KR102034609B1 - Comosition for Detecting Viral Hemorrhagic Septicemia Virus Contiaining Monoclonal Antibody Specific for Viral Hemorrhagic Septicemia virus - Google Patents

Abstract

The present invention relates to a hybridoma cell line (VHSV mAb) that produces a monoclonal antibody exhibiting specific immunoreactivity against viral hemorrhagic septicemia virus (VHSV), a monoclonal antibody that is specifically bound to VHSV produced by the hybridoma cell line, and a VHSV diagnostic reagent comprising the monoclonal antibody. The diagnostic reagent of the present invention can quickly diagnose VHSV before the infection of a fish with a lot of VHSVs, thereby minimizing damage to fishermen with fish farming, and enabling a quarantine agency to respond promptly.

Description

Composition for detecting viral hemorrhagic sepsis virus comprising a monoclonal antibody that specifically binds to viral hemorrhagic sepsis virus

The present invention relates to a composition for detecting viral hemorrhagic sepsis virus comprising a monoclonal antibody that specifically binds to viral hemorrhagic sepsis virus, and more particularly to a monoclonal that exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV). The present invention relates to a hybridoma cell line producing an antibody, a monoclonal antibody specifically binding to a viral hemorrhagic sepsis virus produced by the hybridoma cell line, and a composition for detecting viral hemorrhagic sepsis virus comprising the monoclonal antibody.

Viral Hemorrhagic Septicemia (VHS) was known as a viral disease that caused significant damage in rainbow trout and some freshwater fish species in Europe until the early 1980s, but in 1988, the salmon and the king circulated to the western Pacific coast of the United States. Fish found in salmon and confirmed infection include VHSV from about 50 marine fish and freshwater fish such as flounder, rainbow trout, atlantic salmon, brown trout, silver salmon, king salmon, cod, flounder, turbot, sardine and pollock. Is being separated.

In Korea, cases of viral hemorrhagic sepsis in farmed flounder have been reported in the academic world every year since 2001 in the East Sea and South Sea water at low temperature in winter and spring. It is reported from wild sea fish species and farmed flounder. In Japan, it is separated from wild fish such as halibut, horse mackerel, canary, red snapper and yellowtail, and in Korea, it is separated from mullet, mackerel, yellow snapper and bottlefish. Causative virus of Viral Hemorrhagic Septicemia (VHS) is in the form of a virus with an outer film in the Rhabdoviridae and (科) Novirhabdovirus a RNA virus of the single-stranded, 50 ~ 70nm x size of 180 ~ 240nm, and the viral particles model typical Rhabdovirus Bullet-shaped, one side is round and the other is flat. Glycoprotein (G) gene sequences of VHSV isolated from fresh water are divided into basic genotypes.The genotypes are divided into American type (Genogroup I), British Isles type (Genogroup II), and European type (Genogroup III). Three genotypes are generalized. Genotypes isolated from Korea were found to be similar to viruses isolated from North America and Japan belonging to Genogroup I. Fish cell lines susceptible to VHSV are known as BF-2, RTG-2, EPC, FHM, CHSE-214, but the most susceptible cell line to VHSV originated from seawater is known as EPC. When cultured, the infected cells can be shortened and spheronized and removed from the wall of the culture vessel.

Viral hemorrhagic sepsis is a marine animal infectious disease under Article 2 of the Fisheries Animal Disease Control Act, a viral disease that causes flounder mortality at low temperatures below 15 ° C. The mortality rate is high and contagious. It is a disease that is managed by restricting movement. Viral hemorrhagic sepsis isolated from statutory epidemic has not yet been diagnosed due to the rapid diagnosis in the field.

Therefore, the present inventors have made diligent efforts to develop a technique for rapidly diagnosing VHSV without the help of experts or specialized equipment in the field, and thus, produced a monoclonal antibody that specifically binds to VHSV, and uses the antibody to construct a VHSV diagnostic kit. When manufactured and used, it was confirmed that VHSV can be diagnosed quickly before an infection of many individuals occurs, and the present invention has been completed.

An object of the present invention to provide a composition for the detection of viral hemorrhagic sepsis virus (VHSV).

Another object of the present invention to provide a kit for detecting viral hemorrhagic sepsis virus (VHSV).

It is still another object of the present invention to provide a hybridoma cell line producing monoclonal antibodies exhibiting specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

It is another object of the present invention to provide a monoclonal antibody which exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

In order to achieve the above object, the present invention is a viral hemorrhagic containing a primary antibody for detecting viral hemorrhagic sepsis virus antigen, a detection label, a secondary antibody to which the detection label is bound and a reagent for measuring the activity of the detection label In the composition for detecting sepsis virus (VHSV), the primary antibody for viral hemorrhagic sepsis virus antigen capture is a monoclonal antibody 4A7 produced by a hybridoma cell line deposited with accession number KCTC13807BP, viral Provided is a composition for detecting hemorrhagic sepsis virus (VHSV).

The present invention also provides a kit for detecting viral hemorrhagic sepsis virus (VHSV) comprising the diagnostic composition.

The present invention also provides a hybridoma cell line (KCTC13807BP) producing monoclonal antibody 4A7, which exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

The present invention also provides a monoclonal antibody 2F6 produced by the hybridoma cell line (KCTC13807BP) and exhibiting specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

Provided are hybridoma cell lines (KCTC13806BP) that produce monoclonal antibody 2F6, which exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

The present invention also provides a monoclonal antibody 2F6 produced by the hybridoma cell line (KCTC13806BP) and exhibiting specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

By using the diagnostic kit including the VHSV antibody according to the present invention, it is possible to diagnose VHSV quickly before the infection of many individuals occurs, thereby minimizing the damage of fishers, and promptly responding to the quarantine agency. have.

Figure 1 is a schematic of the monoclonal antibody manufacturing process according to the present invention.
Figure 2 shows hybridoma colonies producing VHSV monoclonal antibodies according to the present invention.
Figure 3 shows the results of detecting the 17 kinds of VHSV virus obtained in the coastal farms of Korea using the VHSV detection kit according to the present invention.

In the present invention, in order to produce a kit for effectively diagnosing viral hemorrhagic sepsis virus (VHSV), mouse splenocytes immunized with VHSV antigen and mouse myeloma-derived cells are fused in the presence of feeder cells to produce VHSV monoclonal antibody. Hybridoma cells were injected to mice with the hybridoma cells to prepare VHSV monoclonal antibodies. The diagnostic kit prepared using the prepared VHSV monoclonal antibody was confirmed to have excellent sensitivity and reproducibility when used for diagnosis using a VHSV infected tissue and a blood sample.

Accordingly, the present invention, in one aspect, viral hemorrhagic sepsis containing a primary antibody for viral hemorrhagic sepsis virus antigen capture, a detection label, a secondary antibody to which the detection label is bound and a reagent for measuring the activity of the detection label In the composition for detecting virus (VHSV), the primary antibody for viral hemorrhagic sepsis virus antigen capture is a monoclonal antibody 4A7 produced by a hybridoma cell line deposited with accession number KCTC13807BP, viral hemorrhagic It relates to a composition for detecting sepsis virus (VHSV).

In the present invention, the secondary antibody may be characterized in that the monoclonal antibody 2F6 produced by the hybridoma cell line deposited with accession number KCTC13806BP.

In the present invention, the detection label biotin, horseradish peroxidase (HRP), alkaline phosphatase (alkaline phosphatase), colloidal gold (colloid gold), poly L-lysine-fluorescein isothiocya nate (FITC) and RITC ( rhodamine-B-isothiocyanate) may be selected from the group consisting of.

In another aspect, the invention is directed to a hybridoma cell line (KCTC13807BP) and a hybridoma cell line (KCTC13807BP) that produce a monoclonal antibody 4A7 that exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV) and a virus. It relates to monoclonal antibody 4A7 which exhibits specific immunoreactivity against sexually hemorrhagic sepsis virus (VHSV).

In another aspect, the present invention is produced by a hybridoma cell line (KCTC13806BP) and the hybridoma cell line (KCTC13806BP) that produces a monoclonal antibody 2F6 that exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV). It relates to a monoclonal antibody 2F6 which exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

The present invention is prepared by fusing Balb / c mouse-derived spleen-derived cells and mouse myeloma-derived cells immunized with VHSV antigen, and hybridoma cells are conventionally known in the art, for example, HAT (Hypoxanthine Aminopterin Thymidine). ) Can be prepared using a selection method or the like (FIG. 1).

Hybridoma cell line producing the 4A7 antibody was deposited with KCTC accession number KCTC13807BP dated January 30, 2019.

The hybridoma cell line producing the 2F6 antibody was deposited with KCTC accession number KCTC13806BP on January 30, 2019.

By “specifically binding” in the present invention is meant a property in which an antibody binds only to a specific antigen, but not to another antigen.

In addition, when a competitive ELISA was performed using the VHSV monoclonal antibody of the present invention, VHSV infected animal-derived tissues and blood VHSV antigens were effectively detected.

Therefore, in another aspect, the present invention relates to a kit for detecting viral hemorrhagic sepsis virus (VHSV) comprising the diagnostic composition.

In the present invention, the detection label and the reagent for measuring the activity of the detection label are various detection labels and respective detection labels used in general immunoassay to measure any one of color development, fluorescence, and luminescence properties by reaction. According to the present invention, a reagent for measuring activity can be used.

VHSV diagnostic reagent of the present invention, the diagnostic antigen, the monoclonal antibody for capturing the diagnostic antigen, the detection label, the monoclonal antibody to which the detection label is bound, and the reagents used for general immunoassay in addition to the reagents for measuring the activity of the detection label. It may be included as. More specifically, antibody immobilized solution, antigen immobilized solution, blocking solution, test sample diluent, wash solution to remove serum reaction and non-specific reaction of antibody, positive and negative control reagent to validate the test, the detection label And a stopper for stopping the reaction for measuring the activity of the. In addition, various solid phase supports used in general immunoassay methods, such as plates, microplate wells, plastic test tubes, glass beads, plastic beads, and the like may also be included.

The VHSV diagnostic kit of the present invention may be a kit comprising the VHSV diagnostic reagent of the present invention.

In the VHSV diagnosis or VHSV neutralizing antibody detection method of the present invention, it is preferable to use Enzyme immunoassay (EIA) or radioimmunoassay (RIA). More preferably, enzyme immunoassay method is used. However, the present invention is not limited to the above-described detection method, and the complex of the monoclonal antibody for detecting the VHSV virus neutralizing antibody, the monoclonal antibody for capturing the antigen, the diagnostic VHSV virus antigen, and the test sample collected from the test subject are reacted with the antigen-. Any method can be used as long as it can detect an antibody reaction.

The test sample means a biological sample collected from a subject. It is preferable to use intercellular body fluid or serum, and a sample obtained by treating the antigen of the VHSV virus or an autoantibody against the VHSV virus is collected and used.

More specifically, the VHSV diagnostic method of the present invention comprises the steps of immobilizing monoclonal antibodies for antigen capture on a solid phase support; Washing and removing antibodies not attached to the solid phase support; Reacting the VHSV virus diagnostic antigen with the solid phase support; Washing and removing antigens not attached to the solid phase support; And reacting the monoclonal antibody bound to the detection label with the VHSV virus diagnostic antigen immobilized on a solid phase support, wherein the monoclonal antibody for capturing the diagnostic antigen is a hybridoma cell line. Monoclonal antibodies produced by VHSV 4A7 and immunoreactive to VHSV are used.

In one aspect of the invention, the detection antibody may be a monoclonal antibody 2F6 or 10F5-3, but is not limited thereto.

The advantages of the VHSV rapid diagnostic kit are lower cost and shorter reading time in 5 ~ 10 minutes than the real-time PCR method using the existing high cost and expensive equipment. The same test results as the technique can be utilized, which has several advantages in field application.

The diagnostic kit of the present invention may be configured in a kit package type for convenience of field use, and the components may include a VHSV quick diagnosis kit, a buffer solution, a simple grinder, and a simple dropper.

The present invention will contribute to strengthening the effective fisheries defense system by minimizing the damage of the aquaculture industry and providing a direction to prepare a response system using new bio materials through the development of a diagnostic system for constant observation and monitoring of infectious diseases. It is expected.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Monoclonal Antibody Construction

1-1: Antigen Boosting to the Mouse

Viral hemorrhagic sepsis virus (VHS2014-5, NCBI BankIt-KY979959, Hwang, JY et al, fish & shellfish Imuun . 26: 293, 2018) isolated from Korea was transferred to EPC (Epithelioma papulosum cyprini) cell line (ATCC®CRL2872 ^TM ) Incubated and purified.

EPC cells were cultured at 20 ° C. using EMEM medium (Earle's salt-based minimal essential medium, Invitrogen, CA) with 1% antibiotic-antimycotic (Gibco BRL, USA) and 10% fetal bovine serum (FBS). Dispense EPC cells into 96-well plates at a concentration of 1x10 ⁴ cells / well, inoculate 200 μL of virus culture solution after 24 hours, adsorb for 30 minutes at room temperature, and add MEM with 2% FBS and 1% antibiotics (Gibco). The medium was added and cultured at 20 ° C., and cytopathic effect (EPC) was observed using an inverted microscope. Virus cultures with CPE were collected, cells were removed by centrifugation, and TCID _{50 was} determined as the supernatant. Normal cells were inoculated with the same amount of PBS and observed under the same conditions.

Purification of the virus was carried out according to the method of Nishizawa et al. (Nishizawa et al. Fish Pathol 26:77, 1991). VHSV was inoculated into 150 cm ² T / C flask, and after cultivation in bulk, the cells were lysed at ultra low temperature of -75 ° C, centrifuged for 4,000xg, 30 min, and only supernatant was taken. Here, 7% polyethylene glycol (PEG-6,000), 2.3 % NaCl was added and reacted overnight with stirring at 4 ° C. The pellet obtained by centrifugation for 20,000xg for 40 minutes was resuspended in a small amount of TNE buffer (0.01 M Tris HCl, 0.1 M NaCl, 0.001 M EDTA), and then layered on a 15% sucrose cushion, and dried for 1 hour at 4 ° C and 115,000xg. Virus pellets were obtained by ultracentrifugation (scien \ -tific WX μLtra 100, Thermo, USA). Final virus was identified by 12% SDS-PAGE and concentration was measured at absorbance 280 nm (NanoVue Plus, Ge, USA).

The abdominal cavity of 6-week-old female BALB / c mice in emulsion after mixing VHSV antigen obtained by the above method as the first immunization with the same amount of antigen (completely 250 μl each) and complete Freund's adjuvant (Sigma). Infused. Six mice were injected with 50 μg of antigen per mouse, and after 2 weeks as a second immunization, the amount of antigen was reduced by 30%, that is, 35 μg, but the volume was injected in the same amount as the first immunization. It was. The third immunization was also carried out in the same manner as the second immunization process, but the dose of the antigen was reduced to 25 μg. Finally, two weeks later, the antigen (the same amount as in the third administration) was mixed with PBS and injected into the vein of the tail (travenous injection, iv). Three days after the last immunization, cell fusion was performed.

1-2: Antibody titering during antigen administration

      Four days after the third immunization, blood was collected from the tail vein and serum was collected. Diluted in 1/1000 in phosphate buffer (PBS, pH 7.4, see Table 1) for enzyme immunoassay (ELISA, Enzyme Linked Immuno-Sorbent Assay). The antibody titer was determined by When the measured absorbance values were good compared to the negative control mice (serum of mice not injected with antigen), the immunization was reported to be successful, and the cells were prepared for fusion.

Phosphate-buffered saline, PBS, 1 liter Name (Formula) Molecular Weight Amount NaCl 58.450 8 g KCl 74.560 0.2 g Na ₂ HPO ₄ 142.00 1.44 g KH ₂ PO ₄ 136.09 0.24 g

1-3: Feeder Cell Preparation

Eighteen hours prior to the fusion of myeloma and spleen cells, the abdominal skin of 12-week-old or older mice (Balb / c females) was removed and an 8 ml of 4 ° C., 11.3% sucrose (Sigma) solution was injected into the abdominal cavity. After injection, massage the abdominal cavity, promote the release of macrophages in the abdominal cavity, recover it and centrifuge at 1300 rpm, wash the pellet using PBS or RPMI-1640 (Sigma) medium, and then 1X HAT (hypoxanthine) It was suspended in aminopterine tymidine, Sigma), 20% FBS (fetal bovine serum, Sigma), RPMI-1640, aliquoted in 96 well plates (Nunc) for cell culture and incubated in 37 ° C CO ₂ incubator (Binder).

      The preliminary preparation of the feeder cells is intended to promote the healing of various cytokines secreted by the feeder cells during various stages of the fusion process and to inhibit growth inhibition due to excessive cell dilution.

1-4: Cell fusion of splenocytes and myeloma cells

Cell fusion was performed according to Khler and Milstein using PEG (polyethylene glycol, Sigma) as fusogen. Two weeks prior to cell fusion, myeloma cells (myeloma, Sp2 / 0-Ag14, HGPRT-, Korea Cell Line Bank) and splenocytes of mice with high antibody titers were prepared in advance and mixed with FBS. After washing with the medium for cell culture without addition, the mixture was fused while stirring with 1 ml of PEG at 37 ° C. over 1 minute. After fusion, the fusion process was completed by adding 1X HAT medium (20% FBS, RPMI-1640) slowly over several minutes. This whole process was carried out using a 37 ℃ water bath (water bath).

Before the prepared feeder cells are crushed 96 well plate with the dilution of the cells at a concentration of about get one colony per well in the HAT is added to the medium by frequency division, and the division 96well plate are incubated 7 days at 37 ℃ CO ₂ incubator, and screening The incubator was incubated without opening until entering step.

1-5: Screening specific wells

      Seven days after the cell fusion, each 96 well plate was taken out and observed by inverted microscopy. Specificity tests were performed by selecting wells whose colony size occupies about 25% of the total well area. The morphological features of the colonies formed are shown in various forms such as complete floating, staphylococci, partial adhesion, and complete adhesion. Therefore, 47 clones are classified and classified in consideration of cell density for each type. Screened (Table 2).

1-6: Specificity test of monoclonal antibodies

Enzyme immunoassay (ELISA) was used to confirm antigen specificity for 47 clones selected in 1-5 .

100 μl of the VHSV antigen solution was dispensed in a 96 well plate at a concentration of 10 μg / ml and reacted at 37 ° C. for 60 minutes. 0.05M sodium bicarbonate solution was used as the diluent, and the reaction solution was removed, and 200 µl of the wash solution (phosphate buffer, 0.05% Tween 20) was added to the well, and then the procedure was repeated three times. After washing, 200 µl of a blocking solution (phosphate buffer containing 1% BSA) was added to each well, followed by reaction at 37 ° C. for 30 minutes. The blocking solution was removed, and the washing solution was added three times to 200 μl per well, and then removed again. 100 μl of the monoclonal antibody (MAb) solution was dispensed per well, and 100 μl of the diluted solution was injected into the negative control well, followed by reaction at 37 ° C. for 60 minutes. Remove the reaction solution and repeat the process of adding the washing solution 200μl per well and then removing again. Dispense 100 μl per well of the reagent fused with anti-mouse IgG to HRP (Horse reddish peroxidase) and react for 60 minutes at 37 ° C. The reaction solution was removed, and the washing solution was added three times to 200 μl per well, and then removed again.

Dissolve 100 μl of enzyme substrate solution (o-phenylenediamine 0.4 mg / ml in phosphate citrate buffer pH 5.0) and react for 30 minutes at room temperature in the dark. Check the absorbance at 492 nm (yellow). The absorbance value of 3 times or more was determined to be negative or positive based on the minimum value of the positive reaction.

As a result, as shown in Table 3, the VHSV mAb was able to confirm a specific response to the autoantigen, and selected 10 clones for which no specific response to the other antigen was confirmed. Of these, IgM cross ELISA was performed on clones in which isotype was duplicated with IgM, and nine monoclonals identified only by IgG were selected.

No. Clone Isotype Sensitivity ELISA Cannon VHSV
Positive SVC
Negative One 1E2 IgG1, IgM + - 2 1G2 IgG2a, IgM + - 3 3B11 IgG2a + - 4 3F4 IgG2a, IgG2b + - 5 3F11 IgG2a + - 6 4A7 IgG2a ++ - 7 5C10 IgG1, IgG2a, IgG2b ++ - 8 7D3 IgG2a + - 9 8B2 IgG2a, IgG2b, IgM + - 10 10F5 IgG2a, IgM +++ -

1-7: Isolation and Purification of Monoclonal Antibodies

0.5-8 ml of pristane was injected into the abdominal cavity of 6-8 week old BALB / c mice and 9 screened at 1-6 (# 1G2, # 3B11, # 3F4, # 3F11-4, # 4A7, # 5C10, # 7D3) , # 8B2, # 10F5-3) VHSV clone cell line was cultured in medium (DMEM, 10% FBS), and the pellet obtained by centrifuging the cell line for 5 minutes at 1300 rpm on the 7th day after Pristane treatment was suspended in an appropriate amount of PBS. After counting. About 1-5 x 10 ⁶ cells per mouse are suspended in 0.5 mll phosphate buffer and injected into the abdominal cavity.After 1-2 weeks, when the mouse swells, the ascites is collected using a syringe. The supernatant was taken by centrifugation at 1500 rpm for 10 minutes. The supernatant taken was filtered using a 0.45 μm syringe filter, passed through a protein G column (GE) to bind only the antibody, and then washed twice with 3 times the volume of the washing solution (0.05M Tris, 0.15M NaCl, 0.1% Tween 20). After passing through the eluate (0.1M glycine, pH 3.0) to the antibody fraction by using a fraction collector. The obtained antibody solution was used after desalting column (GE) was exchanged with various buffers such as phosphate buffer (PBS) according to the experimental purpose. Table 4 shows the yield of the antibody obtained for each clone.

Clone Ascites gained Antibodies
density
(mg / ml) Volume
(ml) Antibody Acquisition
(mg) Yield per 1 ml of ascites
(mg / ml) Yield per horse
(mg / horse) 1G2-1 7.5ml / 3 3.7 3.5 12.8 1.7 4.3 3B11 5.0ml / 2 7.3 3.5 25.55 5.1 12.8 3F4 5.0ml / 2 4.9 3.5 17.08 3.4 8.5 3F11-4 7.5ml / 3 9.1 3.5 31.8 4.2 10.6 4A7-2 5.0 ml / 2 7.3 3 21.81 4.4 11 5C10 7.5ml / 3 2.3 3.5 8.3 1.1 2.8 7D3 7.5ml / 3 6.2 4 24.8 3.3 8.3 8B2-3 7.5ml / 3 10.2 3.5 35.8 4.8 11.9 10F5-3 2.5ml / 1 0.8 0.8 0.6 0.3 0.6 5ml / 3 0.8 2.5 1.9 0.4 0.6

1-7: Cell line  And control of cell lines for production

The fusion cell lines selected in 1-6 were subjected to primary and secondary cloning by limiting dilution, so that the fusion cell population was cloned from one cell. The prepared monoclonal antibody cell line was suspended in medium and grown for 3-4 days at 37 ° C. in a CO ₂ cell incubator. The cell suspension was centrifuged at 1,300 rpm for 10 minutes, and the precipitated cells were then cryoprotectant (RPMI-1640 cell culture solution). In 1% (1X10 ⁶ cells / mL) into a cryopreservation ampoule, record the cell line name, passage number, and passage date, and place them in a freezing container. -70 ℃) overnight and cryopreserved at minus 196 ℃ (liquid nitrogen) was used as a cell line.

The cryopreserved native monoclonal antibody secreting cell line was taken out and quickly thawed in a 37 ° C. constant temperature water bath, suspended in medium, propagated in a 37 ° C. CO ₂ cell incubator for 3-4 days, and then the suspended cell suspension was centrifuged at 1,300 rpm for 10 minutes. After separation, the precipitated cells were suspended in a cryoprotectant, and then dispensed in 1ml (1X10 ⁶ cell / mL) into a cryopreservation ampoule, and recorded the cell line, passage number, passage date, and so on at minus 196 ℃ (liquid nitrogen). Cryopreservation was used as monoclonal antibody secreting cell line for production.

Example 2: Monoclonal Antibody-Based VHSV Diagnostic Kit Construction

2-1: Content Setting of Antibody and Antibody-conjugated Gold in Monoclonal Antibody-based VHSV Diagnostic Kit

VHSV mAb clones of 9 species (# 1G2, # 3B11, # 3F4, # 3F11-4, # 4A7, # 5C10, # 7D3, # 8B2, # 10F5-3) prepared for monoclonal antibody production in Examples 1-7 The ascites of mice obtained by administering was purified by the GE method to obtain mAb. The monoclonal antibody obtained was diluted with each step and tested with Gold. As a result, the antibody value was afforded even at the minimum value, and thus diluted twice with 0.01 M sodium tetraborate buffer and diluted to a concentration of 5.5 mg / ml. As a result of diluting the antibody diluted twice, the appropriate value was confirmed in Gold 100ul / mAb 3ul.

In order to fabricate 40nm CGC (Colloidal gold conjugate), tiitration was carried out for each pH, and in case of # 8B2, precipitation was confirmed under all pH conditions, thus making a kit impossible (Table 5).

Titration Conditions of Nine Anti-VHSV Antibodies mAb. Titration condition # 1G2  pH6.0, 16 ug / ml # 3B11 pH9.0, 18ug / ml # 3F4 pH6.0, 20ug / ml # 3F11-4 pH6.0, 26ug / ml # 4A7 pH6.0, 20ug / ml # 5C10 pH6.0, 28ug / ml # 7D3 pH9.0, 16 ug / ml # 8B2 Not production # 10F5-3 pH6.0, 28ug / ml

2-2: Create Gold and Sample Pads for Diagnostic Kits

10 μL of 8 types of VHSV monoclonal antibodies (# 1G2, # 3B11, # 3F4, # 3F11-4, # 4A7, # 5C10, # 7D3, # 10F5-3), respectively, were determined in 2-1. 16.5 μg) was mixed in an appropriate amount to prepare a gold pad, and sample pad conditions were confirmed to remove the phenomenon of developing the sample and non-specific reactions during kit preparation.

3% to 7% Tween 20 (distilled water) was used, and there was no specific reaction in the kit using the sample pad of 4% Tween 20. The solution used to confirm the nonspecific reaction was 20 mM TBS (pH7.6, 20 mM Tris, 150 mM NaCl) lysis buffer.

2-3: pairing test of 8 antibodies

Eight selected antibodies were subjected to Sandwich pair selection functioning on a quick diagnostic kit. The positive and negative reactivity of 64 pairs of test subjects was confirmed, and the reaction time, sensitivity, specificity, and antigen-antibody binding ability were different in antigen, antibody response, and Sandwich ELISA reaction on the quick diagnosis kit strip. As shown, the search on a valid sandwich pair was run on a quick diagnostic kit.

VHSV antibody was adsorbed at a concentration of 1 mg / ml at the test line position in the nitrocellulose membrane of the test strip, and anti-mouse antibody was adsorbed by aliquoting at the control line position, and then cleaved. Conjugate pads were adsorbed to 3.0 OD of control anti-mouse CGC and VHSV CGC, and each was digested. Conjugate pads and sample pads were assembled on the membrane, and 64 pair strips were used for the test. The test sample was diluted in the sample dilution solution and dispensed into the drop site of the lower end of the device, and was judged 10 minutes.

Capture: 1mg / ml

CGC: 40nm CGC 3.0 OD

Sample pad: Cytosep1662 / 50mM Tris pH8.0 / 1% Tween 20 / 0.2% Casein

Buffer: 1xPBS pH7.4 / 0.8% Tween 20

Sample: VHSV Cannon, SVC

As a result of pairing test of 8 selected antibodies, reactivity with 3 positive standards (VHSV cannon, VHSV I, VHSV IVa) and 3 negative standards (IHNV-10, IHNV-12, SVC) was confirmed. Two pairs of # 4A7 / CGC: # 2F6 pairs and # 4A7 / CGC: # 10F5-3 pairs with no false positives for Buffer were selected (Table 6).

A kit for detecting VHSV was prepared by selecting # 4A7 / CGC: # 2F6 pairs having excellent task results among the two pairs of antibodies.

Hybridoma cell line producing the 4A7 antibody was deposited with KCTC accession number KCTC13807BP dated January 30, 2019.

The hybridoma cell line producing the 2F6 antibody was deposited with KCTC accession number KCTC13806BP on January 30, 2019.

2-3: Evaluation of Monoclonal Antibody-Based VHSV Diagnostic Kits

Using the 4A7 (Capture) and 2F6 (CGC) Pair of the monoclonal antibody pairs determined in 2-1 and 2-2, VHSV kit was prepared by adjusting the antibody content, gold conjugate content, sample pad conditions.

For the testing of the VHSV diagnostic kit, 20mM TBS (pH7.6, 20mM Tris, 150mM NaCl) was determined by the virus lysis buffer composition to confirm nonspecific reactions. Tests were conducted with inactivated VHSV (positive), Khv (negative), Rsiv (negative).

As a result, all of the positive samples, VHSV, could be confirmed as positive, and other viruses except VHSV were confirmed as negative.

17 VHSV viruses obtained from Korea's coastal farms were diluted to 10 ⁶ TCID / ml, 10 ⁵ TCID / ml, 10 ⁴ TCID / ml, and 10 ³ TCID / ml for testing. It was. The strength of the test line was measured using a BIT VALUE reader, and the average of the three repeated experiments was written in the table. In the case of the reader of BIT VALUE company, it was judged to be positive when the value was 50 or more, and to be negative when it was less. As a result of the test, as shown in FIG. 3 and Table 7, the VHSV detection kit using 4A7 (Capture) and 2F6 (CGC) pair was confirmed to have a sensitivity of about 10 ⁵ TCID / ml to 10 ⁴ TCID / ml of virus concentration. The intensity of the test line according to the titer is shown in FIG. 3.

nursery Virus Reader Average for Potency (n = 3) 10 ^ 6 TCID / ml 10 ^ 5 TCID / ml 10 ^ 4 TCID / ml 10 ^ 3 TCID / ml Daecheon Fisheries 1 VH2012-5 2301 754 90 0 Halla Fisheries VH2012-9 2638 1083 116 One Daekyung Fisheries 1 VH2013-2 1716 1520 201 3 Daekyung Fisheries 2 VH2013-9 1088 203 One 4 Sea Dragon Fisheries VH2014-2 2344 866 115 8 Younglim Fisheries VH2016-2 1749 394 9 0 Myeongjinsan VH2012-6 2426 660 90 0 Handong Fisheries VH2012-10 2289 945 112 0 Jinan Fisheries VH2013-3 2677 1069 146 0 Daecheon Fisheries 2 VH2013-1 2184 564 12 0 Sehyeon Fisheries VH2015-2 2286 760 109 0 Southern Tai Fisheries VH2016-3 1456 196 112 0 Oriental Fisheries VH2012-7 2577 971 85 0 JJ Fisheries VH2012-11 2663 1139 151 2 East-West Fisheries VH2013-4 2613 1103 89 0 Yurasu Fisheries VH2014-4 2138 531 15 0 Haiwang Fisheries VH2015-5 2250 477 10 0 Average 2199.71 778.53 86.06 1.06 Standard Deviation 452.64 359.35 58.18 2.16 Standard error (P <0.05) 215.17 170.82 27.66 1.03

As described above in detail specific parts of the present invention, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

In a composition for detecting viral hemorrhagic sepsis virus (VHSV) containing a primary antibody for capturing viral hemorrhagic sepsis virus antigen, a detection label, a secondary antibody to which the detection label is bound, and a reagent for measuring the activity of the detection label ,
The primary antibody for viral hemorrhagic sepsis virus antigen capture is produced by a hybridoma cell line deposited with accession number KCTC13807BP, characterized in that the monoclonal antibody 4A7 showing an immune response specific for viral hemorrhagic sepsis virus, Composition for detecting viral hemorrhagic sepsis virus (VHSV).
The viral hemorrhagic sepsis according to claim 1, wherein the secondary antibody is a monoclonal antibody 2F6 produced by a hybridoma cell line deposited with the accession number KCTC13806BP and exhibits an immunoreactivity specific for the viral hemorrhagic sepsis virus. Composition for detecting virus (VHSV).
The method of claim 1, wherein the detection label is biotin, horseradish peroxidase (HRP), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein isothiocya nate (FITC) and RITC (RITC). rhodamine-B-isothiocyanate).
Viral hemorrhagic sepsis virus (VHSV) detection kit comprising the diagnostic composition of claim 1.
Hybridoma cell line (KCTC13807BP) that produces monoclonal antibody 4A7 that exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).
Monoclonal antibody 4A7 produced by the hybridoma cell line (KCTC13807BP) of claim 5 and showing specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).
A hybridoma cell line (KCTC13806BP) producing monoclonal antibody 2F6 that exhibits specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).
Monoclonal antibody 2F6 produced by the hybridoma cell line (KCTC13806BP) of claim 7 and exhibiting specific immunoreactivity against viral hemorrhagic sepsis virus (VHSV).

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