KR20190138442A - Antibody detecting Infectious pancreatic necrosis virus and use thereof - Google Patents

Abstract

The present invention provides an antibody specifically binding to infectious pancreatic necrosis virus (IPNV); a composition and a kit for diagnosing IPNV infection and comprising the antibody; and a method for detecting IPNV using the same.

Description

Antibody detecting Infectious pancreatic necrosis virus and use

An antibody for detecting infectious pancreatic necrotic virus and a use thereof.

Infectious pancreatic gangrene virus (IPNV) is a virus belonging to the Birnaviridae family and causes acute infectious diseases in several species, including marine fish, mollusks, and shellfish. In particular, it is known to cause infectious pancreatic necrosis in salmon and rainbow trout among various cultured fish species, and the virus has a great influence on the aquaculture industry because it shows a wide spread.

Infection of infectious pancreatic necrosis virus occurs easily in fry of less than 1 g or 8 weeks of age, causing the virus to penetrate into living tissue and destroy cells, eventually leading to death. Infection, the body color darkens at the beginning of the infection and is accompanied by symptoms such as bloating, bulging, and white feces.At the end of the infection, turning movements and sudden death occur, anatomically causing pancreatic, esophagus and stomach ulcers. The intestines are filled with clear mucus.

On the other hand, due to the characteristics of fish that live in groups, and the culture environment in which many fish are farmed in a narrow space, the infection of the infectious pancreatic necrosis virus is very likely to spread to the colony, so whether or not it is infected at an early stage of infection Prompt diagnosis should prevent the spread of disease. Under such technical background, there is a need for a technique capable of quickly and accurately determining whether an infectious pancreatic necrosis virus is infected in a suspicious subject, but it is still inadequate.

One aspect is to provide antibodies for diagnosing infectious pancreatic necrotic virus infection that specifically bind to the infectious pancreatic necrotic virus.

Another aspect is to provide a composition for diagnosing infectious pancreatic necrotic virus infection of an individual, comprising the antibody or antigen-binding fragment thereof.

Another aspect is to provide a kit for diagnosing infectious pancreatic necrotic virus infection, comprising the antibody or antigen-binding fragment thereof.

Another aspect includes contacting a sample isolated from an individual with the antibody or antigen-binding fragment thereof; And it provides a method for detecting an infectious pancreatic necrosis virus comprising the step of detecting a complex formed by binding to the antibody or antigen-binding fragment thereof.

One aspect provides an antibody for diagnosing an infectious pancreatic necrotic virus infection that specifically binds to an infectious pancreatic necrotic virus.

As used herein, the term “antibody” refers to an antibody specific for infectious pancreatic gangrene virus, including a complete antibody, antigen-binding fragment of an antibody molecule, synthetic antibody, recombinant antibody, or antibody hybrid. can do. On the other hand, sequence information regarding the infectious pancreatic gangrene virus is already known in the art. Such antibodies include monoclonal antibodies, bispecific antibodies, non-human antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-binding Fv (sdFv) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the antibodies.

As used herein, the term "specifically binding" has the same meaning as commonly known to those skilled in the art, and the antigen and the antibody may specifically interact to form an antigen-antibody complex, and also may be immune It may mean to make a chemical reaction.

The complete antibody has a structure having two full length light chains and two full length heavy chains, each light chain having a heavy chain constant region and a light chain constant region of a heavy chain and disulfide bond (SS-bond) antibody. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses gamma 1 (γ1), gamma 2 (γ2) , Gamma 3 (γ 3), gamma 4 (γ 4), alpha 1 (α 1) and alpha 2 (α 2). The constant region of the light chain may have kappa (κ) and lambda (λ) types.

As used herein, the term "monoclonal antibody" means that the antibody molecule is made to consist of a single molecule. Monoclonal antibodies have specificity that responds only to antigens with the same epitope, and also show affinity only for specific epitopes.

In one embodiment, the antibody may be produced in the hybridoma cells described in the Examples.

The hybridoma cells can be prepared using methods known in the art. Specifically, the hybridoma cells are immunized with an infectious pancreatic necrosis virus (Whole virus) as an immunogen to the animal, and the hybridoma cells are prepared by fusing B cells, which are antibody-producing cells derived from the immunized animal, with myeloma cells. Next, it can be prepared by a method of selecting hybridomas that produce monoclonal antibodies that specifically bind to infectious pancreatic necrosis virus. The immunized animal can use an animal such as goat, sheep, morphot, rat or rabbit as well as the mouse used in the examples.

As a method for immunizing the immunized animal, a method already known in the art may be used. For example, when immunizing mice, 1 to 100 µg of an immunogen is emulsified at once with an adjuvant such as physiological saline and / or Freund's adjuvant to subcutaneously abdomen of the immunized animal. Or 2-6 inoculations every 2-5 weeks intraperitoneally. After immunizing the immunized animals, spleens or lymph nodes are extracted 3-5 days after the final immunization, and according to cell fusion methods already known in the art, B cells contained in their tissues in the presence of a fusion promoter are myeloma. To fuse with the cell. The fusion promoter may use a material such as polyethylene glycol (PEG), for example. The myeloma cells are described, for example, in P3U1, NS-1, P3x63. Mouse derived cells such as Ag 8.653, Sp2 / 0-Ag14, and rat derived cells such as AG1, AG2 can be used. In addition, the cell fusion method known in the art, for example, B cells and myeloma cells are mixed at a ratio of 1: 1-10: 1, to which a PEG having a molecular weight of 1,000-6,000 is added at a concentration of 10-80%. For example, the method may be performed by incubating at 30-37 ° C. for 1-10 minutes. In addition, hybridomas that produce monoclonal antibodies that specifically bind to the infectious pancreatic gangrene virus are cultured in a selective medium such as HAT medium, in which only hybridomas can survive, and hybridoma culture supernatants The antibody activity in the sample can be measured and selected using a method such as ELISA. Finally, hybridomas that produce monoclonal antibodies that specifically bind to infectious pancreatic necrosis virus are limited, for example, to hybridomas that produce monoclonal antibodies that specifically bind to infectious pancreatic necrosis virus. It can be selected by repeating cloning by a method such as dilution. Meanwhile, the monoclonal antibody may be of IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type.

Another aspect provides a composition for diagnosing infectious pancreatic necrotic virus infection of an individual, comprising the antibody or antigen-binding fragment thereof.

As used herein, the term "antigen binding fragment" refers to a portion of a polypeptide comprising a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin. For example, it can be F (ab ') 2, Fab', Fab, Fv or scFv. Fab in the antigen-binding fragment has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the heavy chain (C _H1 ). Fab 'differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art. Double-chain Fv is non-covalently linked to the heavy chain variable region and light chain variable region. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv. The antigen binding fragments can be obtained using proteolytic enzymes (for example, restriction digestion of the entire antibody with papain can yield Fab and cleavage with pepsin can yield F (ab ') ₂ fragment). In addition, the antigen-binding fragment can be produced through genetic recombination technology.

The composition may be provided in the form of a liquid composition comprising the antibody in a liquid. The liquid may be one capable of dissolving and maintaining the antibody. For example, it may be water or a buffer solution such as PBS, and the composition may further include a substance for keeping the antibody stable.

Another aspect provides a kit for diagnosing infectious pancreatic necrotic virus infection, comprising the antibody or antigen-binding fragment thereof.

The kit may further comprise a detectable moiety. A detectable moiety can include a moiety in which the presence, relative amount and / or location (eg, location on the array) of the moiety can be determined directly or indirectly. Such detectable moieties are well known in the art. For example, a detectable moiety can be detected when exposed to certain conditions, such as fluorescent moieties, luminescent moieties, chemiluminescent moieties, radioactive moieties (eg, radioactive atoms), and enzyme moieties. It may be selected from the group consisting of. For example, fluorescent moieties may need to be exposed to radiation at certain wavelengths and intensities that cause excitation of the fluorescent moieties, thereby detecting at specific wavelengths that can be detected. It can be made to emit as much fluorescence as possible.

The moiety may include metal nanoparticles. The metal nanoparticles may be gold particles. The gold nanoparticles may be red gold nanoparticles or blue gold nanoparticles. The gold nanoparticles may be in a dispersed state, for example colloid.

The kit is provided as a sample pad (1) which is a site to which a liquid sample containing an analyte is applied, and a storage pad (2) which is in fluid communication with the sample pad, and in which the conjugate of gold particles is movably supported. , The conjugate of the gold particles is a conjugate of the first antibody or fragment thereof and the gold particles,

A chromatographic membrane material (3) in fluid communication with the storage pad and to which the liquid sample is moved by capillary movement, wherein the second antibody or fragment thereof is immobilized downstream of the storage pad. A chromatographic membrane material comprising a detection zone;

A hygroscopic pad 4 in fluid communication with the chromatography membrane material; And

It may be provided in the form of a kit for detecting infectious pancreatic necrosis virus, which comprises the sample pad, the storage pad, the chromatographic membrane material and the solid support 5 supporting the hygroscopic pad. The kit may be an immunochromatographic detection device.

The first and second antibodies are, for example, monoclonal antibodies that specifically bind to infectious pancreatic necrosis virus, specifically mAb anti-IPNV 1A # 66, mAb anti-IPNV 1A # 82 according to one embodiment. It can be either. The first antibody and the second antibody may be different.

Figure 3 is a side view showing a kit for detecting pancreatic necrosis virus according to one embodiment, Figure 4 is a schematic diagram showing the detection principle of the kit for detecting pancreatic necrosis virus according to an embodiment.

The kit is supported by a chromatography material membrane (3) having a detection region (T) and a control region (C) on which a second antibody is immobilized on a solid support (5), wherein the specimen pad (1) The storage pad 2 and the moisture absorption pad on which the first antibody and the gold conjugate are supported are overlapped and connected. When the sample is added to the sample pad 1 in the strip, it moves to the storage pad 2 by capillary action so that the antigen in the sample binds to the conjugate of the gold and the first antibody in the storage pad, Capillary movement is made downstream through the direction of the moisture absorption pad (4). When the conjugate of the antigen and the conjugate reaches the detection region (T), the conjugate and the second antibody specifically bind and develop color, and in the absence of the conjugate, no color development occurs. In addition, when the conjugate of the first antibody and the gold particles together with the sample passes through the control region (C), the conjugate of the first antibody and the gold particles is specific to the first antibody and the gold particle conjugate in the control region. In combination with the color development, it is possible to check whether the capillary movement is properly performed.

The kit may be an immunoassay kit using a porous material as a solid carrier of an immunochemical component such as an antigen or an antibody. Sample pads, storage pads, chromatographic membrane materials and hygroscopic pads used in one embodiment of the invention are materials having a porosity and volume sufficient to contain and contain a liquid sample to be analyzed, for example microporous. May be a membrane material. Examples of such microporous membrane materials include nylon, cellulose materials, polysulfones, polyvinylidene difluorides, polyesters and glass fibers. Preferred examples of the microporous membrane material are nitrocellulose membranes. The analysis device may have the form of a strip.

As used herein, the term "in flow communication with" refers to a location (eg storage) due to capillary movement when a liquid sample is applied to one location (eg sample pad). Pads) that are available for use. In addition, the expression “movably supported” means that the conjugate of gold particles is immobilized to the storage pad movably with capillary movement of the liquid sample. In addition, the expression "nondiffusively immobilized" means that the second antibody or fragment thereof is immobilized so as not to diffuse together by capillary movement of the liquid sample in the detection region. The method for non-diffusion immobilizing the antigen and / or antibody can include any method as long as it is capable of non-diffusion immobilizing the chromatography membrane material and the antigen and / or antibody to the detection region of the chromatography membrane material. Examples include, but are not limited to, covalent bonding methods. The detection zone may take any shape, for example in the form of a rectangle and a circle, the area of which is smaller than the area of the chromatography membrane material.

Antibodies and gold conjugates used in the kits according to one embodiment may be prepared by methods well known in the art. For example, it can be prepared by making a gold colloidal solution and covalently binding the gold in the solution with the antibody. Antibodies and gold conjugates can be prepared, for example, by the methods disclosed in US Pat. Nos. 5,514,302 and 4,313,734, which are incorporated herein by reference in their entirety.

In addition, the kit can be used to diagnose infectious pancreatic necrotic virus infection by detecting infectious pancreatic necrosis virus according to an immunoassay method. Such immunoassays can be performed according to various immunoassays or immunostaining protocols developed in the prior art. The immunoassay or immunostaining format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining. One or more selected from immunoaffinity purification, immunochromatography, and combinations thereof.

For example, when the method of the present invention is carried out in accordance with radioimmunoassay methods, antibodies labeled with radioisotopes (eg, C14, I125, P32 and S35) may be used to detect infectious pancreatic necrosis virus. Can be.

In addition, for example, when the method of the present invention is carried out in an ELISA manner, certain embodiments include (i) coating an unknown cell sample lysate to be analyzed on the surface of a solid substrate; (Ii) reacting said cell lysate with a primary antibody against infectious pancreatic gangrene virus; (Iii) reacting the resultant of step (ii) with the secondary antibody to which the enzyme is bound; And (iii) measuring the activity of the enzyme.

Suitable as the solid substrate are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, and may be microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a color reaction, a fluorescence reaction, a luminescence reaction or an infrared reaction. For example, alkaline phosphatase, β-galactosidase, and horse radish fur Oxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as the enzyme binding to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT) naphthol-AS-B1-phosphate (naphthol-AS-) Chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-phosphate) and chromogenic reaction substrates such as enhanced chemifluorescence (ECF) are used and hose radish peroxidase is used. N-methylacridinium nitrate), resorupin benzyl ether, luminol, Amflex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), p-phenylenediamine-HCl and pyrocatechol (HYR), TMB ( tetramethylbenzidine), ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS ( substrates such as phenzaine methosulfate can be used .

When the method is carried out in a capture-ELISA mode, an embodiment of the present invention comprises the steps of: (i) coating the surface of a solid substrate with an infectious pancreatic necrotic virus antibody as a capturing antibody; (Ii) reacting the capture antibody with the sample; (Iii) reacting the product of step (ii) with a detection antibody (eg, an antibody or antigen-binding fragment of the invention) that has a label bound to generate a signal and that specifically reacts to an infectious pancreatic gangrene virus; And (iii) measuring the signal generated from the label.

The detection antibody carries a label which generates a detectable signal. Such labels may include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125, P32 and S35), fluorescent materials (eg, fluorescein), luminescent materials, chemiluminescent and fluorescence resonance energy transfer (FRET).

Measurement of the final enzyme activity or signal in the ELISA method and the capture-ELISA method can be carried out according to various methods known in the art. Detection of these signals allows for qualitative or quantitative analysis of infectious pancreatic gangrene virus infection. If biotin is used as a label, the signal can be easily detected with streptavidin and luciferin if luciferase is used.

Another aspect includes contacting a sample isolated from an individual with the antibody or antigen-binding fragment thereof; And it provides a method for detecting an infectious pancreatic necrosis virus comprising detecting a complex formed by binding to the antibody or antigen-binding fragment thereof.

The method for detecting the infectious pancreatic necrosis virus may include contacting a sample with the above-described antibody or antigen-binding fragment thereof. The contacting step comprises contacting the sample with a plate coated with the infectious pancreatic necrotic virus antibody or antigen-binding fragment thereof (referred to as 'primary antibody or antigen-binding fragment thereof'), followed by infectious pancreatic necrotic virus antibody or antigen thereof. The binding fragment (referred to as a 'secondary antibody or antigen binding fragment thereof') may further comprise contacting the plate. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be the same or different. The primary antibody or antigen-binding fragment thereof and the secondary antibody or antigen-binding fragment thereof may be bound to a detectable moiety. For example, the secondary antibody or binding fragment thereof may be one containing a detectable moiety such as gold. The primary and secondary antibodies or antigen-binding fragments thereof may each be mAb anti-IPNV 1A # 66, mAb anti-IPNV 1A # 82, or vice versa, according to one embodiment.

The method for detecting the infectious pancreatic necrosis virus may comprise detecting a complex formed by binding to the antibody or antigen binding fragment thereof. The detection can detect or quantify the infectious pancreatic necrosis virus by measuring a detectable moiety bound to the complex.

The sample may be a biological material derived from an individual. The subject may be a fish, for example, a fish of aquaculture species such as salmon, rainbow trout and the like. In addition, the sample may be a tissue sample derived from blood or a blood constituent, a bodily fluid, an organ, or may be, for example, a tissue sample derived from the liver, kidney, spleen, or the like. .

Another aspect includes the steps of contacting a sample with said antibody or antigen-binding fragment thereof; Detecting the complex formed by binding to the antibody or antigen binding fragment thereof; And it provides a diagnostic method for infectious pancreatic necrotic virus infection comprising the step of detecting whether the infectious pancreatic necrosis virus infection from the detection result.

The diagnostic method for infectious pancreatic necrosis virus infection may include determining that the individual from which the sample is infected is infected with an infectious pancreatic necrosis virus infection when a complex of an antibody or antigen-binding fragment thereof and an infectious pancreatic necrosis virus is detected. . In addition, when the complex is not detected, the subject from which the sample is derived may include determining that the infectious pancreatic necrosis virus is not infected.

According to an embodiment of the present invention, an antibody specifically binding to an infectious pancreatic necrosis virus, and a composition and kit for diagnosing an infectious pancreatic necrosis virus infection comprising the antibody may be used to efficiently detect whether an infectious pancreatic necrosis virus is infected. This can be useful for the diagnosis of related diseases.

1 is a result of detecting an infectious pancreatic necrosis virus having various virus titers using an IPNV detection kit according to one embodiment.
Figure 2 is a result of confirming the cross-reactivity of the kit for detecting IPNV according to one embodiment.
3 is a view schematically showing the configuration of an IPNV detection kit according to an embodiment.
4 is a diagram schematically illustrating a detection principle of an IPNV detection kit according to an embodiment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Monoclonal Antibodies for Infectious Pancreatic Necrosis Virus (IPNV) Detection

1-1. Preparation of Immunogen

Immunization antigen for the preparation of IPNV antibody was isolated pure virus from virus culture medium using an ultrafast centrifuge, and then immunogen was prepared as follows. The immunogen used for primary immunization was prepared by suspending 1: 1 Complete Freud's Adjuvant (Sigma) and the immunogen used for second- and fourth immunization was 1: 1 suspended with Incomplete Freud's Adjuvant (Sigma). Prepared.

1-2. immune

The immunogen prepared by the above method was immunized to BALB / c mice, 6 weeks old, females as follows. Intraperitoneally inoculated four times at a weekly interval of 200 ul / horse, and a week after the last intraperitoneal injection, the same antigen was diluted in 20% concentration (v / v) in phosphate buffered saline (PBS). 20 μl / time was injected into the tail vein three times daily. After the third intravenous injection, a medium titer test was performed with serum taken from the tail vein. According to the above results, mice obtained with sufficient amounts of antibodies were selected and subjected to cell fusion process.

1-3. Construction of Hybridoma Cell Lines

After IPNV immunization, splenocytes of the antibody-producing mice were removed and filtered through a 70um pore cell strainer (BD falcon). The filtered splenocytes were centrifuged to obtain a precipitate, 5 ml of red blood cell lysis buffer (Sigma) was mixed, and left for 1 minute to remove red blood cells. Splenocytes from which red blood cells were removed were placed in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) and washed three times, and cell counting was performed. As a cell to be fused with splenocytes, a mouse-derived myeloma cell SP2 / 0-Ag 14 (ATCC CRL-1581) cell line was used, and the mixing ratio was 10: 1 (splenocytes: SP2 / 0). The mixed cells were washed twice with DMEM and then these cells were fused using PEG1500 (Roche). Cell fusion was carried out with 1.7 ml of PEG1500 for 1 minute, 30 seconds of standing, 1ml of 1 minute of DMEM, 30 seconds of standing, 2ml of 1 minute of DEME, 6ml of DMEM for 30 seconds, and 10ml of DMEM for 30 seconds Was carried out. Centrifugation of fused cells was used to obtain precipitates and DMEM ('HAT medium') containing 10% of HAT (Gibco), antibiotics (Gibco), and fetal bovine serum (Fetal bovine serum, Hyclone) .), And then aliquoted into 96-well tissue culture plates at a dose of 200 μl / well and grown in incubator for 3 days.

1-4. Cell culture

The cell line after fusion was grown at 37 ° C., 5% carbon dioxide gas, and in a cell incubator maintained at humid conditions, and fused cell lines were selected by replacing HAT medium every two days.

1-5. Screening for Hybridoma Cells Producing Monoclonal Antibodies

Cell lines selected by HAT medium were screened for positive clones by enzyme-linked immunosorbent assay (ELISA). Specifically, 100 μl / well in a 96-well adsorption plate (Costar) coated with a concentration of 2.5 ug / ml of viral IPNV antigen purely separated from the fused cell line culture grown in each well of the 96-well tissue culture plate. It was dispensed at a dose of and reacted at 37 ° C for 1 hour. After reacting for 1 hour, the cells were washed five times, and the anti mouse IgG HRP conjugate and the anti mouse IgM HRP conjugate were dispensed at a dose of 100 μl / well and reacted at 37 ° C. for 30 minutes. After reacting for 30 minutes, it was washed 5 times, the substrate solution was dispensed at a dose of 100 μl / well for 15 minutes of color reaction, and the reaction stop solution of 100 μl / well was added. After stopping the reaction, the absorbance was measured at 450 nm in the reader to select high-positive positive clones IPNV 1A # 66, IPNV 1A # 82 clones. The selected positive clones were transferred to a 24-well tissue culture plate, incubated for 3 days, and subjected to a second search in the same manner as above. That is, limiting dilution of hybridoma cell lines obtained through repeated screening was finally performed on IPNV Single positive clones IPNV 1A # 66, IPNV 1A # 82 that specifically reacted were selected (the final selected monoclonal antibodies were designated mAb anti-IPNV 1A # 66, mAb anti-IPNV 1A # 82).

1-6. Mouse Revenge Collection

The final selected positive clones were transferred to T75 (SPL) dish and incubated for 3 days. Thereafter, 0.5 ml of mice were injected into the abdominal cavity of mice at a cell density of 1 × 10 ⁶ cells / ml, and ascites fluid was collected one week later.

1-7. Antibody purification

The mouse ascites liquid obtained through the above method was precipitated by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the supernatant was mixed with the binding buffer (Binding buffer, Thermo scientific) at a 1: 1 ratio. The reaction was carried out for 1 hour at. After 1 hour, the suspension was precipitated once again by centrifugation (3000 rpm, 10 minutes, 4 ° C.), and the unprecipitated suspension was filtered again using a filter having a 1.2 μm pore size. Ascites fluid was completely removed from the suspension was placed in a column containing Protein G resin (Pierce) to induce binding between the antibody and Protein G. After passing through a plurality of columns to the column washed three times with phosphate buffer solution and eluted using an elution buffer (Elution buffer, Thermo scientific). The eluted antibody was again applied to PD-10 column (GE heathcare) and eluted with phosphate buffer solution for final purification.

Example 2. Detection Experiment of Infectious Pancreatic Necrosis Virus

2-1. Preparation of the kit for IPNV detection

The mAb anti-IPNV 1A # 66 prepared above was immobilized in the detection region (T) of the kit shown in FIG. 3. Meanwhile, mAb anti-IPNV 1A # 82 was conjugated with gold particles in the following manner to prepare a detection solution. First, Gold chloride was dissolved in distilled water at 0.01%, heated for about 10 minutes with the addition of 0.1% sodium citrate solution, and then cooled and refrigerated. Then, the conjugation reaction was induced by reacting the colloidal gold solution for 10 minutes so that the mAb anti-IPNV 1A # 82 became the final concentration of 20 µg / ml. After stabilizing the gold particles, centrifugation was produced at 10,000 g for 30 minutes. The precipitate was again dissolved in physiological saline, filtered with a 0.45 μm filter, and the absorbance was measured at 540 nm and diluted to a final value of 20. The solution was then added to the storage pad 2 of the kit shown in FIG.

2-2. IPNV Detection

Using a kit prepared in Example 2-1 was detecting the infectious pancreatic necrosis virus, subject to a TCID ₅₀ is the measured _{^{IPNV (6.67 × 10 6 TCID 50}} / ml) broth and dilutions thereof were used. Experimental results are as shown in Table 1 and FIG.

IPNV Ag NO Virus titer Dilution Color scale (%) One 6.67 × 10 ⁶ TCID ₅₀ / ml 2 ⁰ 40 2 3.33 × 10 ⁶ TCID ₅₀ / ml 2 ^-1 34 3 1.66 × 10 ⁶ TCID ₅₀ / ml 2 ^-2 20 4 0.83 × 10 ⁶ TCID ₅₀ / ml 2 ^-3 10 5 4.16 × 10 ⁵ TCID ₅₀ / ml 2 ^-4 7-10 6 2.08 × 10 ⁵ TCID ₅₀ / ml ^2-5 5 7 1.04 × 10 ⁵ TCID ₅₀ / ml 2 ^-6 3 8 0.52 × 10 ⁵ TCID ₅₀ / ml ^2-7 One 9 0.02 × 10 ⁵ TCID ₅₀ / ml ^2-8 ± 10 Buffer (Control) 0 0

As described above, the band was not detected in the case of the negative sample (Control, # 10) as a result of detection using the kit for IPNV detection, whereas from the virus titer of 0.52 × 10 ⁵ TCID ₅₀ / ml or more Bands indicating detection of the virus were identified.

2-3. Cross Reactivity Check

Using the kit prepared in Example 2-1, the cross-reactivity of the antibody was confirmed. Samples to check for cross-reactivity include the Burnavirus family IPNV, Viral haemorrhagic septicemia virus (VHSV), Infectious hematopoietic necrosis virus (IHNV), and Hira Merabdovirus (Hirame rhabdovirus: HIRRV) was used.

As a result, as shown in FIG. 2, as in the past, the IPNV detection kit could easily detect IPNV through an antigen-antibody reaction, but it did not show any response to other rhabdoviruses and viruses. .

Claims (9)

Antibodies for diagnosing infectious pancreatic necrotic virus infection, specifically binding to infectious pancreatic necrosis virus (IPNV).
The antibody of claim 1, wherein the antibody is a monoclonal antibody.
A composition for diagnosing infectious pancreatic necrotic virus infection of an individual, comprising the antibody of claim 1 or an antigen-binding fragment thereof.
The diagnostic composition of claim 3, wherein the individual is a fish.
Kit for diagnosing infectious pancreatic necrotic virus infection of an individual, comprising the antibody of claim 1 or an antigen-binding fragment thereof.
The diagnostic kit of claim 5, wherein the subject is fish.
Contacting the antibody of claim 1 or a sample isolated from the individual with an antigen-binding fragment thereof; And
Detecting the complex formed by binding the antibody or antigen-binding fragment thereof.
The method of claim 7, wherein the subject is a fish.
The method of claim 7, wherein the sample is a tissue-derived tissue sample.

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