KR101884069B1 - Monoclonal antibody against nervous necrosis virus and use thereof - Google Patents

Abstract

The present invention provides a fusion cell line of Deposit No. KCLRF-PB-00370, which produces a monoclonal antibody for detecting fish nerve necrosis virus, and a monoclonal antibody Anti-14C3-2 produced therefrom, Diagnostic kits and diagnostic methods are available to diagnose, treat and prevent fish nerve necrosis virus, which causes massive deaths in farms and causes enormous damage.

Description

MONOCLONAL ANTIBODY AGAINST NERVOUS NECROSIS VIRUS AND USE THEREOF <br> <br> <br> Patents - stay tuned to the technology MONOCLONAL ANTIBODY AGAINST NERVOUS NECROSIS VIRUS AND USE THEREOF

The present invention relates to a monoclonal antibody specific to fish nerve necrosis virus and its use, and more particularly, to a method for diagnosing, preventing, and treating fish nerve necrosis virus infection, which is excellent in sensitivity and specificity by using an nerve necrosis virus structural protein as an epitope The present invention relates to a monoclonal antibody specific to fish nerve necrosis virus which can be usefully used.

In recent years, about 40 types of marine fish have been cultivated in Korea in order to reduce natural resources and supply stable aquatic products. Especially, it produces many kinds of fry to establish perfect style from egg to surname. However, in the field of aquaculture, a variety of diseases are occurring along with the development of nursery production technology and breeding technology. In particular, diseases caused by viruses can be caused not only by horizontal infection but also by vertical infection by fertilized eggs.

For example, virus infection caused by yellowtail ascites virus (YAV) occurring in defense, red seabream iridovirus (RSIV) infestation in domdol, infection with VNN caused by nodavirus infection in flounder or flounder nervous necrosis: viral nerve necrosis) have been reported. Among them, viral neuro - necrosis caused by Nodavirus infection has been a threat to seed production and aquaculture since it has occurred in sea fish from all over the world since 1990 and is very lethal. In addition, there are more than 30 species of fish caught in the disease.

Viral neuro-necrosis has been reported in Australian seabass, European seabass, halibut, Japanese dolphin, Japanese ladder, Japanese horse mackerel, marlin, island horse mackerel, and blowfish. It is a fish disease showing mortality rate.

Since 1990, it has been collected from the coasts of Jeju Island, Yeosu, Geomundo, and Geoje Island, and has been reported in the local fisheries, . Especially, it causes serious damage to the production of sea horsetail seedling, and it is caused by fish species such as domes, canine fishes, marine flounder and flounder.

The pathogen responsible for viral neurotoxicity is known as Betanodavirus (Nodaviridae), and according to the International Commission on Vaccination (ICTV), the genotypes based on the genotypes of the nematode necrosis virus (SJNNV) At least four species have been identified, including the flounder nerve necrosis virus (BFNNV), the frequent multiple nerve necrosis virus (TPNNV), and the red nervous nerve necrosis virus (RGNNV), which can infect many other species of fish without being limited to the name.

This neuronal necrosis virus infects the surface of the water without force, does not eat food well, changes color depending on the species, rotates and rotates while dying and sinks, We are dead in full in two weeks. Histologically, neurons in the central nervous system or retinal tissues are necrotic and collapsed to form large pores, and spherical viral particles are observed at high density on the cytoplasm and cell membrane of the cells on an electron microscope.

Neuronal necrosis virus is a virus of 25-30 nm in diameter with no outer membrane and has a single strand RNA virus. It infects nerve cells of fish and necroses nerve tissue to cause mass mortality. As the mortality rate is high, it is highly contagious and horizontal infection is rapid. It is also transmitted by the vertical infection from the mother to the egg to the frog and to the fry. Therefore, Is an effective preventive measure. In case of viral neurotoxicity, most of the seedlings are completely destroyed. As soon as these symptoms are confirmed, disinfection of the culture tank should be carried out and the dead fishes should be incinerated or buried to prevent the virus from spreading to the nearby waters.

Since nerve necrosis virus can survive for up to 60 days in seawater, care must be taken because the viruses that are contaminated with this virus may remain in the seawater and then enter the aquaculture. In addition, since nerve necrosis virus is inactivated by ultraviolet rays, sterilization of inflow water by ultraviolet rays is effective for prevention of viral nerve necrosis.

However, once the nerve necrosis virus is infected, the infectious rate and the mortality rate due to the vertical infection and the horizontal infection are very high. Therefore, in the nursery, it is more necessary to manage the infection diagnosis of the infant.

Korean Patent No. 10-1414009 discloses a vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant or an extract thereof containing a gene coding for a coat protein derived from Nodavirus and a fish virus A feed additive for preventing sexual neurotoxicity and a method for preventing fish viral neurotoxicity comprising orally administering the vaccine composition or the feed additive to fish. Korean Patent No. 10-1273264 discloses a polypeptide comprising an epitope of a fish nodavirus protein and a method for immunizing a vaccine, an antibody or a fish using the polypeptide, and a method for diagnosing fish nodavirus disease using the polypeptide, B2, a recombinant expression vector comprising the polynucleotide encoding the B2, a transformed host cell and a recombinant protein produced by the host cell, the vaccine composition comprising the recombinant protein, and the vaccine composition, To provide a method for immunizing a female. Korean Patent Laid-Open No. 10-2006-01301931 discloses a method for preventing or treating diseases such as Nodavirus infection of fish or viral neuro-necrosis caused by Nodavirus infection, in which aquavirnavirus is inoculated percutaneously into fish Or a method in which a fish is immersed in a liquid containing an aquavirinavirus or a mixed feed of an aquavirina virus is fed. Korean Patent No. 10-1273264 discloses a polypeptide comprising an epitope of a fish nodavirus protein, a recombinant vaccine using the polypeptide, a method of immunizing a fish with the polypeptide, and a method of diagnosing fish nodavirus disease. However, the above-mentioned inventions provide a fusion cell line 14C3-2 of Accession No. KCLRF-PB-00370, which produces a monoclonal antibody for detecting fish nerve necrosis virus, and a monoclonal antibody Anti-14C3-2 produced therefrom, The composition and effect of the monoclonal antibody specific for fish nerve necrosis virus of the present invention and its use which provide a diagnostic kit and a diagnostic method capable of diagnosing neural necrosis virus are different.

The present invention has high susceptibility and specificity to fish nerve necrosis virus, which is a causative agent of a fish disease with a high mortality rate of 100% in a larval stage at the time of seedling production, and can be easily used for diagnosis, treatment and prevention of fish nerve necrosis A monoclonal antibody against a fish nerve necrosis virus and a fusion cell line producing the same.

The present invention provides a fusion cell line 14C3-2 of Accession No. KCLRF-PB-00370, which produces a monoclonal antibody for detecting fish nerve necrosis virus, and a monoclonal antibody Anti-14C3-2 produced therefrom, The present invention provides a diagnostic kit and diagnostic method capable of diagnosing necrotizing virus. The diagnostic kit is a fish nerve necrosis virus diagnostic kit for detecting an antigen-antibody complex by a color particle binding method, and the diagnostic kit comprises an antibody condensed with a marker that reacts with a substrate and develops color; And a chromogenic substrate, wherein the marker is HRP (Horse-radish peroxidase) and the chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl-benzidine) Provide a kit.

The present invention relates to a step (1) of diluting a monoclonal antibody Anti-14C3-2 specific to fish nerve necrosis virus produced by a fusion cell line of accession number KCLRF-BP-00370 with a coating buffer solution, ; A step (2) of reacting the plate of (1) with the target tissue-washing solution to be examined; (3) washing the plate of (2) with a phosphate buffer solution; (4) reacting the plate of (3) with HRP-condensed neurotropic virus monoclonal antibody; (5) adding TMB to the plate of (4) to develop color; (6) stopping the enzyme reaction with sulfuric acid when the coloring reaction of (5) is over; And (7) measuring the absorbance at 405 nm of the color development degree of the above (6).

The above-mentioned diagnostic kit is a strip type using immunochromatography. The strip-type diagnostic kit comprises a monoclonal antibody Anti-14C3-2 specific to fish nerve necrosis virus produced by a fusion cell line of accession number KCLRF-BP-00370 A goldfish conjugate, a membrane coated with an antibody specific to mouse immunoglobulin, and a strip to which a test piece absorbent pad is attached, is provided by a rapid immunochromatography method.

The monoclonal antibody against fish nerve necrosis virus according to the present invention and the fused cell line producing the same can be used for production of diagnostic reagents, diagnostic kits, prophylactic agents and therapeutic agents having high sensitivity and specificity to fish nerve necrosis virus, You can perform the form.

Figure 1 is a photograph showing the band of virus observed in CsCl at a density of 1.35 g / cm &lt; ² &gt;.
Figure 2 is a photograph showing the structural protein band of purified NNV observed on SDS-PAGE.
Figure 3 is a table showing isotyping results for monoclonal antibodies.
FIG. 4 shows Western blotting results for confirming the NNV recognition site of a monoclonal antibody (M: marker (kDa), +: anti-NNV polyclonal antibody, negative control (primary antibody: 2% Skim milk).
Figure 5 is a Western blotting photograph showing the specificity of monoclonal antibodies for NNV isolates.
FIG. 6 is a graph showing the results of ELISA confirming the specificity of monoclonal antibodies against NNV isolates (antigen 1: GemudoJI08, 2: Gemudo06, 3: GemudoBJ08, 4: SgNag05, 5: SSN- 1).
7 is a graph showing ELISA results of confirming the specificity of the monoclonal antibody against fish viruses.
8 is a graph showing an ELISA result confirming the specificity of the monoclonal antibody against the NNV-infected fish.
Figure 9 shows the deposit.

The present invention relates to a monoclonal antibody specific for fish nerve necrosis virus, which is useful as a diagnostic, preventive and therapeutic agent for fish nerve necrosis virus infection because of its excellent sensitivity and specificity as an epitope of a fish nerve necrosis virus structural protein, Fusion cell lines and uses thereof. Hereinafter, the present invention will be described in detail with reference to specific examples.

< Example  1> Culture of virus

Viral nervous necrosis virus (NNV), which was used in the experiment, was isolated in 2008 from a fish farm in Yeosu, Jeollanam - do. In order to cultivate a large amount of isolated virus (Yeosu08), a striped snake head cell line (SSN-1) was cultured in a 75 cm 2 tissue culture flask (Nunc, Denmark) and viruses were inoculated and cultured at 25 ° C for 10 days CPE was observed. The cells were incubated at 4 ° C for 30 minutes at 12,000 rpm for 30 minutes to remove cell debris, and the supernatant was separated. The isolated virus supernatant was stored at -80 ° C until used in the experiment.

&Lt; Example 2 > Concentration and purification of virus

To the viral culture, polyethylene glycol (PEG) -6000 (Sigma, USA) and NaCl were added at 7.5% (w / v) and 2.3% (w / v), respectively. The PEG-treated viral culture was centrifuged at 12,000 rpm for 30 minutes at 4 ° C, and the pellet was suspended in PBS. The suspension was subjected to ultracentrifugation at 30,000 rpm for 2 hours and the pellet was resuspended in PBS to concentrate the virus. To purify the virus, CsCl was added to the concentrated virus (1 g CsCl / 2044 바이러스 virus solution) and ultracentrifugation was carried out at 30,000 rpm for 14 hours. The band formed at the lower part was taken using a syringe, suspended in PBS, and ultracentrifuged at 30,000 rpm for 2 hours. The precipitate obtained after centrifugation was resuspended in PBS buffer and stored at -80 ° C until used in the experiment.

< Example  3> Structural protein analysis of virus

SDS-PAGE was performed to analyze the structural proteins of the purified virus. The same amount of SDS-sample buffer was added to the purified virus, followed by heat treatment at 100 ° C for 3 minutes, followed by SDS-PAGE. After electrophoresis, the gel was stained with 0.2% coomassie brilliant blue and the results were confirmed.

< Example  4> virus immunity and hybridoma  making

Immunization was performed by mixing 1: 1 ratio of purified virus (18 ㎍) and complete freunds adjuvant to the peritoneal cavity of BALB / c mice. After 2 weeks, blood was drawn from the tail of the mouse, ELISA was performed to measure antibody titer, and secondary immunization was performed using only the virus. Three days later, the mouse spleen was isolated and fused with myeloma cells using PEG. The cells were divided into 96-well plates using HT medium and cultured at 37 ° C in a CO ₂ incubator.

Example 5 Screening of Hybridoma

ELISA was performed to screen hybridomas producing antibodies that specifically react with viruses. Antigens were coated with 50 μl (200 ng / well) of purified virus solution in 96 well plate at 4 ° C overnight or 37 ° C for 2 hours. 200 μl of 2% skim milk was blocked, washed once with TBST, and 50 μl of the hybridoma culture supernatant as the primary antibody was dispensed at 37 ° C for 2 hours. After washing three times with TBST, goat anti-mouse IgG labeled with HRP was diluted 5,000 times with 2% skim milk as a secondary antibody, and 50 μl of each was diluted at 37 ° C for 1 hour. After washing 5 times with TBST, 50 μl of tetramethylbenzidine base was added to each well to develop color. 50 μl of 1N H ₂ SO ₄ was added to each well to stop the color reaction, and the absorbance at 450 nm was measured with an ELISA plate reader.

<Example 6> Monoclonal antibody reaction using Western blot

Western immunoblot analysis was performed to confirm the ability of the antibody to recognize the antigen. SDS-PAGE was performed in the same manner as above. After the electrophoresis using the purified virus, the protein in the gel was blotted on a nitrocellulose membrane for 1 hour at 144 mA using a transblot device. After blotting, the cells were blocked with 2% skim milk, reacted for 1 hour, and then washed with buffer 1 for 15 minutes. As the primary antibody, hybridoma supernatant prepared in this study was reacted for 1 hour and then washed in the same manner as above. As a secondary antibody, goat polyclonal anti-mouse IgG antibody labeled with AP was diluted 1,000-fold with 2% skim milk, reacted for 1 hour, washed 5 times, developed with a coloring agent and visually confirmed. After completion of color development, a coloring stop solution was added.

< Example  7> Monoclonal antibody Isotyping

ELISA was performed according to the manual using a rapid ELISA mouse mAb isotyping kit to confirm the heavy and light chain subtypes of the prepared monoclonal antibody.

Example 8: Investigation of the specificity of monoclonal antibodies against NNV isolates

To investigate whether monoclonal antibodies against NNV Yeosu08 isolates respond to various NNV isolates, we used NNV isolates isolated from Gemundo06 in 2008, GemundoJI08 and GemundoBJ08 in 2008, and Nagano (SgNag05) in 2005 western blot and ELISA. SSN-1 cells were inoculated with 3 NNV isolates (Yeosu08, Gemundo06, and SgNag05) on the Western blot. Cells were lysed by SDS-sample buffer for 3 days and then heat-treated at 100 ° C for 3 minutes. Respectively. ELISA was performed using Yeosu08 (10 ^8.3 TCID ₅₀ / ml), Gemundo 06 (10 ^8.3 TCID ₅₀ / ml), SgNag05 (10 ^8.55 TCID ₅₀ / ml) supernatant in the following manner.

NNV isolates were diluted 320 times with distilled water, and 50 μl each was dispensed into 96-well ELISA plates, followed by overnight at 37 ° C to coat the antigen. After washing 3 times with T-PBS, 380 μl of 5% skim milk was added and blocked at 25 ° C for 1 hour. After washing three times with T-PBS, the monoclonal antibody prepared in this study was diluted 2-fold with 5% skim milk, and 50 μl was added to two wells per sample. Respectively. After washing three times with T-PBS, goat anti-mouse IgG labeled with HRP was diluted 1,000 times with 5% skim milk, and 50 ㎕ per well was dispensed and reacted at 25 ° C for 1 hour. After washing 5 times with T-PBS, 50 μl of ELISA was added to each well. 50 μl of 1N H ₂ SO ₄ was added to each well to discontinue the color reaction, and the absorbance was measured at 490 nm.

Example 9: Investigation of specificity for fish viruses

ELISA was performed using NNV, VHSV, MABV, HIRRV, SVCV, IHNV, and IPNV in order to investigate whether monoclonal antibodies against Yeosu08 isolates responded to various fish viruses. ELSIA is _{^{NNV Yeosu08 (108 .3 TCID 50 /}} ㎖), VHSV (10 8.05 TCID 50 / ㎖), MABV (10 9.3 TCID 50 / ㎖), HIRRV (10 8.3 TCID 50 / ㎖), SVCV (10 7.55 TCID 50 / Ml), IHNV (10 ^7.3 TCID ₅₀ / ml), IPNV (10 ^9.55 TCID ₅₀ / ml) supernatant. ELISA was performed using the method used to investigate specificity for NNV isolates.

< Example  10> NNV - Infected fish  Specificity for

To investigate whether the mAb to Yeosu08 isolate responded to the in situ samples (NNV-infected fish), NNV infection experiments were performed. Ten livers were placed in two 20 L water baths maintained at 25 ° C. NNV was injected intraperitoneally at a concentration of 10 ^3.8 TCID ₅₀ / fish in one tank, and HBSS (Control). After the NNV-infected and frozen groundnut and brains of healthy lethal fishes were harvested, they were mixed with 1: 10 (0.1 g / ml) of HBSS and centrifuged at 6,000 rpm for 30 minutes. ELISA Respectively.

Example 11 NNV-Infection Diagnostic Kit

The present invention provides a diagnostic kit capable of diagnosing infection of fish nerve necrosis virus comprising the monoclonal antibody of the present invention. In particular, the present invention provides a diagnostic kit for detecting a fish-nerve necrosis virus by an antigen-antibody complex by a color particle binding method. The diagnostic kit of the present invention comprises a monoclonal antibody of the present invention, an antibody condensed with a marker that reacts with a substrate, and a chromogenic substrate. Specifically, the diagnostic kit was prepared by diluting the monoclonal antibody of the present invention with a carbonate (pH 9.4), plating on a plate coated with a monoclonal antibody diluted to a final concentration of 10 μg / ml, HRP-condensed fish nerve necrosis diluted 1: Virus monoclonal antibody, phosphate buffered solution, TMB and 1N H ₂ SO ₄ .

Methods for diagnosing neuroinflammatory virus infection of suspected fish of fish nerve necrosis virus infection using the diagnostic kit of the present invention are as follows. 100 μl / well of the tissue markers collected from the monkeys on a plate coated with a fish nerve necrosis virus suspected or randomly selected from the farm were added at 25 ° C for 60 minutes, &Lt; / RTI &gt; wash the plate three times.

HRP (horse-radish peroxidase) -conjugated neuropeptide viral monoclonal antibody is added to each well of the plate at 100 μl, reacted at 25 ° C for 60 minutes, and the plate is washed three times with phosphate buffer solution. Then, 100 μl of TMB (3,3 ', 5,5'-tetramethyl-benzidine), which is a substrate of HRP, is added to each well of the plate and reacted in a dark place for 30 minutes to induce a color reaction. Then, 1N H ₂ SO ₄ is added to each well of the plate at 100 μl to stop the enzyme reaction. The plate thus treated with the diagnostic kit of the present invention is measured for its absorbance at 405 nm using an ELISA reader. When the absorbance is 0.2 or more, it is determined to be positive.

<Example 12> NNV-infectious rapid diagnostic kit using immunochromatography

The present invention provides a strip-type NNV-infectious rapid diagnostic kit using an immunochromatographic method capable of rapidly diagnosing infection of fish nerve necrosis virus, comprising the monoclonal antibody of the present invention. The anti-NNV antibody prepared above was diluted with PBS to a concentration of 0.58 μg / strip and adsorbed on a nitrocellulose membrane inspection line (T: detection line of NNV) to be used as a coating solution. In addition, 0.75 ㎍ / strip of antibody recognizing mouse Ig was coated at the position of the control line (C).

Anti-14C3-2 antibody was also conjugated with gold colloid. Gold chloride was reduced with a sodium citrate solution to prepare plain gold. Then, the antibody was added, and gold particles bound with antibody of 40 nm in size were prepared so as to have an absorbance of 10 +/- 1 at 532 nm . Polyethylene glycol (PEG) solution was treated to stabilize bound antibody.

Each gold conjugate with the gold particles and antibody was mixed and wetted to polyester or glass fiber to be 8.3 ㎕ / strip and dried to prepare a gold pad. The hygroscopic pad and the sample pad were prepared by drying so that the reaction solution could be absorbed well. Thereafter, 1) sample pad, 2) gold pad, 3) membrane (T: inspector position, C: tumbler position), 4) hygroscopic pad, and 5) plastic card are superimposed and cut to 4.45 mm / The cut strips were placed in the bottom plate of the final device and covered with the top plate on which the sample application holes were formed to prepare the NNV-infectious rapid diagnostic kit.

To diagnose fish nerve necrosis virus, dilute the tissue lysis fluid or serum of the black fish and apply it to the sample application hole. In this case, if fish nerve necrosis virus is present in the sample, it binds to the anti-14C3-2 antibody bonded to the gold particle of the gold pad in a primary way, and then this conjugate moves along the membrane by the immunochromatographic principle, NNV antibody at the inspecting line position (T), while forming an antibody-antigen-antibody complex while exhibiting a purple band. However, if no fish nerve necrosis virus is present, only the control band (C) appears.

< Test Example  1> Fish Nerve necrosis virus ( NNV ) refine

Fig. 1 is a photograph showing the band of virus observed in CsCl at 1.35 g / cm &lt; ² &gt; density, and Fig. 2 is a photograph showing a structural protein band of purified NNV observed in SDS-PAGE. After concentrating with PEG using NNV, which was mass-cultured in SSN-1 cells, CsCl was added to the concentrated virus (1 g CsCl / 2044 μl virus solution) and ultracentrifugation was carried out. .

<Test Example 2> Hybridoma preparation and screening of monoclonal antibodies

Hybridoma was prepared by fusing Sp2 / 0Ag14 myeloma cells with PEG to spleen tissue of mouse immunized with purified NNV. Antibodies generated from Hybridoma were screened by ELISA, and the results were positive in 7 samples. Seven samples were cloned three times by ELISA and finally one clone (14C3-2) could be selected. In subsequent experiments, we performed a specificity study using antibodies produced from one clone (using the same name as the antibody name: clone).

< Test Example  3> Monoclonal  Antibody isotyping

Figure 3 is a table showing isotyping results for monoclonal antibodies. Using the rapid ELISA mouse mAb isotyping kit for monoclonal antibodies, the heavy and light chain subtypes of IgG were identified as IgG2a and L-chain, respectively.

< Test Example  4> NNV For Monoclonal  Investigation of virus recognition site of antibody

FIG. 4 is a Western blotting result showing confirmation of the NNV recognition site of the monoclonal antibody (+: positive control, primary antibody: anti-NNV polyclonal antibody, negative control, primary antibody: 2% Skim milk). To examine the NNV recognition site of the mAb, western blot was performed using purified NNV. The monoclonal antibody recognized the 41 kDa NNV coat protein.

&Lt; Test Example 5 > Specificity of NNV isolates

FIG. 5 is a Western blotting photograph showing the specificity of a monoclonal antibody against NNV isolates (M: marker (kDa), 1: anti-NNV polyclonal antibody, 2: Yeosu08, 3: Gemundo06, 4: SgNag05 , 5: SSN-1 cells).

FIG. 6 is a graph showing the results of ELISA confirming the specificity of monoclonal antibodies against NNV isolates (antigen 1: GemudoJI08, 2: Gemudo06, 3: GemudoBJ08, 4: SgNag05, 5: SSN- 1). ELISA was performed using NNV culture broth (Gemudo06, GemudoJI08, GemudoBJ08, SgNag05), SSN-1 medium and SSN-1 markers. The monoclonal antibody reacted to all NNV isolates (OD value: 0.2-0.46 ) And did not react with SSN-1 medium and SSN-1 medium.

< Test Example  6> Other To fish viruses  Investigation of specificity for

7 is a graph showing ELISA results of confirming the specificity of the monoclonal antibody against fish viruses. ELISA was performed using the culture media of viruses (NNV, VHSV, IHNV, HIRRV, MABV, SVCV) and SSN-1. As a result, the monoclonal antibody reacted only with NNV and VHSV, IHNV, HIRRV, MABV, SVCV and SSN- I did not respond.

&Lt; Test Example 7 > Specificity of NNV-infected fishes

8 is a graph showing an ELISA result confirming the specificity of the monoclonal antibody against the NNV-infected fish. ELISA was performed using NNV-infected and lethal (four samples) and healthy (n = 3) samples, and the monoclonal antibody reacted with the brain liquor of NNV-infected fish (OD value: 0.56 - 1.3), but did not respond to brain erosion of normal fish (OD value: 0.1 or less).

The present invention provides a fusion cell line for detecting fish nerve necrosis virus and a monoclonal antibody produced therefrom, and provides a diagnostic kit and a diagnostic method capable of diagnosing fish nerve necrosis virus using the same, It can be used in the diagnosis, treatment and prevention of fish nerve necrosis virus, which is suffering a great damage, and thus is industrially applicable.

<Access number>

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00370

Funding Date: 2016816

Claims (8)

Monoclonal antibody specific for fish nerve necrosis virus produced by a fusion cell line of accession number KCLRF-BP-00370 Anti-14C3-2, a conjugate of gold, a membrane coated with an antibody specific to mouse immunoglobulin, and a sample absorbing pad A kit for diagnosing a fish nerve necrosis virus by a rapid immunochromatography method comprising a strip adhered thereto.
delete delete Diluting a monoclonal antibody Anti-14C3-2 specific to fish nerve necrosis virus produced by a fusion cell line of accession number KCLRF-BP-00370 with a coating buffer solution and dispensing it on a plate to coat the plate (1);
(2) a step of reacting the plate of (1) with a test brain eraser to be tested; (3) washing the plate of (2) with a phosphate buffer solution;
(4) reacting the plate of (3) with HRP-condensed neurotropic virus monoclonal antibody; (5) adding TMB to the plate of (4) to develop color;
(6) stopping the enzyme reaction with sulfuric acid when the coloring reaction of (5) is over; And (7) measuring the absorbance at 405 nm of the color development degree of the above (6).
delete delete Monoclonal antibody produced in the fusion cell line of accession number KCLRF-BP-00370 Anti-14C3-2.
Fusion cell line of accession number KCLRF-BP-00370.

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