KR102384570B1 - Specimen sample composition for point of care testing (POCT) kit and method for detection of hirame rhabdovirus (HIRRV) using the composition - Google Patents

Abstract

The present invention relates to a technique for diagnosing hirame rhabdovirus (HIRRV) disease that causes death of Hippoglossus stenolepis and, more specifically, to a specimen composition for a point-of-care testing kit capable of improving the accuracy of diagnosis by improving the sensitivity of existing point-of-care testing kits, and to a point-of-care diagnosis method using the composition, wherein the existing point-of-care testing kits can determine whether a person is infected with a HIRRV virus within 20 minutes.

Description

Specimen sample composition for point of care testing (POCT) kit and method for detection of hirame rhabdovirus (HIRRV) ) using the composition}

The present invention relates to a technology for diagnosing halibut rhabdovirus disease that causes the death of halibut, and more specifically, by improving the sensitivity of a point-of-care diagnostic kit capable of diagnosing halibut rhabdovirus infection within 20 minutes, thereby improving the accuracy of diagnosis It relates to a sample composition for an on-site inspection diagnostic kit for halibut rhabdovirus disease infection, and a method for on-site diagnosis of halibut rhabdovirus disease using the composition.

Hirame rhabdoviral disease (Hirame rhabdoviral disease) was first isolated from the causative virus when a large amount of flounder ( Paralichthys olivaceus ) died in Japan in the early 1984's. In Korea, viruses were isolated from halibut in 1998, and after 2013, viruses were also isolated from black sea bream ( Acanthopagrus schlegeli ) and point sea bass ( Lateolabrax mac ulatus ).

Halibut rhabdovirus disease develops at a water temperature of 2 to 18°C and mainly occurs in halibut with a body weight of 100 to 700 g. Low water temperature (5~10℃) causes mortality in halibut farms, and the lower the water temperature, the higher the mortality rate. Halibut rabidovirus disease is known as a viral disease that causes damage not only in Korea but also in Japan.

Hirame rhabdovirus (HIRRV), the causative agent of halibut rhabdovirus disease, is a single-stranded RNA virus and belongs to the Rhabdoviridae family and the genus Novirhabdovirus .

HIRRV is a negative-strand RNA virus with approximately 11,000 bp of 6 genes, nucleocapsid (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), non-virion protein (NV) and polymerase (L) genes. are in the order of 3'-N-P-M-G-NV-L-5'.

Conventionally known methods for testing HIRRV include a separation culture method using chemotactic fish cells, a molecular biological method using genes [reverse transcription-polymerase chain reaction (RT-PCR), real time PCR, etc.], an immunological method using an antibody [enzyme -linked immunosorbent assay (ELISA), fluorescent antibody techniques (IFAT), etc.] are being used.

Looking at the pros and cons of these test methods, first, the isolation and culture method using chemotactic fish cells: The virus isolation and culture method using chemotactic fish cells (CHSE-214, EPC, FHM) is widely used worldwide, but in the field It is not easy to use for direct detection, it takes at least 10 days or more, so the detection time may be slightly longer, it requires specialized personnel and facilities, it is impossible to process multiple samples at once, and when testing multiple samples The disadvantage is that it costs a lot.

In the case of molecular biological methods, the PCR method targeting the HIRRV genome is widely used worldwide due to its excellent specificity and sensitivity, but it is not easy to use for direct detection in the field, If necessary, it takes at least one day, so the overall detection time may be slightly longer. Also, professional personnel and facilities are required, it is impossible to process a large number of samples at a time, and it is expensive to test a large number of samples. Not only that, but even if the pathogen is detected through PCR test, there is a problem in that it is impossible to determine the infection status of the pathogen in the fish body.

In the case of immunological methods, immunological techniques (ELISA, IFAT, etc.) using antibodies that specifically respond to HIRRV have excellent specificity and sensitivity, and have the advantage of being able to process a large number of samples, but they cannot be used in the field. In addition, as time is required for pre-processing of the specimen, it takes at least one day or more, which may lengthen the overall detection time. inspection is possible.

As such, a new diagnostic technology capable of rapidly detecting halibut rhabdovirus disease at the aquaculture site is required for a quick response at the aquaculture site. There is a need to develop a technology that can improve even the sensitivity of a diagnostic kit developed for rapid detection.

Domestic Patent Registration No. 10-1865878

Therefore, it is an object of the present invention to improve the sensitivity of a diagnostic kit developed to rapidly detect halibut rhabdovirus disease in a culture field using a monoclonal antibody with sensitivity and specificity to HIRRV. It is to provide a sample composition for a diagnostic kit for on-site inspection of whether flounder raptor infection with viral disease is possible.

Another object of the present invention is to provide a point-of-care diagnostic method capable of accurately diagnosing halibut rhabdovirus disease within 20 minutes using a spot inspection diagnostic kit for halibut rhabdovirus disease infection with improved sensitivity by the sample composition. will be.

Objects of the present invention are not limited to the objects mentioned above, and other objects not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, first, the present invention provides a sample composition for a field test diagnostic kit for flounder rhabdovirus infection, which includes a liquid sample derived from flounder spleen tissue.

In a preferred embodiment, it further comprises flounder slime.

In a preferred embodiment, the flounder slime is included in 0.5 to 3% by volume.

In addition, the present invention includes the steps of contacting any one of the above-described sample composition with a spot test diagnostic kit for flounder rhabdovirus infection; and detecting a positive reaction of the test line member of the diagnostic kit.

In a preferred embodiment, after the sample composition is brought into contact with the sample pad member of the diagnostic kit, a positive reaction of halibut rhabdovirus disease is detected within 20 minutes to detect whether halibut infection is detected.

In a preferred embodiment, the diagnostic kit comprises: a strip-shaped porous thin film member; an absorbent pad member formed at one end in the longitudinal direction of the porous thin film member; A monoclonal that is laminated on the other longitudinal end of the porous thin film member and is produced by hybridoma cells deposited with accession number KCLRF-BP-00513 and specifically binds to hirame rhabdovirus (HIRRV) Antibody conjugate pad member containing antibody 11-2D9; a sample pad member laminated on some or all regions of the antibody conjugate pad member; Flounder wraps produced by hybridoma cells deposited with an accession number KCLRF-BP-00514 and formed with a predetermined width on a surface positioned between the antibody conjugate pad member and the absorbent pad member among the surfaces of the porous thin film member a test line member to which the monoclonal antibody 15E10 specifically binding to hirame rhabdovirus (HIRRV) is immobilized; and a control line member formed with a predetermined width on a surface of the porous thin film member and positioned between the test line member and the absorbent pad member, to which the secondary antibody binding to the monoclonal antibody is fixed.

In a preferred embodiment, the monoclonal antibody 11-2D9 is formed on the antibody conjugate pad member to be movable according to the flow of the liquid sample passing through the sample pad member in the form of an antibody-label conjugate formed by conjugation with a label. do.

In a preferred embodiment, the label is Horseradish peroxidase (HRP), alkaline phosphatase, gold nanoparticles, poly L-lysine-fluorescein isothiocyanate (FITC), Rhodamine-B-isothiocyanate (RITC), Cy3 , Cy5, and any one selected from the group consisting of rhodamine.

In a preferred embodiment, the diagnostic sensitivity of the test line member is 10 ^5.2 TCID ₅₀ /100 μl.

In a preferred embodiment, the secondary antibody immobilized on the control line member is an anti-mouse IgG derived from a group of mammals other than mice.

In a preferred embodiment, the porous thin film member is any one selected from the group consisting of a nitrocellulose film, a PVDF film, a polyvinyl resin film, or a polystyrene resin film, and has a pore size of 5 to 12 µm.

In a preferred embodiment, it further comprises a strip-shaped support member formed on the lower surface of the porous thin film member.

According to the sample composition for the point-of-care diagnostic kit of the present invention described above, it is possible to improve the accuracy of diagnosis by improving the sensitivity of the diagnostic kit developed to quickly detect halibut rhabdovirus disease at the farm site.

In addition, according to the point-of-care diagnostic method of the present invention, by using the on-site inspection diagnostic kit for halibut rhabdovirus disease infection with improved sensitivity by the sample composition, it is possible to accurately diagnose flounder rhabdovirus disease within 20 minutes. Because of its excellent performance, it is possible to perform safe halibut farming by minimizing the damage caused by this disease through rapid disease diagnosis, prevention and response.

Effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 is a schematic diagram showing the usage of the flounder rhabdovirus infection on-site inspection diagnostic kit used in the flounder rhabdovirus disease on-site diagnosis method according to an embodiment of the present invention.
Figure 2 shows the results of Western blotting showing the confirmation of the HIRRV recognition site of the monoclonal antibody used in the field test diagnostic kit for halibut rhabdovirus disease infection in Figure 1 [M: marker (kDa), 1: monoclonal antibody 11-2D9, 2: monoclonal antibody 15E10].
3 shows the results of measuring the diagnostic sensitivity to the HIRRV culture of the flounder rhabdovirus disease point-of-care diagnostic kit used in the method for point-of-care diagnosis of halibut rhabdovirus disease according to an embodiment of the present invention.
4 shows HIRRV and other types of fish viruses (VHSV, IHNV, IPNV, MABV, NNV) specificity measurement results.

As for the terms used in the present invention, general terms that are currently widely used are selected as possible, but in certain cases, there are also terms arbitrarily selected by the applicant. So the meaning should be understood.

As used herein, the term “diagnosis” refers to ascertaining the presence or characteristics of a pathological condition. For the purpose of the present invention, diagnosis is to determine whether or not infection with hirame rhabdovirus (HIRRV).

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numbers used to describe the invention throughout the specification refer to like elements.

The technical feature of the present invention is to develop a monoclonal antibody that specifically binds to HIRRV and a fusion cell that produces the monoclonal antibody, and using it, it is developed so that it can be diagnosed within 20 minutes of flounder rhabdovirus infection. There is a sample composition for the on-site inspection diagnostic kit for halibut rhabdovirus infection, which enables more accurate inspection by improving the sensitivity of the on-site inspection diagnostic kit, and a method for on-site diagnosis of halibut rhabdovirus disease using the same.

That is, the conventionally known flounder rhabdovirus infection detection method takes at least one day or more and requires specialized personnel and facilities, so field applicability is very poor. We developed a fusion cell that produces an antibody and the monoclonal antibody, and using it, we developed a point-of-care diagnostic kit that can confirm whether or not a flatfish rhabdovirus infection is present within 20 minutes, as well as improving the sensitivity of the point-of-care diagnostic kit. This is because a sample sample composition and a method for on-site diagnosis of halibut raptor virus disease using the sample composition were developed.

Therefore, the sample composition for the flounder rhabdovirus disease diagnostic kit of the present invention includes a liquid sample derived from flounder spleen tissue. Here, the liquid sample derived from flounder spleen tissue is prepared including flounder spleen tissue. When flounder is infected with flounder rhabdovirus (HIRRV), as will be described later, compared to brain and heart tissue, HIRRV is particularly high in spleen tissue. This is because the distribution was confirmed. Based on these findings, the sample composition of the present invention includes a liquid sample derived from flounder spleen tissue for easy detection. It may be a supernatant obtained after mixing and grinding in a weight ratio of

However, when the sample composition for the flounder rhabdovirus disease diagnostic kit of the present invention consists only of a liquid sample derived from flounder spleen tissue, the detection sensitivity of the diagnostic kit is 10 ^5.2 TCID ₅₀ /100 μl or more, but the infection value is 10 ^{5.55 - 6.05} There was a problem in that a sample of ₅₀ /100 μl of TCID showed a negative result. In order to improve the sensitivity of the flounder rhabdovirus disease diagnostic kit, the composition was changed so that the sample composition further contains flounder mucus.

In other words, as a new diagnostic technology that can quickly detect halibut rhabdovirus disease at the aquaculture site for quick response at the aquaculture site, we have developed a field inspection diagnostic kit for halibut rhabdovirus infection. It showed a diagnostic sensitivity of 10 ^5.2 TCID ₅₀ /100 μl with respect to the flounder rhabdovirus (HIRRV) culture medium, and the prepared sample composition {the spleen tissue of the flounder was extracted and the tissue: POCT sample lysis buffer [20 mM Tris, 25 mM NaCl, 2.5 mM EDTA (ethylene-diamine-tetraacetic acid), 0.05% sodium dodecyl sulfate (SDS), pH 8.0] was mixed in a weight ratio of 1:10 and ground and then left to stand. This is because, as a result of checking the results 10 minutes after dropping, about 30% of the samples were negative despite the presence of 10 ^5.2 TCID ₅₀ /100 μl or more of virus in the spleen tissue.

As a result, the sample composition of the present invention may further contain flounder mucus, which is obtained from the skin surface of flounder and may further contain 0.5 to 3% by volume based on a liquid sample derived from flounder tissue. This is because, when halibut mucus is included in less than 0.5% by volume, the sensitivity improvement effect of the diagnostic kit is insufficient, and when it exceeds 3% by volume, there is no further sensitivity improvement effect and the possibility of other side effects increases.

Next, the method for on-site diagnosis of halibut rhabdovirus disease of the present invention comprises the steps of contacting any one of the above-described sample compositions with a field inspection diagnostic kit for flounder rhabdovirus infection; and detecting a positive reaction of the test line member of the diagnostic kit.

Here, the point-of-care diagnostic kit includes at least one of monoclonal antibody 11-2D9 and monoclonal antibody 15E10. The point-of-care diagnostic kit uses an immune response as necessary, and uses wells, stains, and enzyme-labeled antibodies for detection. , wash solution, antibody diluent, sample diluent, enzyme substrate, enzyme substrate diluent, and other reagents.

In particular, the monoclonal antibody "11-2D9 antibody" or "monoclonal antibody 11-2D9", which is a monoclonal antibody that specifically binds to HIRRV included in the point-of-care diagnostic kit developed in the present invention, is deposited under the accession number KCLRF-BP-00513. refers to the monoclonal antibody produced by the hybridoma cells, and "15E10 antibody" or "monoclonal antibody 15E10" refers to the monoclonal antibody produced by the hybridoma cells deposited under the accession number KCLRF-BP-00514. it means.

Monoclonal antibodies can be prepared by a technique for generating an immortalized cell line by fusion known to those skilled in the art [Koeher and Milstein, Nature, 256:495 (1975)]. The manufacturing method is briefly described as follows. First, about 20 µg of pure protein is obtained and immunized to mice, or peptides are synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing B lymphocytes isolated from mice are fused with human or mouse myeloma cells to generate immortalized hybridomas. Next, the production of monoclonal antibodies in hybridoma cells is checked by ELISA (Enzymelinked immunosorbent assay), positive clones are selected and cultured, and the antibody is isolated and purified or injected into the abdominal cavity of mice to collect ascites.

First, mice required for the production of antibody-producing hybridoma cells were immunized. Specifically, after culturing and concentrating and purifying HIRRV, the purified viral structural protein is diluted in phosphate buffered saline (PBS) and mixed with the same volume of complete Freund's adjuvant (Sigma) to emulsion. After preparing, the emulsion was first injected into the abdominal cavity of a mouse. Two weeks after the primary immunization, blood was collected from the mice and antibody titers were measured by ELISA. In order to increase the immunity of the mice, only purified viruses were used for secondary immunization. Three days after the second immunization, spleen tissues were harvested from the immunized mice. The extracted spleen tissue and myeloma cells, Sp2/0Ag14 cells, were mixed in a ratio of 10:1 based on the number of cells, and polyethylene glycol (PEG) was added to the cells, followed by cell fusion and culture.

Among the group of fused cells, fusion cells that respond specifically to HIRRV are selected, the fusion cells are diluted using a limiting dilution method so that the selected fusion cells become monoclonal, and the fusion cells proliferated from one cell. 8 monoclones with the best absorbance were selected by confirming the absorbance (OD value) by ELISA. Each of the selected 8 monoclones was injected into mice, and monoclonal antibodies 1B3, 5G6, 11-2D9, 15E10, 15-1G9, 17F11, 24-1C6, and 36D3 that specifically bind to HIRRV were isolated from the serum of the mouse. did Thereafter, as in the experimental example to be described later, a pair test for the HIRRV rapid diagnostic kit was performed on eight monoclonal antibodies, and fusion cells producing two monoclonal antibodies finally selected according to the results, i.e., hybridoma cells, were isolated from each other. They were named "HIRRV 11-2D9" and "HIRRV 15E10", and were deposited with the Korea Cell Line Research Foundation on May 4, 2021 and were given accession numbers KCLRF-BP-00513 and KCLRF-BP-00514, respectively.

In one embodiment, the point-of-care diagnostic kit used in the point-of-care diagnostic method of the present invention comprises: a strip-shaped porous thin film member; an absorbent pad member formed at one end in the longitudinal direction of the porous thin film member; A monoclonal that is laminated on the other longitudinal end of the porous thin film member and is produced by hybridoma cells deposited with accession number KCLRF-BP-00513 and specifically binds to hirame rhabdovirus (HIRRV) Antibody conjugate pad member containing antibody 11-2D9; a sample pad member laminated on some or all regions of the antibody conjugate pad member; Flounder wraps produced by hybridoma cells deposited with an accession number KCLRF-BP-00514 and formed with a predetermined width on a surface positioned between the antibody conjugate pad member and the absorbent pad member among the surfaces of the porous thin film member a test line member to which the monoclonal antibody 15E10 specifically binding to hirame rhabdovirus (HIRRV) is immobilized; and a control line member formed with a predetermined width on the surface of the porous thin film member and positioned between the test line member and the absorbent pad member, to which the secondary antibody binding to the monoclonal antibody is fixed; .

All known techniques can be applied as long as the porous thin film member functions as a fixture for the antigen-antibody binding reaction and allows the liquid sample for analysis to be moved from one end to the other through capillary action. It may be a strip formed of any one material selected from the group consisting of a nitrocellulose film, a PVDF film, a polyvinyl resin film, or a polystyrene resin film. The size of the strip for the porous thin film member is not particularly limited in size, but is not limited in size, as long as it is large enough to transport an appropriate amount of analysis sample and observe the degree of reaction between the sample in the analysis sample and the analysis strip, but approximately 5 mm in width or less , it may have a size of 100 mm or less in length. In addition, since a large number of micropores with a diameter of 5 to 12 μm are formed inside the porous thin film member, the liquid sample containing HIRRV can be easily absorbed and moved into the porous thin film member together with the monoclonal antibody-labeled conjugate. It can be configured to

The antibody conjugate pad member is a component that is laminated on one end in the longitudinal direction of the porous thin film member. In particular, when the liquid sample that has passed through the sample pad member passes through the antibody conjugate pad member, a monoclonal antibody is conjugated with a label to form an antibody. -The label conjugate may be included in the liquid sample to be movable according to the flow of the liquid sample. As a result, the liquid sample injected into one end of the porous thin film member and passing through the sample pad member passes through the antibody conjugate pad member while the antibody-labeled body conjugate contained in the antibody conjugate pad member is contained at the other end of the porous thin film member. It has a structure that moves to the absorbent pad member laminated on the . Here, the antibody-labeled conjugate is produced by the hybridoma cell line deposited under accession number KCLRF-BP-00513, monoclonal antibody 11-2D9 that specifically binds to HIRRV, Horseradish peroxidase (HRP), and alkaline phosphatase (Alkaline phosphatase), gold nanoparticles, FITC (Poly L-lysine-fluorescein isothiocyanate), RITC (Rhodamine-B- isothiocyanate), Cy3, Cy5, and any one marker selected from the group consisting of rhodamine conjugate (conjugate) ) may have been Therefore, when HIRRV is present in the liquid sample, the monoclonal antibody of the antibody-labeled conjugate and the HIRRV antigen protein are bound, that is, "HIRRV-antibody-labeled conjugate", especially "HIRRV antigen protein M-antibody-labeled conjugate." " is formed and moves along the flow of the liquid sample, and if HIRRV is not present in the liquid sample, only the "antibody-labeled conjugate" moves.

The absorbent pad member is a component formed at the other end in the longitudinal direction of the porous thin film member, and as long as it constitutes an absorbent pad capable of inducing the flow of the liquid sample through the porous thin film member, the material and structure are not limited, but one implementation Cellulose may be used as an example.

All known configurations can be used as long as the sample pad member is laminated on some or all regions of the antibody conjugate pad member to filter the liquid sample containing the crushed tissue of the sample for analysis or diagnosis to some extent. For example, it may be made of a glass fiber material.

The inspection line member is positioned between the antibody conjugate pad member and the absorbent pad member laminated on both ends of the porous thin film member, and is formed in a certain width on the surface of the exposed porous thin film member, and deposited under the accession number KCLRF-BP-00514. The monoclonal antibody 15E10, which is produced by the hybridoma cell line and specifically binds to HIRRV, especially the antigen protein N of HIRRV, is immobilized. Any known technique can be used as long as it does not move with the flow of the sample. If necessary, a certain amount of a nonionic surfactant such as BSA, sucrose or Tween 20 may be used as a stabilizer.

The control line member is positioned between the test line member and the absorbent pad member and formed in a predetermined width on the exposed surface of the porous thin film member, and a secondary antibody binding to the monoclonal antibody is fixed thereto. When the control line member is formed, the porous thin film member is formed. All known techniques can be used as long as the secondary antibody is immobilized on the surface and does not move with the flow of the liquid sample. If necessary, a certain amount of a nonionic surfactant such as BSA, sucrose or Tween 20 may be used as a stabilizer. In addition, the secondary antibody immobilized on the control line member is an anti-mouse IgG derived from a non-mouse mammal group (goat, sheep, rabbit, etc.), and can bind to a monoclonal antibody of an antibody-labeled conjugate.

The use of the HIRRV infection point test diagnostic kit of the present invention configured as described above is very simple. That is, when the sample composition for analysis is input to the sample pad member of the diagnostic kit of the present invention, the analysis is performed through the color reaction of the test line member and the control line member according to the following principle according to the presence or absence of HIRRV in the sample composition. This is because it is possible to visually confirm whether the sample composition is infected with HIRRV.

Specifically, a liquid sample composition including a liquid sample in which the tissue of the sample to be analyzed is ground is prepared and then put into the sample pad member of the diagnostic kit of the present invention. The liquid sample composition injected into the sample pad member passes through the sample pad member and is filtered so that the crushed tissue of the sample contained in the sample sample composition exists only in an appropriate size, so that the sample sample composition can pass through the porous thin film member uniformly. At this time, if HIRRV is present in the sample composition, the sample composition that has passed through the sample pad member passes through the antibody conjugate pad member located below it and is present in the antibody-labeled conjugate and the sample composition. The HIRRV-antibody-labeled conjugate is formed and, in particular, the HIRRV antigen protein M-antibody-labeled conjugate is formed and contained in the sample composition. Since the HIRRV antigen protein M-antibody-marker conjugate binds to the monoclonal antibody (recognition of antigen protein N of HIRRV) immobilized on the test line member and reacts to develop a color reaction, HIRRV infection can be easily confirmed. On the other hand, if HIRRV is not present in the sample composition for analysis input to the sample pad member, HIRRV is not present in the sample composition that has passed through the sample pad member. Antibody-labeled conjugates present in HIRRV do not form M-antibody-label conjugates of HIRRV and are included in the sample composition in the form of antibody-labeled conjugates. The antibody-labeled conjugate included in the product does not bind to the monoclonal antibody immobilized on the test line member, so that the color reaction does not occur.

In addition, regardless of whether the liquid sample contains HIRRV, when the antibody-labeled conjugate contained in the liquid sample passes through the antibody conjugate pad member and reaches the control line member through the test line member, the secondary antibody fixed to the control line member Since it is combined with , a color reaction always occurs in the control line member. Through such a control line member, it can be confirmed that the diagnostic kit for on-site inspection of HIRRV infection of the present invention operates normally.

According to the on-site diagnosis method of the present invention having the above-described configuration, a positive reaction of halibut rhabdovirus disease is detected within 20 minutes after the sample composition is brought into contact with the sample pad member of the diagnostic kit to determine whether or not flounder rhabdovirus infection is present. As one embodiment, as shown in FIG. 1, after extracting the spleen tissue of halibut, grinding it with a tissue:buffer ratio of 1:10, and then leaving 100 μl of the supernatant obtained by dropping it into the kit. After 10 minutes, the result is checked, and if a red line appears on the test line, it can be judged positive.

Example 1

A hybridoma cell line producing a monoclonal antibody that specifically binds to hirame rhabdovirus (HIRRV) was prepared by the following method.

1. Virus Culture

In order to culture halibut rhabdovirus (HIRRV) in large quantities, epithelioma papilosum cyprine (EPC) was cultured as a single layer in a 75cm2 tissue culture flask (Nunc, Denmark), then the virus was inoculated and cultured at 15℃ for 10 days for cytopathic effect ( CPE) was observed. The cell culture medium in which the cells were infected with the virus and more than 90% dissolved was centrifuged at 4° C. at 12,000 rpm for 30 minutes to remove the cell residue, and then the supernatant was separated. The isolated virus supernatant was stored at -80°C until used in experiments.

2. Concentration and purification of virus

Polyethlene glycol (PEG)-6000 (sigma, USA) and NaCl at 7.5% (w/v) and 2.3% (w/v), respectively, were added to the virus culture medium and incubated at 4°C overnight. After centrifuging the PEG-treated virus culture solution at 4°C at 12,000 rpm for 30 minutes, the pellet was buffered with phosphate buffer saline (PBS: 0.13M NaCl, 2.7 mM KCl, 4.3mM Na ₂ HPO ₄ , 1.4mM KH ₂ PO ₄ ) suspended in solution. The suspension was ultracentrifuged at 30,000 rpm for 2 hours, and the pellet was resuspended in PBS to concentrate the virus. To purify the virus, the concentrated virus was placed on a step sucrose gradient [20%, 35%, 50% sucrose solution (w/v)], followed by ultracentrifugation at 30,000 rpm for 2 hours. A band presumed to be a virus formed at the boundary of 20% and 35% 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.

3. Virus immunity and hybridoma production

For immunization, purified virus (about 100 μg) and complete freunds adjuvant were mixed in a 1:1 ratio, and primary immunization was performed in the abdominal cavity of BALB/c mice. After 2 weeks, blood was collected from the mice to measure the antibody titer by performing ELISA, and secondary immunization was performed using only purified virus. After 3 days of secondary immunization, the mouse spleen tissue was isolated and fused with myeloma cells (Sp2/0Ag14) using PEG 1500 (Roche, USA), and then hypoxanthine, thymidine (HT) (gibco, USA) medium was used. Hybridomas were prepared by aliquoting into 96 well plates (TPP, Switzerland) and culturing at 37° C. in a CO ₂ incubator.

4. Hybridoma screening

An enzyme-linked immunosorbent assay (ELISA) was performed to screen hybridomas that produce antibodies that react specifically to viruses. For antigen, 50 μl (250 ng/well) of the purified virus solution was dispensed per well in a 96 well plate (corning, USA) and coated at 4°C overnight or at 37°C for 2 hours. After blocking by dispensing 200 μl of 2% skim milk/TBST (Tris-buffered saline-Tween 20), wash once with TBST, and as the primary antibody, 50 μl of hybridoma culture supernatant per well was dispensed at 37°C for 2 hours reacted. After washing 3 times with TBST, goat anti-mouse IgG (Pierce, USA) labeled with horseradish peroxidase (HRP) as a secondary antibody was diluted 5,000 times with 2% skim milk/TBST, and 50 μl per well was dispensed. The reaction was carried out at 37°C for 1 hour. After washing 5 times with TBST, 50 μl of tetramethylbenzidine base (TMB, colorreagents) (surmodics, TMBC) was dispensed per well for color development. After 50 μl of 1N H ₂ SO ₄ was added to each well to stop the color reaction, the absorbance was measured at 450 nm with a microplate photometer (Multiskan, USA).

As a result of screening the antibody generated from the Hybridoma prepared as described above by ELISA, finally 8 clones (1B3, 5G6, 11-2D9, 15E10, 15-1G9, 17F11, 24-1C6, 36D3) were selected. As described below, a pair test of the HIRRV rapid diagnostic kit was performed as in Experimental Example 1 using 8 clones, and the finally selected fusion cells, that is, hybridoma cells, were used as "HIRRV 11-2D9" and "HIRRV 15E10". They were named and deposited with the Korea Cell Line Research Foundation on May 4, 2021, and were given accession numbers KCLRF-BP-00513 and KCLRF-BP-00514, respectively.

Using the antibody produced from the deposited clone (the name of the antibody: use the same name as the clone), the specificity was checked, and as a result, it was confirmed that it was a monoclonal antibody that specifically binds to HIRRV. Thereafter, a HIRRV point-of-care diagnostic kit was prepared using the antibody produced from the deposited clone.

Example 2

The antigen recognition sites of 11-2D9 and 15E10 pairs of monoclonal antibodies that specifically bind to HIRRV were investigated by the following method.

1. SDS-PAGE

To analyze the structural protein of the purified virus, sodium dodecyle sulfate poly acrylamide gel electrophoresis (SDS-PAGE) was performed. SDS-sample buffer [1.59% SDS, 0.5 M Tris-HCl (pH 6.8), 14% glycerol (w/v), 0.01% bromophenol blue (w/v), 4% 2-mecaptoethanol ( v/v)] and heat-treated at 100° C. for 3 minutes, followed by SDS-PAGE (12% acrylamide separating gel, 4% acrylamide stacking gel, 30 mA, 2 hours). After electrophoresis, the gel was stained with 0.2% coomassie brilliant blue R-250 to confirm the results.

2. Westen blot

Western blot analysis was performed to confirm the recognition ability of the antibody against HIRRV. SDS-PAGE was performed in the same manner as above. After electrophoresis using purified HIRRV, the protein in the gel was blotted on a nitrocellurose membrane at 144 mA for 1 hour using a transblot device. After blotting, blocking with 2% skim milk and reacting for 1 hour, followed by reaction for 1 hour, washed with buffer 1 (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 15 minutes. As the primary antibody, the culture supernatant of the hybridoma cells of the monoclonal antibody used in this study was diluted twice with 2% skim milk, reacted for 1 hour, and washed in the same manner as above. As the secondary antibody, alkaline phosphatase (AP)-labeled goat anti-mouse IgG antibody was diluted 1,000-fold with 2% skim milk, allowed to react for 1 hour, washed 5 times, and a coloring agent [100 mM Tris-HClm 100 mM NaCl, 50 mM MgCl ₂ solution (pH 9.5) 20 ml, NBT (75 mg/ml 4-nitrotetrazolium blure chloride) 90 μl, BCIP (50 mg/ml 5-Bromo-4-choloro-3-indolyl phosphate p-toluidine salt/ dimethyl-formamide) 7 μl] and visually confirmed. After color development was completed, a color development stop solution (1 mM DETA, 10 mM Tris-HCl) was added.

The deposited hybridoma cells KCLRF-BP-00513 and KCLRF-BP-00514 and the monoclonal antibodies 11-2D9 and 15E10 produced therefrom, respectively, had high binding specificities for HIRRV antigen proteins M and N, respectively.

Example 3

After laminating the porous thin film member on the plastic supporting membrane member, the antibody conjugate member, the absorbent pad member, and the sample pad member are laminated at a predetermined position, and then the inspection line member and the control line member are formed to infect the halibut scab virus disease as follows. A point-of-care diagnostic kit was prepared.

1. Monoclonal Antibody Preparation

In Example 1, monoclonal antibodies 11-2D9 and 15E10 that specifically bind to HIRRV were prepared in pairs.

2. Preparation of porous thin film member, absorbent pad member and support member

For the porous thin film member, a nitrocellulose membrane having a size of about 4 mm and about 70 mm in width and length having a pore size of 5 to 12 μm was prepared, and for the absorbent pad member, cellulose (4 mm × 18 mm) was prepared. . As the support member, a commercially available strip-type plastic support membrane was prepared in the same size as the porous thin film member.

3. Preparation of the antibody conjugate member

① Preparation of antibody-labeled conjugate

The monoclonal antibody 11-2D9 prepared in Example 1 was added to a gold nanoparticle (40 nm) solution to confirm binding, and an antibody-labeled conjugate was prepared as follows. The reaction with monoclonal antibody 11-2D9 was accelerated while adding 0.2MK ₂ CO ₃ to the gold nanoparticles. When binding to the monoclonal antibody 11-2D9 was confirmed, it was reacted at 25°C for 2 hours, and then blocked by adding 10% bovine serum albumin (BSA). After washing with washing buffer by centrifugation at 10,000 rpm for 20 minutes, filtration through a 0.45 μm filter, monoclonal antibody 11-2D9-gold nanoparticle conjugate was stored at 4° C. until used in the experiment.

② Preparation of the antibody conjugate member

20 ml of the monoclonal antibody 11-2D9-gold nanoparticle conjugate prepared on a PE pad (4 mm × 6 mm) was dispensed per 20 cm × 30 cm size and dried for one day to prepare a gold pad as an antibody conjugate member.

4. Preparation of sample pad member

After dispensing 35 ml of Tris buffer (0.1 M Tris, pH 8.5) per 20 cm × 30 cm size on a glass fiber material (4 mmㅧ18 mm), spread evenly so that the buffer is sufficiently absorbed, and then dry the sample at room temperature for one day. The pad was prepared.

5. Production of on-site inspection diagnostic kit for halibut virus infection

A nitrocellulose film was laminated on the prepared strip-type plastic support film in a form of sandwiching the plastic support film. A gold pad was laminated with an adhesive to one end of the strip on which the nitrocellulose film was laminated, and an absorbent pad member was laminated to the other end with an adhesive. The prepared sample pad was laminated with an adhesive to almost cover the gold pad. After that, a test line member and a control line member were respectively formed on the surface of the nitrocellulose film exposed because the gold pad and the absorbent pad were not laminated. The test line member was formed by dispensing monoclonal antibody 15E10 (1 mg/ml) with a biodot micro-volume airjet at a location about 11 mm away from the gold pad and drying it. The monoclonal antibody contained in the test line member and control line member is formed by dispensing goat anti-mouse IgG (2.5 mg/ml) with a biodot micro-volume airjet at an interval of 5 mm from the test line member on the surface of the cellulose membrane and drying it. And the secondary antibody was fixedly formed on the surface of the nitrocellulose membrane so as not to move according to the flow of the liquid sample.

Example 4

Flounder spleen tissue obtained through infection experiment: POCT sample lysis buffer was mixed to a ratio of 1:9 (0.1 g: 900 μl), ground and left for 2 minutes, and the resulting supernatant was obtained as Specimen Sample Composition 1. Here, the composition of the POCT sample lysis buffer is as follows: 20 mM Tris, 25 mM NaCl, 2.5 mM EDTA (ethylene-diamine-tetraacetic acid), 0.05% sodium dodecyl sulfate (SDS), pH 8.0. The same applies to the following.

Example 5

Normal halibut spleen tissue [HIRRV (10 ^6.8 TCID ₅₀ /100 μl)]: POCT sample lysis buffer was mixed to be 1:9 (0.1 g: 900 μl) and ground, and then left for 2 minutes. got 2

Example 6

Normal halibut spleen tissue [HIRRV (2 × 10 ^6.55 TCID ₅₀ /100 μl)]: POCT sample lysis buffer 1:9 (0.1 g: 900 μl) [HIRRV (2 × 10 ^5.55 TCID ₅₀ /100 μl) diluted] After mixing and grinding, the resulting supernatant was left for 2 minutes to obtain a sample sample composition 3.

Example 7

Using sterilized forceps, halibut mucus was collected from the flounder body surface and diluted with sterile distilled water to prepare a flounder mucus buffer with a concentration of 10%.

Thereafter, the flounder mucus buffer was diluted to 1% flounder mucus concentration in POCT sample lysis buffer containing HIRRV 2×10 ^6.55 TCID ₅₀ /100 μl to prepare a buffer solution.

Normal halibut spleen tissue: After mixing and grinding the buffer solution to 1:9 (0.1 g: 900 μl) [diluted with HIRRV (2×10 ^5.55 TCID ₅₀ /100 μl), the upper layer obtained after leaving it for 2 minutes A liquid was obtained as a sample sample composition 4.

Comparative Examples 1 to 55

Comparative Examples Diagnostic Kits 1 to 55 were prepared in the same manner as in Example 2, except that a monoclonal antibody pair was used in the combination as shown in Table 1 below when manufacturing the diagnostic kit.

Experimental Example 1

In order to determine in which case the test sensitivity is better when the same monoclonal antibody or different monoclonal antibody pairs are used for the antibody-conjugating member and the test line member in the diagnostic kit for on-site inspection of the present invention, prepared in Example 2 After dropping 100 μl of HIRRV (10 ^8.5 TCID ₅₀ /100 μl) into the diagnostic kit for on-site inspection of flounder rhabdovirus infection and the diagnostic kits 1 to 55 of Comparative Examples 1 to 55, the results were obtained 10 minutes later. A pair test was conducted to confirm, and the results are shown in Table 2.

As shown in Table 2, as a result of the pair test, it can be seen that the diagnostic kit for on-site inspection of halibut rhabdovirus disease infection prepared in Example 2 shows the best sensitivity, so monoclonal antibodies 11-2D9 and 15E10 pair are the most effective. It can be confirmed that it is an excellent antibody pair.

Experimental Example 2

The antigen recognition sites of 11-2D9 and 15E10 pairs of monoclonal antibodies specifically binding to HIRRV were investigated, and the results are shown in FIG. 2 .

2 is a Western blotting result showing the HIRRV recognition loci of monoclonal antibodies [M: marker (kDa), 1: 11-2D9, 2: 15E10]

As can be seen from FIG. 2, the monoclonal antibodies 11-2D9 and 15E10 had high binding specificity to the antigenic proteins M and N of HIRRV, respectively.

Experimental Example 3

To investigate the sensitivity of the prepared point-of-care diagnostic kit, the HIRRV culture medium was diluted to confirm the sensitivity of the point-of-care diagnostic kit as follows, and the results are shown in FIG. 3 .

To investigate the sensitivity of the diagnostic kit, the HIRRV (10 ^7.5 TCID ₅₀ /100 μl) culture medium was diluted with a buffer (10 ^7.5 , 10 ^6.5 , 10 ^5.5 , 10 ^5.2 , 10 ^4.5 TCID ₅₀ /100 μl) in each kit. After dropping 100 μl each, the result was confirmed 10 minutes later.

As shown in FIG. 3 , as the test line appeared red up to a concentration of 10 ^5.2 TCID ₅₀ /100 μl, the diagnostic sensitivity of the rapid diagnostic kit could be determined as 10 ^5.2 TCID ₅₀ /100 μl.

Experimental Example 4

To investigate the specificity of the diagnostic kit for on-site inspection, the specificity of the diagnostic kit for on-site inspection was confirmed by using HIRRV culture medium and culture medium of five fish viruses (VHSV, IHNV, IPNV, MABV, NNV) as follows. and the results are shown in FIG. 4 .

To investigate the specificity of the diagnostic kit, HIRRV (10 ^7.5 TCID ₅₀ /100 μl) and [viral hemorrhagic septicemia virus (VHSV: 10 ^7.05 TCID ₅₀ /100 μl), infectious hmatopoietic necrosis virus, IHNV: 10 ^7.8 TCID ₅₀ /100 μl), infectious pancrearic necrosis (IPNV: 10 ^7.3 TCID ₅₀ /100 μl), marine birnavirus (MABV: 10 ^7.3 TCID ₅₀ /100 μl) , nervous necrosis virus (NNV: 10 ^7.55 TCID ₅₀ /100 μl)] The culture solution was dropped into the kit by 100 μl, and the results were checked 10 minutes later.

As shown in FIG. 4 , it was confirmed that a red line appeared on the test line in the HIRRV culture (positive), and a red line appeared on the test line in the culture of five fish viruses (VHSV, IHNV, IPNV, MABV, NNV). did not (voice). From this result, it was confirmed that the diagnostic kit for on-site inspection of halibut rhabdovirus disease infection produced in the present invention exhibits a specific response to HIRRV.

Experimental Example 5

The effectiveness evaluation and the virus infection value of the flounder rhabdovirus infection on-site inspection kit (rapid diagnostic kit) prepared in Example 3 were compared as follows with the sample sample composition 1 obtained in Example 4, and the results are shown in Table 3 shown in

1. Infection experiment

Infection experiments were conducted at 6° C. using flounder fry (about 25 g). After acclimatization of 20 animals in a 20 L tank, the experimental group was intraperitoneally infected with HIRRV (10 ^6.5 TCID ₅₀ /fish), and the control group was intraperitoneally injected with hanks' balanced salt solution (HBSS). Halibut were randomly sampled for 20 days after infection.

2. Infectious value measurement

Infectious values were measured using some of the spleen tissues of 20 halibut obtained through the infection experiment. To measure the infectivity, the spleen tissue was mixed with HBSS in a ratio of 1:9, ground, and left for 30 minutes to recover the obtained supernatant to prepare a virus sample. HIRRV-sensitive epithelioma papilosum cyprine (EPC) was subcultured and cultured as a monolayer in a 96-well cell culture plate, and the cells were attached to the bottom of the plate. The virus sample was diluted by a 10-fold serial dilution method using HBSS, and the cytopathic effect (CPE) was observed after inoculating the cells with 50 μl of each diluted virus solution. TCID ₅₀ was calculated by the method of Reed and Muench.

3. On-site inspection diagnostic kit test

Spleen tissue of the same individual as the virus infectivity value measurement for 10 out of 20 halibut obtained through the infection experiment: 100 μl of each sample composition 1 obtained in the same manner as in Example 4 using POCT sample lysis buffer was dropped into the on-site inspection diagnostic kit and the results were checked 10 minutes later. If a red line appeared on the test line, it was judged to be positive.

^* On-site inspection diagnostic kit detection sensitivity: 10 ^5.2 TCID ₅₀ /100 μl

Table 3 above shows the results obtained by comparing the virus infection value and the point-of-care diagnostic kit using the spleen tissues of 10 halibut obtained through the infection experiment. From Table 3, the spleen tissue infected with HIRRV showed an infectivity value of 10 ^{5.55 - 6.8} TCID ₅₀ /100 μl, and the point-of-care diagnostic kit showed positive values in subjects with an infectivity value of 10 ^6.3 TCID ₅₀ /100 μl or more, but 10 ^6.05 It can be seen that the TCID _50/100 μl or less showed negative results.

Therefore, although the detection sensitivity of the point-of-care diagnostic kit is 10 ^5.2 TCID ₅₀ /100 μl or more, the sample with an infectivity value of 10 ^{5.55 - 6.05} TCID ₅₀ /100 μl shows a negative result, and the sample composition is a liquid sample derived from spleen tissue. It was confirmed that some false negatives were exhibited in the case of including only .

Experimental Example 6

Among the organs of HIRRV-infected halibut, the most experimentally distributed HIRRV is the spleen tissue. The non-distributed brain and heart and reactivity to the point-of-care diagnostic kit as follows were investigated, and the results are shown in Table 4.

Each of 10 normal flounder organs (brain, spleen, heart) and HIRRV culture medium (10 ^6.8 TCID ₅₀ /100 μl POCT sample lysis buffer) were 1:9 (0.1 g:900 μl) [HIRRV (10 ^5.8 TCID ₅₀ ) /100 μl)), mixed and ground, and then, 100 μl of the obtained supernatant was dropped into a point-of-care diagnostic kit, respectively, and the results were checked 10 minutes later. If a red line appeared on the test line, it was judged as positive, and if there was no red line on the test line, it was judged as negative. Here, the supernatant containing the spleen tissue is the sample sample composition 2 obtained in Example 5.

^* On-site inspection diagnostic kit detection sensitivity: 10 ^5.2 TCID ₅₀ /100 μl

Table 4 shows the results of comparing the response between the on-site inspection diagnostic kit and flounder organs (brain, spleen, heart). 10 normal flounder organs (brain, spleen, heart) and HIRRV culture medium + POCT sample lysis buffer were used for 100 μl (10 ^5.8 TCID ₅₀ /100 μl) each. 100% [10/10 (number of positive samples/total number of samples)] were positive, but 30% (3/10) of spleen tissues were positive. In fact, compared to the brain and heart tissue, which is hardly distributed even when flounder is infected with HIRRV, the spleen tissue contains a large number of red blood cells, hematopoietic cells, and spleen parenchyma cells, so even if HIRRV is widely distributed, antigen- It is predicted that the detection sensitivity will be lowered even in point-of-care diagnostic kits with a detection sensitivity of 10 ^5.2 TCID ₅₀ /100 μl or more by inhibiting the antibody reaction. Therefore, it can be confirmed that the removal of these factors is necessary in order to accurately diagnose halibut rhabdovirus disease by improving the sensitivity of the point-of-care diagnostic kit.

Experimental Example 7

The effectiveness of the point-of-care diagnostic kit was investigated by varying the dilution factor of the POCT sample lysis buffer in the flounder spleen tissue as follows. In the control group, the ratio of the spleen tissue to the POCT sample lysis buffer was 1:9 (tissue: POCT sample lysis buffer), and the experimental group was 1:19. As a control, the HIRRV culture medium (2×10 ^6.55 TCID ₅₀ /100 μl POCT sample lysis buffer) was 1:9 (0.1 g: 900 μl) in the spleen tissue of 10 normal halibut [HIRRV (2×10 ^5.55 TCID ₅₀ ) /100 μl)], and after grinding, 100 μl of the supernatant was dropped into the rapid diagnosis kit, and the result was checked 10 minutes later. Here, the control is the sample sample composition 3 obtained in Example 6 as the supernatant containing the spleen tissue. In the experimental group, the HIRRV culture solution (2×10 ^6.55 TCID ₅₀ /100 μl POCT sample lysis buffer) was 1:19 (0.1 g: 1,900 μl) in the spleen tissue of 10 normal halibut (the same object as the control group) [HIRRV ( 10 diluted with ^5.55 TCID ₅₀ /100 μl)], mixed and ground, 100 μl of the supernatant was dropped into the rapid diagnosis kit, and the result was checked 10 minutes later. If a red line appeared on the test line, it was judged as positive, and if there was no red line on the test line, it was judged as negative.

Table 5 shows the results of comparing the reaction according to the dilution factor of the POCT sample lysis buffer using the on-site inspection diagnostic kit.

^a Tissue: POCT sample lysis buffer dilution factor of 1:9 Infectious value: 2×10 ^5.55 TCID ₅₀ /100 μl

^b Tissue: POCT sample lysis buffer dilution factor 1:19 Infectious value: 10 ^5.55 TCID ₅₀ /100 μl

Table 5 investigates the reaction according to the dilution factor of the POCT sample lysis buffer under two conditions. The spleen tissue of 10 halibut and the dilution factor of the POCT sample lysis buffer are 1:9 (2×10 ^5.55 TCID ₅₀ /100 μl) was confirmed to be 20% (2/10 rats) positive in , and 80% (8/10 rats) was confirmed to be positive when the dilution factor was 1:19 (10 ^5.55 TCID ₅₀ /100 μl). This is because as the dilution factor increases, the factors that inhibit antigen-antibody reaction existing in the tissue are also diluted, and it is predicted that the positive reaction of the point-of-care diagnostic kit is higher under the dilution condition of 1:19 than at the dilution condition of 1:9. . Therefore, when the dilution factor of the sample and POCT sample lysis buffer was 1:19, false negatives were reduced compared to 1:9, but false negatives were not completely eliminated.

Experimental Example 8

By giving a change (addition of halibut mucus) to the composition of the POCT sample lysis buffer, that is, using the sample composition 4 obtained in Example 7, the effectiveness of the point-of-care diagnostic kit was investigated as follows. As a control, the sample composition 3 using only the POCT sample lysis buffer obtained in Example 6 was used, and as the experimental group, the sample composition 4 obtained in Example 7 was used.

Using 10 normal flounder spleen tissues, 100 μl of Specimen Sample Composition 3 and Specimen Sample Composition 4 prepared by the methods of Examples 6 and 7, respectively, were dropped into a point-of-care diagnostic kit, and the results were checked 10 minutes later. did. If a red line appeared on the test line, it was judged as positive, and if there was no red line on the test line, it was judged as negative.

Table 6 below shows 10 sample composition 3 containing spleen tissue and POCT sample lysis buffer, and 10 sample composition 4 containing spleen tissue, POCT sample lysis buffer and halibut mucus using a point-of-care diagnostic kit. The results of comparing the effectiveness of on-site inspection diagnostic kits are shown.

^* Point-of-care diagnostic kit detection sensitivity: 10 ^5.2 TCID ₅₀ /100 μl

Table 6 shows that sample composition 3 containing only spleen tissue and POCT sample lysis buffer showed a positive reaction in 20% (2/10 animals), whereas specimen composition 4 containing spleen tissue, POCT sample lysis buffer and halibut mucus 4 was positive in 100% (10/10 animals).

From Table 6, it can be seen that when the flounder mucus is further included in the sample composition, false negatives appearing when only the spleen tissue and POCT sample lysis buffer are present are completely eliminated.

That is, when halibut mucus is included in the sample composition of the present invention, lysozyme, immunoglobulin, lectin, glycosaminoglycans, complement, etc. contained in the flounder mucus present in the spleen tissue are antigen-antibody reaction inhibitory factors, such as a large number of red blood cells and hematopoietic cells. This is because it can be seen that the sensitivity of the point-of-care diagnostic kit is improved by improving the antigen-antibody reaction that occurs in the point-of-care diagnostic kit, despite parenchymal cells of the spleen, etc.

From the above experimental results, the present invention develops novel monoclonal antibodies 11-2D9 and 15E10 that specifically bind antigen proteins M and N of hirame rhabdovirus (HIRRV) and hybridoma cells producing them, and , using this pair of monoclonal antibodies, not only produced a point-of-care diagnostic kit that enables rapid on-site diagnosis, but also confirmed that the spleen tissue was the most distributed HIRRV tissue when halibut was infected with HIRRV. Some false negatives were found as a result of preparing and using the sample composition containing the sample composition in the developed on-site inspection diagnostic kit. By changing the composition of the sample sample composition, even a slight false negative was resolved, that is, even when the infectivity value was ^6.05 TCID ₅₀ /100 μl or less. It can be seen that the sensitivity is improved so that the test diagnostic kit can accurately detect it. Therefore, the method for diagnosing halibut rhabdovirus disease on-site using the flounder rhabdovirus infection field inspection diagnostic kit of the present invention can be used quickly and immediately in the field of halibut farming, so Prevention and countermeasures will make a significant contribution to safe halibut farming.

Although the present invention has been illustrated and described with reference to preferred embodiments as described above, it is not limited to the above-described embodiments, and those of ordinary skill in the art to which the present invention pertains within the scope not departing from the spirit of the present invention Various changes and modifications will be possible.

Korea Cell Line Research Foundation KCLRFBP00513 20210504 Korea Cell Line Research Foundation KCLRFBP00514 20210504

Claims (12)

Specimen sample composition for on-site inspection diagnostic kit for halibut rhabdovirus disease infection, characterized in that it contains a liquid sample derived from flounder spleen tissue and flounder mucus.
delete The method of claim 1,
The flounder mucus is contained in 0.5 to 3% by volume, Specimen sample composition for an on-site inspection diagnostic kit for infection with halibut rhabdovirus disease.
A step of contacting the sample composition of claim 1 or 3 with a field test diagnostic kit for flounder rhabdovirus infection; and detecting a positive reaction of the test line member of the diagnostic kit, wherein the diagnostic kit includes:
a strip-shaped porous thin film member;
an absorbent pad member formed at one end in the longitudinal direction of the porous thin film member;
A monoclonal that is laminated on the other longitudinal end of the porous thin film member and is produced by hybridoma cells deposited with accession number KCLRF-BP-00513 and specifically binds to hirame rhabdovirus (HIRRV) Antibody conjugate pad member containing antibody 11-2D9;
a sample pad member laminated on some or all regions of the antibody conjugate pad member;
Flounder wraps produced by hybridoma cells deposited with an accession number KCLRF-BP-00514 and formed with a predetermined width on a surface positioned between the antibody conjugate pad member and the absorbent pad member among the surfaces of the porous thin film member a test line member to which the monoclonal antibody 15E10 specifically binding to hirame rhabdovirus (HIRRV) is immobilized; and
and a control line member formed with a predetermined width on a surface of the surface of the porous thin film member positioned between the test line member and the absorbent pad member and to which the secondary antibody binding to the monoclonal antibody is fixed. A method for on-site diagnosis of halibut rhabdovirus disease.
5. The method of claim 4,
Flounder rhabdovirus disease site, characterized in that after contacting the sample composition with the sample pad member of the diagnostic kit, a positive reaction of halibut rhabdovirus disease is detected within 20 minutes to detect whether halibut rhabdovirus infection is present diagnostic method.
delete 5. The method of claim 4,
The monoclonal antibody 11-2D9 is an antibody-label conjugate formed by conjugation with a label, characterized in that it is formed on the antibody conjugate pad member to be movable according to the flow of the liquid sample passing through the sample pad member. A method for on-site diagnosis of rhabdovirus disease.
8. The method of claim 7,
The label is Horseradish peroxidase (HRP), alkaline phosphatase, gold nanoparticles, poly L-lysine-fluorescein isothiocyanate (FITC), Rhodamine-B-isothiocyanate (RITC), Cy3, Cy5, and rhodamine. A method for on-site diagnosis of halibut rhabdovirus disease, characterized in that it is any one selected from the group consisting of.
5. The method of claim 4,
The diagnostic sensitivity of the test line member is 10 ^5.2 TCID ₅₀ /100 μl.
5. The method of claim 4,
The secondary antibody fixed to the control line member is an anti-mouse IgG derived from a group of mammals other than mice.
5. The method of claim 4,
The porous thin film member is any one selected from the group consisting of a nitrocellulose film, a PVDF film, a polyvinyl resin film, or a polystyrene resin film, and has a pore size of 5 to 12 µm. .
5. The method of claim 4,
The flatfish rhabdovirus disease on-site diagnosis method further comprising a strip-shaped support member formed on the lower surface of the porous thin film member.

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