KR102163241B1 - Monoclonal antibody against immunoglobulin m of rock bream and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to dolphin immunoglobulin M and a hybridoma cell line producing the same, wherein the monoclonal antibody of the present invention specifically binds to dolphin immunoglobulin M, It can be preferably used for antibody detection

Description

A monoclonal antibody specific for immunoglobulin M of Doldom and its use {MONOCLONAL ANTIBODY AGAINST IMMUNOGLOBULIN M OF ROCK BREAM AND USE THEREOF}

The present invention is stone sea bream ( Olegnathus fasciatus ) to immunoglobin M (Immunoglobulin M, Ig M) specific monoclonal antibody and its use, and in more detail, a fusion cell line that produces monoclonal antibody specific to immunoglobin M of Doldom and monoclonal antibodies produced therein It relates to a method for detecting the antibody contained in the serum of the dolphin by using.

Along with the vitalization of the fish breeding industry around the world, the damage caused by fish diseases has increased remarkably, which has emerged as a major problem in the aquaculture industry. In particular, the mortality rate due to viral diseases is estimated to account for more than 30% of fish production, but there is no effective treatment so far, so rapid diagnosis and preventive measures remain a major task.

In Korea, in order to systematically manage aquatic organisms infectious diseases and minimize damage caused by diseases, the Aquatic Products Disease Management Act has been implemented since 2008 to conduct quarantine and quarantine for 21 infectious diseases, and these infectious diseases are of importance according to their risk. By dividing the list and the quarantine guidelines for each disease occurrence situation, quarantine measures such as killing, quarantine, and movement restrictions are implemented.

Among the aquatic material diseases subject to these quarantine and quarantine, viral diseases account for 67%, and the diagnosis method for viral infectious diseases used for quarantine and quarantine in Korea is mainly performed by PCR method targeting the viral genome. . In addition, the separation culture method using fish coined cells and the histopathological method are also used in parallel. In the case of fish, the PCR method is mainly used, and the PCR method and the histopathological method are used in combination with shellfish and crustaceans. However, immunological methods are rarely used because they do not yet possess specific antibodies against pathogenic viruses.

The diagnostic method by PCR is widely used worldwide as a virus detection method because it allows rapid diagnosis.However, quarantine of seafood imported from foreign countries, epidemiological investigation in aquaculture, pathogenicity assessment, and periodic identification of virus infection status in fish Since there are many unsuitable points for use in quarantine, there are limits to general application in the field. In other words, since the PCR method directly detects pathogens, even aquatic organisms with high product value, such as koi, can be tested only by killing them. In addition, it is difficult to process a large number of samples at once, and it has a disadvantage that it takes a lot of cost to inspect a large number of samples. In addition, because the PCR test targets genes, even if the pathogen is inactivated, the gene is detected, and if two or more viruses are mixed and infected in farmed fish, accurate diagnosis is difficult.

In contrast, immunological diagnosis is a method of diagnosing pathogen infection using antibodies. Immunological diagnostic methods include a method of directly detecting a pathogen using a specific antibody of a pathogen, and an indirect method of diagnosing a pathogen infection by detecting a specific antibody present in blood.

Immunological diagnosis by the direct method is a method that directly detects pathogens, and has the rapidity of disease testing within 6 hours. In addition, the sensitivity and specificity are very high, it is easy to trace pathogens in the fish, and it is possible to confirm the infected cells and the degree of infection. When it is possible to stably produce specific antibodies against pathogens, diseases can be easily diagnosed even at aquaculture sites through the production of diagnostic kits.

Immunological diagnosis by the indirect method detects specific antibodies present in the blood. It is possible to process a large number of samples at low cost, and the detection sensitivity and reliability of the results are high. It is widely used for monitoring and validation of vaccine efficacy.

Immunological diagnosis by indirect method does not need to kill the organism to be tested, and diagnosis is possible only by collecting a small amount of serum. In addition, a large number of samples can be quickly diagnosed at low cost, so if the antibody detection ELISA method is used, more than 300 samples can be processed at a time, and more than 15 times the sample can be processed at about 100 times lower cost than PCR. In addition, it is possible to diagnose unexpressed infected fish and to identify the history of viral infection, and is useful for determining the efficacy of vaccines and after conducting quarantine.

In order to use the immunological diagnostic method by such an indirect method, a specific antibody against IgM in fish is required. However, unlike mammalian IgG, fish antibodies are known to consist of IgM, which has a low affinity, and when antibody detection ELISA is applied to fish, a high background is caused by nonspecific adsorption of fish IgM and blocking agents. Problems that do not appear in mammals have been reported. For this reason, the International Waters Office (OIE) does not accept antibody detection ELISA as a diagnostic method because of its low reproducibility.

However, in recent studies, a method of inhibiting non-specific adsorption between fish IgM and blocking agents has been proposed. Therefore, there is an urgent need for research on ELISA to produce and isolate specific antibodies for fish IgM and to use them for diagnosis of pathogenic infections of aquatic organisms with low non-specific adsorption with blocking agents.

KR 10-2018-0023250 A KR 10-2018-0023248 A

The present invention is to provide a monoclonal antibody that specifically binds to Doldom immunoglobulin M and can be preferably used for detection of a specific antibody against fish antigens, a fusion cell line that produces the monoclonal antibody, and a method using the same. The purpose.

The present invention provides a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M) of Oplegnathus fasciatus , and a diagnostic kit and diagnostic kit that can be used to detect a specific antibody against fish antigens using this It provides a composition for use.

Specifically, the present invention relates to a fusion cell line that produces a monoclonal antibody specific for immunoglobin M (Immunoglobulin M, Ig M) of Oplegnathus fasciatus . The fusion cell line may be a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437. In this aspect, the present invention may specifically be a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 that produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M) of Oplegnathus fasciatus . have.

In another aspect, the present invention is a monoclonal produced in a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 that produces a monoclonal antibody specific for immunoglobin M (Immunoglobulin M, Ig M) of Oplegnathus fasciatus It relates to antibodies. More specifically, the present invention provides a monoclonal antibody produced by the parrot fish immune globin M (Immunoglobulin M, Ig M) Accession No. KCLRF-BP-00437 fusion cell line (RB IgM 20A2) for producing a specific monoclonal antibody to the (Oplegnathus fasciatus) Can be

In still another aspect, the present invention relates to a kit for diagnosing infectious diseases of dolphin, comprising a monoclonal antibody specific for immunoglobin M of dolphin.

In detail, the kit for diagnosing an infectious disease of doldom may include a monoclonal antibody specific for immunoglobin M of doldom produced by a fusion cell line that produces a monoclonal antibody specific for immunoglobin M of doldom. The fusion cell line may be a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437.

In this aspect, the present invention is preferably produced by the fusion cell line (RB IgM 20A2) of deposit number KCLRF-BP-00437 that produces a monoclonal antibody specific to the immunoglobin M of the dolphin. It may be a kit for diagnosing an infectious disease of a dolphin containing a monoclonal antibody specific to the immunoglobin M of dolphin.

The diagnostic kit includes detecting an antigen-antibody complex by a color particle binding method, preferably an antibody condensed with a label that reacts with a substrate to develop color; And a chromogenic substrate, wherein the marker is HRP (Horse-radish peroxidase), and the chromogenic substrate may be TMB (3,3',5,5'-tetramethyl-benzidine).

In addition, the diagnostic kit may be a strip type using an immunochromatography method.

The strip-type diagnostic kit includes a monoclonal antibody specific for immunoglobin M of dolphin produced by a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 that produces a monoclonal antibody specific for immunoglobin M of dolphin. It may be obtained by rapid immunochromatography including a gold conjugate, a membrane coated with an antibody specific for mouse immunoglobulin, and a strip attached with a moisture absorption pad for a subject.

In yet another aspect, the present invention may relate to a composition for detecting a specific antibody of dolmens against fish antigens.

In addition, the composition for detecting a specific antibody of dol dome against fish antigen is a monoclonal produced by a fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 that produces a monoclonal antibody specific for immunoglobin M of dolphin. It may contain an antibody.

The monoclonal antibody of the present invention specifically binds to Doldom immunoglobulin M and can be preferably used for detection of a specific antibody of Doldom against fish antigens.

1 is an SDS-PAGE photograph of purified Dolphin IgM according to an embodiment of the present invention.
FIG. 2 is a table showing the isotyping results of a monoclonal antibody (20A2) against dolphin IgM (PA IgM) purified using a Protein A affinity column according to an embodiment of the present invention.
3 is an ELISA result showing the response of a monoclonal antibody to PA IgM according to an embodiment of the present invention.
FIG. 4 is a graph showing the detection result of a specific dolphin antibody against bovine serum albumin (BSA) using a monoclonal antibody according to an embodiment of the present invention.

The present invention has excellent sensitivity and specificity to the immunoglobin M (Ig M) of dolphin and is specific to the immunoglobin M (Ig M) of dolphin. It relates to specific monoclonal antibodies and fusion cell lines producing the same, and uses thereof. Hereinafter, the present invention will be described in detail with reference to specific examples.

Example

<Experiment method>

1. Serum separation of stone sea bream

Doldom serum used in the experiment was collected from the caudal vein of Doldom (weight: 300 450 g) purchased from a seafood wholesaler located in Yeosu City, and the blood was coagulated at room temperature for 30 minutes and 1 hour, and then centrifuged at 6,000 rpm for 20 minutes. The serum was separated. Serum was stored at -80°C until use in the experiment.

2. Stone sea bream IgM tablet

IgM of stone sea bream was purified using Protein A resin (GE Healthcare, UK). After adding PBS (pH 7.4) to the Protein A column, add stone dome serum and bind. After washing with PBS, 0.1M citric acid (pH 2.5 3.0) was added to collect doldom IgM, and then neutralized with 1M Tris (pH 9.0). Purified Doldom IgM (PA lgM) was stored at -80°C until use in the experiment.

3. Analysis of Structural Proteins of Doldom IgM

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

4. Doldom IgM immunity and hybridoma production

For immunity, purified stone sea bream IgM (PA IgM, 100 ㎍) and complete Freund's adjuvant (Gibco, USA) were first inoculated into the abdominal cavity of two BALB/c mice at 100 uL by mixing the same amount. Second inoculation with IgM. One week later, after the third inoculation with purified Dole Dome IgM, the spleen was separated from the mouse, fused with SP2/0 myeloma cells using polyethylene glycol (Roche, Germany), and then 10% fetal bovine serum was added to HAT. After suspension with medium (0.1 mM hypoxanthine, 4×10 ^-4 mM aminoprotein, 1.6×10 ^-2 mM thymidinein Dulbecco's modifiedeaglemedium), it was dispensed into 96 well plates and cultured in a CO ₂ incubator set at 37°C.

5. Hybridoma Screening

ELISA was performed to screen for hybridomas that produce antibodies that react specifically to dolphin IgM. For antigen, 50 µl (250 ng/well) per well was dispensed into a 96 well plate with purified Dole Dome IgM, and coated at 4°C overnight or 37°C for 2 hours. 200 µl of 2% skim milk was dispensed and blocked, washed three times with TBST, and 50 µl per well of the hybridoma culture supernatant as a primary antibody were dispensed for 2 hours at 37°C. After washing three times with TBST, as a secondary antibody, goat anti-mouse IgG labeled with HRP was diluted 5,000 times with 2% skim milk, and 50 µl per well was dispensed and reacted at 37°C for 1 hour. After washing 5 times with TBST, 50 µl of a color developing solution (TMB, color reagents) was dispensed per well to develop color. 50 µl of 1N H ₂ SO ₄ was added to each well to stop the color reaction, and the absorbance was measured at 450 nm with a microplate photometer.

6. Monoclonal Of antibodies Isotyping

ELISA was performed according to the manual using the Rapid ELISA mouse mAb isotyping kit (Pierce, USA) in order to confirm the subtype of the heavy and light chain of the produced monoclonal antibody.

7. Using ELISA Monoclonal Investigation of antibody reaction

ELISA was performed to confirm the response of the monoclonal antibody to PA IgM. About 300 ng of PA IgM was dispensed into 96 well ELISA microplates (Greiner-bio-one, Germany) by 50 μl each, and then overnight at 37° C. to coat the antigen. Washed three times with T-PBS (0.05% Tween-20/PBS (v/v)), and 380 μl of 5% skim milk was dispensed and blocked at 25° C. for 1 hour. After washing three times with T-PBS, 50 µl of the monoclonal antibody produced in this study was dispensed into two wells per sample and reacted at 25° C. for 1 hour as the primary antibody. After washing three times with T-PBS, goat anti-mouse IgG (Youngin, Korea) labeled with horseradish peroxidase (HRP) was diluted 1,000 times with 5% skim milk, and 50 µl per well was dispensed at 25°C. It reacted for time. After washing 5 times with T-PBS, ELISA color development solution (100 mM Na ₂ HPO ₄ ,50mMcitric acid, 1 mg/ml o-phenylen diamine, 0.1% H ₂ O ₂ ) was dispensed in 50 µl per well to develop color. 50 µl of 1N H ₂ SO ₄ was added to each well to stop the color reaction, and the absorbance was measured at 490 nm.

8. Detection of specific dolphin antibody against bovine serum albumin (BSA)

1 mg/200 µl of BSA was injected into the peritoneal cavity to a dolphin (260 345 g) purchased from a seafood wholesaler located in Yeosu City, and then housed in a 300 L water tank (water temperature: 17-20°C) and reared for 22 days. After the BSA inoculation, about 1 ml of blood was collected from the caudal vein of the dolphin on days 0, 11, and 22, and then centrifuged (12,000 rpm, 4°C, 10 minutes) to separate the serum. The separated serum was stored at 20°C until use in the experiment.

In order to detect specific doldom antibodies against BSA, ELSIA was performed using the serum obtained above. After dispensing 5 μg BSA/50 μl into each well on ELISA plates, the antigen was coated overnight at 37°C. Washed three times with T-PBS (0.05% Tween-20/PBS (v/v)), and 380 μl of 5% skim milk was dispensed and blocked at 25° C. for 1 hour. After washing three times with T-PBS, as the primary antibody, dolphin serum collected from the stomach was diluted 40 times with 5% skim milk and reacted for 1 hour, and then 50 µl per sample was dispensed into two wells. As for the furnace, 50 µl of the monoclonal antibody produced in this study was used, and as the tertiary antibody, horseradish peroxidase (HRP) diluted 1,000 times with 5% skim milk was labeled goat anti-mouse IgG (Youngin, Korea). ) Was dispensed in 50 μl each. Each antibody reaction was reacted at 25° C. for 1 hour. After washing 5 times with T-PBS, ELISA color development solution (100 mM Na ₂ HPO ₄ ,50mMcitric acid, 1 mg/ml o-phenylen diamine, 0.1% H ₂ O ₂ ) was dispensed in 50 µl per well to develop color. 50 µl of 1N H ₂ SO ₄ was added to each well to stop the color reaction, and the absorbance was measured at 490 nm.

<Experiment result>

1. Stone sea bream IgM refine

1 is an SDS-PAGE photograph of purified Doldom IgM. As a result of SDS-PAGE after purifying Doldom IgM using a Protein A affinity column, heavy chain (75 kDa) and light chain (27.2 kDa) were observed.

2. Hybridoma Production and Monoclonal Antibody screening

A hybridoma was fabricated by fusion of the spleen tissue of a mouse immunized with Doldom IgM (PA IgM) purified by Protein A affinity column and Sp2/0Ag14 myeloma cells with PEG. As a result of screening antibodies generated from hybridoma by ELISA and western blot, 42 samples showed a positive reaction. As a result of cloning four times by ELISA for 42 samples, one clone (20A2) was finally selected.

The clone 22A3 (hybridoma) was deposited with the Korea Cell Line Research Foundation with 000000 letters, and was given the deposit number 0000000 (fusion cell line, Dole Dome IgM 20A2, Rock bream IgM 20A2).

In subsequent experiments, specificity was investigated using an antibody produced from one clone (name of antibody: same as the name of clone).

3. Monoclonal Of antibodies isotype

2 is a table showing the isotyping results of a monoclonal antibody (20A2) against PA IgM. As a result of investigating subtypes of heavy and light chains of IgG using a rapid ELISA mouse mAb isotyping kit for monoclonal antibodies produced with PA IgM, H-chains were identified as IgG1 and L-chains as κ.

4. Monoclonal Antibody dome IgM reaction

3 is an ELISA result showing the response of a monoclonal antibody to PA IgM. 20A2 showed an OD value of about 1.4 for PA IgM. No OD value was observed in the control (antigen: skim milk).

5. Detection of specific dolphin antibody against BSA

4 is a graph showing the detection result of a specific doldom antibody against BSA using a monoclonal antibody. In order to investigate the specificity of the monoclonal antibody produced in the present invention, BSA was immunized to Doldom, and serum was isolated on days 0, 11, and 22 to measure the antibody titer against BSA. BSA-immunized dolphin serum showed an increase in antibody titer over time. In the control dome (control 1, control 2), an increase in antibody titer was not observed. That is, in BSA-immunized stone dome (BSA 1, BSA 2, BSA3), antibody titers were measured on days 11 and 22 in BSA 1 and BSA 3 excluding BSA2.

Korea Cell Line Research Foundation KCLRFBP00437P 20180614

Claims (8)

Stone sea bream ( Olegnathus fasciatus ) immunoglobin M (Immunoglobulin M, Ig M)-specific monoclonal antibody produced by the fusion cell line (RB IgM 20A2) of KCLRF-BP-00437, which produces a monoclonal antibody specific to Immunoglobulin M (Ig M) Kit for diagnosing infectious diseases of stone dome comprising a. The method of claim 1,
The diagnostic kit is a kit for diagnosing infectious diseases of stone dome for detecting an antigen-antibody complex by a color particle binding method. The method of claim 2,
The diagnostic kit includes an antibody condensed with a label that reacts with a substrate to develop color; And a chromogenic substrate, wherein the marker is HRP (Horse-radish peroxidase), and the chromogenic substrate is TMB (3,3',5,5'-tetramethyl-benzidine). Diagnostic kit. The method of claim 1,
The diagnostic kit is a kit for diagnosing an infectious disease of doldom, which is a strip type using immunochromatography. The method of claim 4,
The strip-type diagnostic kit is a conjugate of gold and a monoclonal antibody specific for immunoglobin M of dolphin produced by a fusion cell line that produces a monoclonal antibody specific for immunoglobin M of dolphin, and an antibody specific for mouse immunoglobulin A kit for diagnosing infectious diseases of stone dome by rapid immunochromatography comprising a coated membrane and a strip with a moisture absorbing pad attached to the subject. Stone sea bream ( Olegnathus fasciatus ), which produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M), a fish antigen containing a monoclonal antibody produced by the fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 A composition for detecting a specific antibody of. Stone sea bream ( Olegnathus fasciatus ) monoclonal antibody produced in the fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 that produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M). Stone sea bream ( Olegnathus fasciatus ) fusion cell line (RB IgM 20A2) of accession number KCLRF-BP-00437 producing a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M).

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