaks during large-scale eel farming, causing significant economic losses to farmers. Anguillid herpesvirus (AngHV) has been confirmed to be the causative agent of DSS in eels. This disease can cause clinical symptoms such as mucus shedding and bleeding from the skin, bleeding from fin rays, and bloody ascites in the abdominal cavity. The disease is characterized by high morbidity, a short course, rapid transmission, and a high cumulative mortality rate, posing a serious threat to the sustainable development of the eel farming industry.

[0003] AngHV (Anguilla hyopneumoniae) is one of the most destructive viruses and is ubiquitous in eel aquaculture. Since its initial isolation from Japanese eels (Anguilla japonica) farmed in Japan, AngHV has been subsequently isolated from various regions around the world, including the major farmed species, the European eel (Anguilla anguilla) and the American eel (Anguilla rostrata). Serological and molecular studies have confirmed that these strains are the same virus. AngHV is an enveloped, linear, double-stranded DNA virus belonging to the family Heteroherpesviridae (Heteroherpesviridae) of the order Herpesvirales. Proteomic analysis has identified the AngHV virion structure as containing seven nucleocapsid proteins, 11 envelope proteins, and 22 tegument 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 prevalence in juvenile eels and is latent, transmissible through water, and easily activated and erupting by exogenous or endogenous stimuli, with a mortality rate as high as 30%. However, to date, there is still a lack of effective vaccines or antiviral drugs to prevent and control AngHV. Therefore, early diagnosis of AngHV infection in eels is crucial to prevent the spread of "de-adhesion septicemia 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 paper (RAA-LFD), loop-guided isothermal amplification (LAMP), and real-time quantitative loop-guided isothermal amplification (qLAMP).

[0006] However, existing technologies for detecting AngHV all rely on molecular diagnostic methods, which are cumbersome and time-consuming, require high 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 that secretes anti-eel herpes virus monoclonal antibodies, anti-eel herpes virus monoclonal antibodies and their applications. The anti-eel herpes virus monoclonal antibodies have the function of specifically recognizing AngHV, can be used for the preparation of eel herpes virus detection products, and are simple and fast to operate.

[0008] The present invention provides a hybridoma cell line AngHV-10G8 secreting an anti-anguilla herpes virus monoclonal antibody, with a deposit number of CCTCC NO: C2024410.

[0009] The present invention also provides an anti-eel herpes virus monoclonal antibody 10G8-9F4, which 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 comprising 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 polyclonal antibody to anguilla herpesvirus is obtained by immunizing animals with the envelope structural protein ORF95 of 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 herpesvirus monoclonal antibody, with a deposit number of CCTCC NO: C2024410. Furthermore, the present invention provides an anti-eel herpesvirus monoclonal antibody 10G8-9F4 secreted by the hybridoma cell line AngHV-10G8. The present invention uses purified AngHV as an immunogen, and the resulting monoclonal antibody has high specificity and affinity, can specifically recognize AngHV, and can be used for the detection of eel herpesvirus, 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. The double-antibody sandwich ELISA detection kit is prepared using the anti-eel herpes virus monoclonal antibody as the capture antibody and the anti-eel herpes virus polyclonal antibody as the detection antibody. 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 requires the collection of a small amount of samples including blood, body fluids, tissues or cells for detection. It 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, at 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 for use 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 are as follows;

[0026] Figure 2 The results of Western-blot identification of the specificity of the anti-anguilla herpesvirus monoclonal antibody in Example 1; wherein, M: Marker; 1: supernatant of EO cells infected with AngHV; 2: supernatant of EO cells not infected with AngHV;

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

[0028] Figure 4 These are 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 present invention provides a hybridoma cell line AngHV-10G8 that secretes an anti-anguilla herpes virus monoclonal antibody. The deposit number of the hybridoma cell line 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 the anti-eel herpes virus monoclonal antibody 10G8-9F4 after purification. Furthermore, the anti-eel herpes virus monoclonal antibody 10G8-9F4 also falls within the scope of protection 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 a product for detecting eel herpes virus and / or in molecular biology research on eel herpes virus. As one embodiment, the product of the present invention is a product prepared based on an immunoassay technique, such as immunofluorescence technology, blotting detection technology, ELISA detection technology, flow cytometry 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 comprising a capture antibody and a detection antibody, the capture antibody being an anti-eel herpes virus monoclonal antibody, the anti-eel herpes virus monoclonal antibody being the anti-eel herpes virus monoclonal antibody 10G8-9F4 described in the above technical solution; and the detection antibody being an anti-eel herpes virus polyclonal antibody.

[0034] In one embodiment, the anti-eel herpesvirus polyclonal antibody is obtained by immunizing animals with the envelope structural protein ORF95 of the eel herpesvirus as an immunogen. The present invention does not specifically limit the preparation process of the anti-eel herpesvirus polyclonal antibody, and conventional polyclonal antibody preparation methods 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 expression, and immunizes New Zealand rabbits with the AngHV-ORF95 recombinant expression protein as an immunogen to obtain anti-eel herpesvirus polyclonal antibodies with high immunogenicity and specificity.

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

[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 fluids, tissues and cells.

[0037] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection 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 cell) 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 used for virus propagation; when 80% of the cells produced cytopathic When the cell membrane is exposed to the viral load (CPE), the supernatant and cells are collected and centrifuged at 15,000 × g for 60 minutes; the supernatant is ultracentrifuged at 200,000 × g for 2 hours; the supernatant is removed and the pellet is resuspended with sterile PBS; the pellet is layered according to a 20%-60% discontinuous sucrose gradient and ultracentrifuged at 200,000 × g for 2 hours; the interlayer bands are collected, diluted with PBS, and centrifuged at 200,000 × g for 1 hour; the residual sucrose is removed and the viral 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. Multiple subcutaneous injections were performed, and 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 mice with the highest titer and the boosted immunization were selected, their eyes were enucleated and blood was collected, and the serum was separated as a positive control serum for testing; the mice were killed by cervical dislocation, and the spleens were aseptically removed and placed in a sterile dish containing basal culture medium for cleaning, and the connective tissue on the membrane was peeled off; the spleen was transferred to a filter membrane in another dish containing basal culture medium, and the spleen was punctured with the curved needles on two syringes. Then, one curved needle was used to fix the filter membrane, and the other curved needle was used to squeeze the filter membrane to completely release the spleen cells into the basal culture medium in the dish; the cells were pipetted with a sterile dropper to make a single cell suspension, and the spleen cell suspension was harvested; the spleen cell suspension in the dish was transferred to a centrifuge tube, centrifuged, the supernatant was discarded, and the cell was centrifuged and washed once with cell culture medium; the separated spleen cells were fused with myeloma cells that had been resuscitated and prepared in advance using PEG, centrifuged, the supernatant was discarded, and the cell was centrifuged and washed once with cell culture medium, and the cell culture medium containing HAT was added and pipetted to make a single cell suspension, which was then plated into a 96-well cell culture plate.

[0048] Seven days after cell fusion, visible colonies appear at the bottom of the culture plate wells. Initially, all wells are screened indirectly by ELISA, and strongly positive wells are recorded. Wells that test positive in the initial screening should be promptly expanded and cloned in 24-well cell culture plates. The cell suspension is serially diluted by limiting dilution until statistically only a single cell is present in each well, then transferred to a 96-well plate for culture. After several cloning operations, when the positive rate for all cloned wells reaches 100%, it is determined that hybridoma cell lines NA-1, NA-2, and NA-3 secreting monoclonal antibodies have been obtained. The positive hybridoma cells NA-1, NA-2, and NA-3 obtained are expanded in cell culture flasks and then frozen for ascites preparation.

[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 6The hybridoma cells were collected after the mice were observed for 7-10 days. When the abdominal cavity of the mice was obviously swollen, the ascites was collected and the supernatant was the ascites, which was divided for use. The ascites was centrifuged at 4000 rpm for 15 minutes at room temperature, the supernatant was taken, and saturated ammonium sulfate was slowly added dropwise to half saturation under stirring at 4°C, and stirring was continued for 30 minutes. The supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS (0.01M, pH 7.4). Saturated ammonium sulfate was slowly added dropwise to 33% under stirring at 4°C, and stirring was continued for 30 minutes. The supernatant was discarded. The precipitate was dissolved in an appropriate amount of PBS (0.01M, pH 7.4) and dialyzed at 4°C overnight. After ammonium sulfate precipitation, the protein was purified using a Protein G column. The column was first passed through with 5 mL of ultrapure water, and then the purification column was equilibrated with 5 mL of 0.4M PB buffer (pH 7.0). 10 mL The purification column was equilibrated with 0.4 M PB buffer (pH 7.0); the antibodies on the binding sites were eluted with 5 mL of 0.1 M glycine-HCl buffer (pH 2.7), and glycine was neutralized by adding 1 M Tris-HCl (pH 8.0) to maintain a neutral pH 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 microplate 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 (1:1000; 1:2000; 1:4000; 1:8000; 1:1000) with PBS. : 16000; 1:32000; 1:64000; 1:128000; 1:256000; 1:512000; 1:1024000) as the primary antibody, incubated at 37°C for 1 hour; washed the wells three times with PBST, anti-mouse IgG diluted 1:3000 with PBST was used as the secondary antibody, 100 μL per well, incubated at 37°C for 1 hour; washed the wells three times with PBST, washed the plate thoroughly to remove unbound conjugates, and then added 100 μL of OPD substrate solution to each well, incubated at 37°C for 15 minutes in the dark, and detected by xMark TMAbsorbance was read at 595 nm using a microplate spectrophotometer. A positive result was determined when the absorbance ratio (P / N) between the test group and the control group was > 2.1. The results are shown in Table 2. The titers of all three monoclonal antibodies were greater 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, adenomatous virus 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 the figure. Figure 1 shown.

[0056] Depend on Figure 1 It can be concluded that all three monoclonal antibodies showed positive reactions 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 immunoblotting (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 hour; imaging was performed using Li-Cor, 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 them, 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. 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 hour, then remove the excess virus solution and replace it with fresh L15 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin); after 48 hours, wash the cells with PBS, fix them with pre-cooled paraformaldehyde (4%) for 10 minutes at room temperature, permeabilize them with TritonX-100 (0.1%) for 10 minutes, and then wash and block them with goat serum (5%) for 2 hours; 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 hour; 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. The signal of 10G8-9F4 is the strongest, followed by 3G10-5G1, and the signal of 9E8-7E9 is relatively weak. No red fluorescence signal is 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 conserved sequence and high gene abundance, making it a suitable target site for virus detection. In preliminary laboratory studies, recombinant AngHV-ORF95 protein was obtained by cloning and expression. It was then used as an immunogen to immunize New Zealand rabbits, resulting in a highly immunogenic and specific polyclonal antibody 9NA. The polyclonal antibody 9NA was identified by reference to the literature [Chen Xi, Yang Jinxian, Chen Hua, et al. Identification, expression and characterization 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 the ORF95 gene of eel herpesvirus and its expression in Escherichia coli [J]. Journal of Fujian Agricultural Sciences, 2012.] and used as an immunogen to immunize New Zealand rabbits to obtain a polyclonal antibody 9NA with high immunogenicity and specificity. The ORF95 primer sequences are: 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 most preferred detection antibody in the double antibody sandwich ELISA test.

[0067] (3) Preparation method

[0068] 100 μL of capture antibody (optimally 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; washed 3 times with PBST; AngHV-infected samples were added to each well and incubated at 37°C for 1 h; washed 3 times with PBST, 100 μL of detection antibody (optimally 1:500 dilution) was added to each well and incubated at 37°C for 1 h; washed 3 times with PBST, 100 μL of enzyme-labeled secondary antibody (goat anti-rabbit, optimally 1:20,000 dilution) was added to each well and incubated at 37°C for 1 h; washed 3 times with PBST, 100 μL of OPD substrate solution was added to each well and incubated at 37°C for 15 min; stop buffer (2MH2SO4) was added and the absorbance at 495 nm was measured to detect the OD of the sample and the negative control sample. 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 test the specificity of the double antibody sandwich ELISA detection kit, cross-reaction tests were performed with 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. Large yellow croaker iridovirus LYCIV was reported in the literature [XHChen, KBLin and XWWang. Outbreaks of large yellow croaker iridovirus disease in mariculture, Larimichthys crocea (Richardson), in China [J]. 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. Monoclonal antibody 10G8-9F4 was used as the primary antibody and goat anti-mouse as the secondary antibody. Indirect ELISA detection was performed, and a P / N > 2.1 was considered positive. The results are as follows Figure 4 As shown in Figure A, the detection kit has good specificity and does not cross-react with other aquatic viruses.

[0072] To analyze the detection rate of the double-antibody sandwich ELISA kit for different eel tissues, tissues from eels infected with AngHV were collected for testing, including the heart, liver, spleen, kidney, gills, intestine, muscle, mucus, and fin rays. The specific testing steps were as follows: A 96-well plate was coated with the capture monoclonal antibody 10G8-9F4 (500 ng / well) and incubated at 37°C for 5 hours. After blocking with 2% BSA overnight at 4°C, the homogenates of the different tissues were added to the wells and incubated at 37°C for 1 hour. The plates were washed three times with PBST. The ORF95 polyclonal antibody 9NA (1:500) was then added to each well and incubated at 37°C for 1 hour. After washing, an HRP-conjugated goat anti-mouse polyclonal antibody (1:20,000) was added to each well and incubated at 37°C for another 1 hour. Subsequently, the plate was washed, 100 μL of TMB was added, and the plate 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 in medium B 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 Ang HV viral load was diluted to different copy numbers (10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 copy / μL), and detect 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 double antibody sandwich ELISA detection kit was 10 4 Viral copy number.

[0074] Example 3

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

[0076] To determine the clinical efficacy of the double-antibody sandwich ELISA test kit, nine intestinal clinical samples from different eel farms in Fujian and surrounding areas were collected in this example and tested for the presence of AngHV according to the above-described procedures, using PBS as a blank control. Simultaneously, qPCR was used to validate the same samples, and the consistency between 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 had a compliance rate of 88.89% (8 / 9), has higher sensitivity, and can be used for clinical detection.

[0078] Table 3 Comparison of the results of qPCR and DAS-ELISA 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 herpesvirus, and their specificity and affinity are confirmed by WB and IFA experiments, providing 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 herpesvirus, which has good specificity and sensitivity and is suitable for the detection of AngHV in eel tissues and body fluids. It is simple to operate and low in cost, and can provide technical support for the clinical diagnosis and epidemiology of AngHV.

[0081] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection of the present invention.

Claims

1. A hybridoma cell line AngHV-10G8 secreting anti-anguilla herpesvirus monoclonal antibodies, with a deposit number of CCTCC NO: C2024410.

2. An anti-eel herpesvirus monoclonal antibody 10G8-9F4, characterized in that: The anti-eel herpesvirus monoclonal antibody 10G8-9F4 is obtained by secretion from the hybridoma cell line AngHV-10G8 according to claim 1.

3. Use of the hybridoma cell line AngHV-10G8 according to claim 1 or the anti-eguillemot herpesvirus monoclonal antibody 10G8-9F4 according to claim 2 in the preparation of a product for detecting eguillemot herpesvirus.

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 product includes a reagent or a kit.

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 body fluids, tissues and cells.

11. The use according to claim 10, characterized in that The sample to be tested includes blood.

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