KR20180023250A - Monoclonal antibody against immunoglobulin m of olive flounder and use thereof - Google Patents

Abstract

The present invention provides a fusion cell line of Accession No. KCLRF-BP-00368 which produces a monoclonal antibody specific to immunoglobulin M of Paralichthys olivaceus, and a monoclonal antibody Anti-24B12 produced therefrom, and also provides a detection kit capable of detecting an antigen of fish by using the same, thereby being used for the diagnosis, treatment and prevention of infectious diseases of Paralichthys olivaceus, which has caused massive deaths in farms and caused enormous damage.

Description

MONOCLONAL ANTIBODY AGAINST IMMUNOGLOBULIN M OLIVE FLOUNDER AND USE THEREOF <br> <br> <br> Patents - stay tuned to the technology MONOCLONAL ANTIBODY AGAINST IMMUNOGLOBULIN M OLIVE FLOUNDER AND USE THEREOF

The present invention relates to a monoclonal antibody specific to immunoglobulin (Ig) M of a flounder ( Paralichthys olivaceus ) and a use thereof, and more particularly, to a fusion cell line which separates IgM of a flounder and produces a monoclonal antibody specific thereto The present invention relates to a method for detecting an antibody contained in a serum of a flounder using a monoclonal antibody produced herein.

Along with the revitalization of the fish breeding industry worldwide, the damage caused by fish diseases has become a major problem in the aquaculture industry. In particular, the mortality rate due to viral diseases is expected to account for more than 30% of the fish production. However, as there is no effective treatment until now, the rapid diagnosis and preventive measures of the disease remain a major challenge.

In order to systematically manage marine infectious diseases and to minimize the damage caused by diseases, the Ministry of Agriculture, Forestry and Fisheries has enforced the Marine Biological Disease Control Act in 2008 and carried out quarantine and quarantine for 21 infectious diseases. The list is divided into preventive guideline for each disease occurrence situation, and stepwise prevention measures such as disposal, quarantine, and movement restriction are carried out.

Among these aquatic organisms that are subject to quarantine and quarantine, virus disease accounts for 67%. In Korea, the method of diagnosis of viral infectious diseases used in quarantine and prevention is mainly performed by PCR method targeting viral genome . In addition, the isolation culture method and the histopathological method using fish coin cells are performed in parallel. In case of fish, PCR method is mainly used, and shellfish and crustacean are combined with PCR method and histopathological method. However, immunological methods are rarely used because they do not yet have specific antibodies against the hospital virus.

The PCR-based diagnosis method is widely used worldwide as a virus detection method because it can detect rapidly. However, it can be used for quarantine of aquatic products from foreign countries, epidemiological investigation in aquaculture field, pathological emotion, There is a limit to apply in general in the field. In other words, since the PCR method is a method of directly detecting a pathogen, it is possible to perform an inspection only by killing a marine organism having a high commercial value such as Nishikigoi. Also, it is difficult to process a large number of samples at once, and it is disadvantageous that a large number of samples are expensive to be inspected. In addition, since the PCR test targets the gene, the gene is detected even if the pathogen is inactivated, and it is difficult to accurately diagnose if the virus is mixed with two or more viruses.

In contrast, immunological diagnosis is a method of diagnosing pathogen infection using antibodies. Immunological diagnosis includes direct detection of pathogens using specific antibodies of pathogens and indirect detection of pathogen infection by detecting specific antibodies present in the blood.

Immunological diagnosis by direct method is a method of directly detecting pathogens and has a rapidity to test disease within 6 hours. It is also highly sensitive and specific, and it is easy to trace pathogens in the fish body, and the infected cells and the degree of infection can be checked. Once a specific antibody against a pathogen is produced stably, diagnosis can be easily diagnosed at aquaculture site through the production of a diagnostic kit.

Indirect immunoassay is a method to detect specific antibodies in the blood. It can treat a large number of samples at low cost. It has high detection sensitivity and reliability of results. Therefore, in human and livestock, Monitoring, and verifying the efficacy of vaccines.

Immunological diagnosis by indirect method does not need to kill the organism to be tested, and it can be diagnosed only by a very small amount of serum. In addition, it is possible to rapidly diagnose a large number of samples at a low cost. By using the antibody detection ELISA method, more than 300 samples can be processed at once, and 15 times more samples can be processed at a cost about 100 times lower than that of PCR. In addition, it is possible to diagnose infectious infectious diseases and to detect the history of virus infection, and it is useful for determining efficacy after determination of vaccine efficacy and prevention.

In order to use this indirect immunological assay, specific antibodies against fish IgM are required. However, fish antibodies are known to be composed of low affinity IgM, unlike mammalian IgG. In case of applying ELISA for detection of fish, high background is caused by nonspecific adsorption of fish IgM and blocking agent Problems that do not occur in mammals are reported. For this reason, the International Bureau of OIE (OIE) has not accepted antibody detection ELISA as a diagnostic method because of its low reproducibility.

However, in recent studies, a method for suppressing nonspecific adsorption of fish IgM and blocking agent has been proposed. Therefore, it is urgent to use low specific adsorption with blocking agent in ELISA, and to produce and isolate specific antibody against fish IgM for diagnosis of aquatic organism hospital infection.

In Korean Patent Laid-Open No. 10-2008-0008802, immunoglobulin (Ig) isolated and purified from Sebastes schlegeli and a method for producing the same are disclosed. The monoclonal antibody against immunoglobulin isolated / purified from Sebastes schlegeli And a method for producing the same, a monoclonal antibody against a purified immune globulin, and a method for producing the monoclonal antibody against immune globulin, which provides useful information necessary for vaccine development. Korean Patent No. 10-1414009 discloses a vaccine composition for preventing fish viral neurotoxicity comprising a transgenic plant or an extract thereof containing a gene encoding a coat protein derived from Nodavirus, a fish virus comprising the vaccine composition A feed additive for preventing sexual neurotoxicity and a method for preventing fish viral neurotoxicity comprising orally administering the vaccine composition or the feed additive to fish. Domestic Patent No. 10-0257319 discloses a fusion cell line IP324 of a mouse and a method for producing the same, which can fundamentally prevent IPNV from infecting a host, regardless of the serotype thereof, thereby reducing enormous economic loss of aquaculture caused by massive deaths And can be used as a tool for researching the mechanism of neutralizing IPNV receptors and viruses, as well as being able to fundamentally block the ongoing annual damage of IPNV. Infectious pancreatic necrosis virus A monoclonal antibody against an antigenic protein of the present invention and a preparation method thereof. Korean Patent No. 10-1273264 discloses a polypeptide comprising an epitope of a fish nodavirus protein, a recombinant vaccine using the same, a method of immunizing a fish with the polypeptide, and a method of promoting fish nodavirus disease. However, the above-mentioned inventions are different from the present invention in providing the monoclonal antibody specific for immunoglobulin M and its use in its constitution and effect.

The present invention relates to a fusion cell line which produces a monoclonal antibody against IgM for the purpose of easily diagnosing, treating and preventing an infectious disease of a flounder, a typical cultured fish of Korea, and a monoclonal antibody of the flounder IgM produced thereby .

The present invention provides a fusion cell line 24B12 of Accession No. KCLRF-BP-00368, which produces a monoclonal antibody specific for the immunoglobulin M of flounder, and the monoclonal antibody Anti-24B12 produced therefrom, and uses this to diagnose the immunoglobulin of the flounder The present invention provides a detection kit and a detection method that can be used. Wherein the fish antigen is selected from the group consisting of fish nerve necrosis virus, fish viral hemorrhagic septicemia virus, flounder lavovirus, lymphocystis, streptococcus, Edwards bacterium, Vibrio sp. An antigen, an attenuated pathogen, an inactivation pathogen, a recombinant protein, and an epitope that is an epitope (antigen).

The detection kit of the flounder that recognizes the fish antigen is a kit for detecting a specific antibody of a flounder against a fish antigen that detects an antigen-antibody complex by a color particle binding method, and the detection kit comprises a marker which reacts with a substrate, Conjugated antibodies; And a chromogenic substrate, wherein the marker is Horse-radish peroxidase (HRP) and the chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl-benzidine) Specific antibody detection kit.

(1) diluting the fish antigen with a coating buffer solution and dispensing the same on a plate to coat the plate; (2) a step of allowing the plate of (1) to react with the serum of the serum to be examined; (3) washing the plate of (2) with a phosphate buffer solution; A step (4) of reacting a conjugate of an enzyme with a monoclonal antibody Anti-24B12 specific to a flounder immunoglobulin M produced by a fusion cell line of Accession No. KCLRF-BP-00368 on the plate of the above (3); Adding (5) the coloring substrate of (4) to color; (6) stopping the enzyme reaction with sulfuric acid when the coloring reaction of (5) is over; And (7) measuring the absorbance at 405 nm of the color development degree of (6). The present invention also provides a method for detecting an antigen using the immunoglobulin monoclonal antibody of flounder.

The present invention provides a diagnostic kit and a vaccine effective for diagnosis, treatment and prevention of infectious diseases of flounder by providing a fusion cell line producing a monoclonal antibody specific for the flounder's IgM and a monoclonal antibody specific for the IgM of the flounder produced thereby And thus contribute to efficient and stable aquaculture.

Figure 1 is an SDS-PAGE photograph of purified flounder IgM.
FIG. 2 is a table showing the results of isotyping of the monoclonal antibody (24B12) against a flounder IgM (PL IgM) purified using a Protein L affinty column.
Figure 3 shows ELISA results showing the reaction of monoclonal antibodies against PL IgM and MBP IgM.
4 is a graph showing the results of detection of a specific flounder antibody against BSA using a monoclonal antibody.
Figure 5 shows the haze of a monoclonal antibody of the present invention.

The present invention relates to a method of isolating and isolating Immunoglubulin M (IgM), an immunological protein of flounder, and producing a monoclonal antibody having specificity thereto, to provide a method for diagnosis and treatment and prevention of pathogenic diseases in fish, Monoclonal antibodies and uses thereof. Hereinafter, the present invention will be described in detail with reference to specific examples.

Example 1 Serum Separation of Flounder

The flounder serum used in the experiment was collected from the arrhythmia of the flounder (weight: 1-1.5 kg) and the flounder (500-800 g) purchased from the fishery wholesaler located in Yeosu city at the fisheries science institute of Chonnam National University. Min for 1 hour, and the serum was separated by centrifugation at 6,000 rpm for 20 minutes. Serum was stored at -80 ° C until use in the experiment.

 Example 2: Flounder IgM purification

1) Protein L affinity column

The IgM of the flounder was purified using Protein L bead (GE Healthcare, UK). Add PBS (pH 7.4) to the Protein L column, and add the serum to the plate. After washing with PBS, the flounder IgM was collected by adding 0.1 M citric acid (pH 2.5 - 3.0) and then neutralized with 1M Tris (pH 9.0). Purified flounder IgM (PL IgM) was stored at -80 ° C until used in the experiment.

2) Mannan binding protein (MBP) affinity column

IgM in the serum of the flounder was purified using MBP affinity column (ImmunoPure IgM Purification Kit, Pierce, USA). ImmunoPure MBP column was washed with 5 ml of preparation buffer and equilibrated with 20 ml of ImmunoPure IgM binding buffer. Flounder serum and ImmunoPure IgM binding buffer are mixed 1: 1 and then incubated at 4 ℃ for 30 minutes. To remove the unbound protein, 42 ml of ImmunoPure IgM binding buffer was added to the column and washed with water. ImmunoPure IgM elution buffer (3 ml) was added to the column and reacted at room temperature for 3 hours. Then, flounder IgM was collected. Purified flounder IgM (MBP IgM) was stored at -80 ° C until used in the experiment.

<Example 3> Structural protein analysis of flounder IgM

SDS-PAGE was performed to analyze the structural proteins of the purified flounder IgM. SDS-PAGE (12% acrylamide separating gel, 4% acrylamide stacking gel) was applied to the purified flounder IgM by the same amount of SDS-sample buffer and heat-treated at 100 ° C for 3 minutes. After electrophoresis, the gel was stained with coomassie brilliant blue.

<Example 4> Immunization and Hybridoma production of flounder IgM

Immunity was first immunized with BALB / c mouse peritoneal fluids by mixing 1: 1 ratio of flounder IgM (100 ug) purified with protein L affinity column and complete freunds adjuvant. After 2 weeks, blood was drawn from the tail of the mouse, ELISA was performed to measure antibody titer, and IgM and PBS were mixed to perform secondary immunization. Three days later, the mouse spleen was isolated and fused with myeloma cells using PEG. The cells were divided into 96-well plates using HT medium and cultured at 37 ° C in a CO ₂ incubator.

Example 5 Screening of Hybridoma

ELISA was performed to screen hybridomas producing antibodies that specifically react to flounder IgM. Antigens were coated with 50 μl (250 ng / well) of purified flounder IgM in a 96-well plate at 4 ° C overnight or 37 ° C for 2 hours. 2% skim milk (200 μl) was blocked, washed once with TBST, and 50 μl of the hybridoma culture supernatant as the primary antibody was dispensed at a rate of 25 ° C for 2 hours. After washing 3 times with TBST, goat anti-mouse IgG labeled with HRP was diluted 5,000 times with 2% skim milk and 50 ㎕ per well was reacted at 25 ℃ for 1 hour. After washing 5 times with TBST, coloring solution (TMB, color reagents) was added at a rate of 50 μl per well. 50 μl of 1N H ₂ SO ₄ was added to each well to discontinue the color reaction and the absorbance was measured at 450 nm using a microplate photometer.

Example 6 Isotyping of Monoclonal Antibodies

ELISA was performed using a Rapid ELISA mouse mAb isotyping kit (Pierce, USA) to confirm the heavy and light chain subtypes of the prepared monoclonal antibodies.

<Example 7> Examination of monoclonal antibody response using ELISA

ELISA was performed to confirm the reactivity of monoclonal antibodies against PL IgM and MBP IgM. Approximately 300 ng of PL IgM and MBP IgM were dispensed into each well of 96 well ELISA microplates (Greiner-bio-one, Germany) at 50 μl each, and then coated with antigen at 37 ° C overnight. The cells were washed 3 times with T-PBS (0.05% Tween-20 / PBS (v / v)) and blocked with 5% skim milk at 38 ° C for 1 hour at 25 ° C. After washing three times with T-PBS, 50 μl of the monoclonal antibody prepared in this study was dispensed into two wells per sample and reacted at 25 ° C for 1 hour. After washing three times with T-PBS, goat anti-mouse IgG (HRP) labeled horseradish peroxidase (HRP) was diluted 1,000 times with 5% skim milk and 50 ㎕ per well was dispensed. Time reaction. After washing 5 times with T-PBS, 50 μl of ELISA color development solution (100 mM Na ₂ HPO ₄ , 50 mM citric acid, 1 mg / ml o-phenylen diamine, 0.1% H ₂ O ₂ ) was added to each well. 50 μl of 1N H ₂ SO ₄ was added to each well to discontinue the color reaction, and the absorbance was measured at 490 nm.

<Example 8> Detection of specific flounder antibody against bovine serum albumin (BSA)

BSA (1 mg / 200 μl) was injected intraperitoneally into flounder (800-1,000 g) purchased from a marine wholesaler located in Yeosu City and housed in a 300 L water tank (water temperature: 17-20 ° C) for 21 days. After BSA inoculation, approximately 1 ml of blood was collected from the arrhythmia of flounder on days 0, 7, 14, and 21, and serum was separated by centrifugation (6,000 rpm, 4 ° C, 20 minutes). Separated serum was stored at -20 ° C until used in the experiment.

In order to detect specific flounder antibodies against BSA, ELSIA was performed using the serum obtained above. 5 μg of BSA / 50 μl was dispensed into each well of the ELISA plates, and the antigen was coated overnight at 37 ° C. The cells were washed 3 times with T-PBS (0.05% Tween-20 / PBS (v / v)) and blocked with 5% skim milk at 38 ° C for 1 hour at 25 ° C. After washing three times with T-PBS, the flounder serum collected from the above was diluted 40 times with 5% skim milk and reacted for 1 hour. Then, 50 μl of each antibody was dispensed into two wells per sample. 50 μl of each of the monoclonal antibodies prepared in this study was used. As a tertiary antibody, goat anti-mouse IgG labeled with horseradish peroxidase (HRP) diluted 1,000-fold with 5% skim milk (Youngin, Korea ) Were dispensed in 50 μl aliquots. Each antibody reaction was reacted at 25 ° C for 1 hour. The cells were washed 5 times with T-PBS, and 50 ㎕ of each ELISA coloring solution (100 mM Na ₂ HPO ₄ , 50 mM citric acid, 1 mg / 1 o- phenylenediamine, 0.03% H ₂ O ₂ ) And then developed at 25 ° C. 50 ㎕ of 2N H ₂ SO ₄ was added to each well to discontinue the color reaction, and the absorbance was measured at 490 nm.

Example 9 Detection kit for a specific antibody against an antigen (pathogen) in a flounder serum using a monoclonal antibody of an immunoglobulin M of a flounder

The present invention provides a specific antibody detection kit for an antigen in a flounder serum using an immunoglobulin M of a flounder including a monoclonal antibody of the present invention. In particular, the present invention can be used for an antigen detection kit of various known pathogenic viruses, bacteria, parasites and the like, and the detection kit can be a detection kit for detecting an antigen-antibody complex by a color particle binding method. Wherein said known antigen is selected from the group consisting of fish nerve necrosis virus, fish viral hemorrhagic sepsis virus, flounder virus, limpocystis, streptococcus, Edwards fungi, Vibrio fungi, An antigen, an attenuated pathogen, an inactivated pathogen, a recombinant protein, or an epitope.

The detection kit of the present invention includes a known antigen, a monoclonal antibody of the present invention, an antibody condensed with a marker that reacts with a substrate, and a chromogenic substrate. Specifically, the detection kit was prepared by diluting sera from NNV, VHSV, etc., diluted at a final concentration of 10 占 퐂 / ml, onto serum-coated plates, , And the presence or absence of immunoglobulin for the specific antigen in the serum of the black fish is determined by reacting the monoclonal antibody against the flounder immunoglobulin M according to the present invention.

The detection kit of the present invention comprises a plate coated with a specific antigen dilution solution, a monoclonal antibody specific for immunoglobulin M of horse-radish peroxidase (HRP) -conjugated flounder diluted 1: 2000, phosphate buffer solution, TMB and 1N H ₂ SO ₄ .

A method for detecting immunoglobulin of a flounder using the detection kit of the present invention is as follows. To the plate coated with the antigen, 100 μl of the serum collected from the black flounder is added to each well, reacted at room temperature for 60 minutes, and the plate is washed three times with phosphate buffer solution. Add 100 μl of the monoclonal antibody specific for the immunoglobulin M of the HRP-condensed flounder to each well of the plate, incubate at 20 ° C for 60 minutes, and wash the plate three times with phosphate buffer solution. Next, 100 μl of TMB (3,3 ', 5,5'-tetramethyl-benzidine), which is a substrate of HRP, is added to each well of the plate and reacted in a dark place for 30 minutes to induce a color reaction. Then, 1N H ₂ SO ₄ is added to each well of the plate at 100 μl to stop the enzyme reaction. The plate thus treated with the detection kit of the present invention is measured for its absorbance at 405 nm using an ELISA reader. When the absorbance is 0.2 or more, it is determined to be positive.

Test Example 1: Flounder IgM purification

Figure 1 is an SDS-PAGE photograph of purified flounder IgM. Both the heavy chain (76 kDa) and the light chain (26.5 kDa) were detected by SDS-PAGE after purification of the flounder IgM using Protein L affinity column and MBP affinty column.

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

Hybridoma was prepared by fusing Sp2 / 0Ag14 myeloma cells with PEG in mouse immunized with flounder IgM (PL IgM) purified with Protein L affinity column. Antibodies generated from Hybridoma were screened by ELISA and western blot, and the results were positive in 6 samples. Six clones were cloned three times by ELISA and finally one clone (24B12) was selected. In subsequent experiments, we performed a specificity study using antibodies produced from one clone (using the same name as the antibody name: clone).

&Lt; Test Example 3 > Isotype of monoclonal antibody

2 is a table showing the results of isotyping of the monoclonal antibody (24B12) against PL IgM. The heavy and light chain subtypes of IgG were examined by immunoblotting with a rapid ELISA mouse mAb isotyping kit. The H-chain was identified as IgG1 and the L-chain as κ.

&Lt; Test Example 4 > IgM response of a monoclonal antibody to a flounder

Figure 3 shows ELISA results showing the reaction of monoclonal antibodies against PL IgM and MBP IgM. Both ODs (PL IgM and MBP IgM) showed an OD value of about 1.8. No OD value was observed in the control (antigen: skim milk).

Test Example 5 Detection of specific flounder antibody against BSA

4 is a graph showing the results of detection of a specific flounder antibody against BSA using a monoclonal antibody. In order to investigate the specificity of the monoclonal antibody produced in the present invention, sera were isolated at 0, 7, 14, and 21 days after immunization with BSA in flounder, and antibody titers to BSA were measured. Serum of BSA immunized flounder was found to increase with time. That is, in the case of flounder 1 (BSA 1), antibody titer was measured at 14 days and 21 days, and that of flounder 2 (BSA 2) was measured at 21 days. No elevation of antibody titer was observed in the control flounder.

The present invention provides a diagnostic kit and a vaccine effective for diagnosis, treatment and prevention of infectious diseases of flounder by providing a fusion cell line producing a monoclonal antibody specific for the flounder's IgM and a monoclonal antibody specific for the IgM of the flounder produced thereby Therefore, it can be utilized in aquaculture industry, diagnostic kits, and preventive vaccines. Therefore, there is a possibility of industrial application.

<Access number>

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00368

Funding Date: 2016816

Claims (8)

A fish antigen detection kit comprising a monoclonal antibody Anti-24B12 specific for a flounder immunoglobulin M produced by a fusion cell line of Accession No. KCLRF-BP-00368.
The method according to claim 1, wherein the fish antigen is selected from the group consisting of fish nerve necrosis virus, fish viral hemorrhagic septic virus, flounder virus, lymphocystis, streptococcus, Edwards fungus, Vibrio fungus, A kit for detecting a specific antibody of a flounder recognizing a selected one or more isolated antigens, attenuated pathogens, inactivation pathogens, recombinant proteins, epitopes which are epitopes (antigens).
The kit for detecting a specific antibody of a flounder according to claim 1 or 2, wherein the specific antibody detecting kit for detecting the fish antigen is a kit for detecting a specific antigen of a flounder against a fish antigen by detecting an antigen-antibody complex by a color particle binding method.
[Claim 4] The method according to claim 3, wherein the detection kit comprises: an antibody condensed with a labeling substance that reacts with a substrate; And a chromogenic substrate, wherein the marker is Horse-radish peroxidase (HRP) and the chromogenic substrate is TMB (3,3 ', 5,5'-tetramethyl-benzidine) Specific antibody detection kit.
Diluting the fish antigen with a coating buffer solution and dispensing it on the plate to coat the plate (1);
(2) a step of allowing the plate of (1) to react with the serum of the serum to be examined;
(3) washing the plate of (2) with a phosphate buffer solution;
A step (4) of reacting a conjugate of an enzyme with a monoclonal antibody Anti-24B12 specific to a flounder immunoglobulin M produced by a fusion cell line of Accession No. KCLRF-BP-00368 on the plate of the above (3);
Adding (5) the coloring substrate of (4) to color;
(6) stopping the enzyme reaction with sulfuric acid when the coloring reaction of (5) is over; And
(7) measuring the absorbance at 405 nm of the color development degree of (6), and detecting the absorbance of the oligonucleotide using the immunoglobulin monoclonal antibody of flounder.
The method according to claim 5, wherein the fish antigen is selected from the group consisting of fish nerve necrosis virus, fish viral hemorrhagic septic virus, flounder virus, lymphocystis, streptococcus, Edwards fungus, Vibrio fungus, A kit for detecting a specific antibody of a flounder recognizing a selected one or more isolated antigens, attenuated pathogens, inactivation pathogens, recombinant proteins, epitopes which are epitopes (antigens).
Monoclonal antibody Anti-24B12 produced in the fusion cell line of accession number KCLRF-BP-00368.
Fusion cell line of accession number KCLRF-BP-00368.

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