KR102163242B1 - Monoclonal antibody against immunoglobulin m of rainbow trout and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody specifically binding to rainbow trout immunoglobulin M and a hybridoma cell line producing the same, wherein the monoclonal antibody of the present invention specifically binds to rainbow trout immunoglobulin M, It can be preferably used for detection of specific antibodies of rainbow trout.

Description

Monoclonal antibody specific to immunoglobulin M of rainbow trout and its use {MONOCLONAL ANTIBODY AGAINST IMMUNOGLOBULIN M OF RAINBOW TROUT AND USE THEREOF}

The present invention is rainbow trout ( Oncorhynchus) mykiss ) to immunoglobin M (Immunoglobulin M, Ig M) specific monoclonal antibody and its use, and more specifically, a fusion cell line that produces monoclonal antibodies specific to immunoglobin M of rainbow trout and the monoclonal produced therein It relates to a method for detecting an antibody contained in the serum of rainbow trout using an antibody.

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 provides a monoclonal antibody that specifically binds to rainbow trout immunoglobulin M and can be preferably used for detection of a specific antibody of rainbow trout against fish antigens, a fusion cell line producing the monoclonal antibody, and a method using the same. It aims to do.

The present invention is rainbow trout ( Oncorhynchus mykiss ) provides a specific monoclonal antibody to immunoglobin M (Immunoglobulin M, Ig M), and provides a diagnostic kit and a composition for diagnostic kit that can be used to detect specific antibodies of rainbow trout against fish antigens. .

Specifically, the present invention is a rainbow trout ( Oncorhynchus mykiss ) relates to a fusion cell line that produces monoclonal antibodies specific for immunoglobin M (Immunoglobulin M, Ig M). In this aspect, the present invention is specifically a fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M) of rainbow trout ( Oncorhynchus mykiss ). I can.

In another aspect, the present invention is a rainbow trout ( Oncorhynchus mykiss ) to immunoglobin M (Immunoglobulin M, Ig M) specific monoclonal antibody is produced in the fusion cell line (RT IgM 44H10) of the accession number KCLRF-BP-00438 producing a monoclonal antibody. Specifically, the present invention is a rainbow trout ( Oncorhynchus It may be a monoclonal antibody produced in a fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M) of mykiss ).

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

In detail, the kit for diagnosing infectious diseases of rainbow trout may include a monoclonal antibody specific to immunoglobin M of rainbow trout produced by a fusion cell line that produces a monoclonal antibody specific to immunoglobin M of rainbow trout. . The fusion cell line may be a fusion cell line (RT IgM 44H10) with accession number KCLRF-BP-00438.

In this aspect, the present invention is preferably in the fusion cell line (RT IgM 44H10) of the accession number KCLRF-BP-00438 producing a monoclonal antibody specific to the immunoglobin M of rainbow trout, preferably the kit for diagnosing infectious diseases of rainbow trout It may be a kit for diagnosing an infectious disease of rainbow trout containing a monoclonal antibody specific to immunoglobin M of rainbow trout produced by.

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 is a monoclonal immunoglobin M-specific immunoglobin M produced by a fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a monoclonal antibody specific to the immunoglobin M of rainbow trout. It may be obtained by rapid immunochromatography including a conjugate of an antibody and gold, a membrane coated with an antibody specific for mouse immunoglobulin, and a strip with a moisture absorbing pad attached to the subject.

In another aspect, the present invention may relate to a composition for detecting a specific antibody of rainbow trout against a fish antigen.

Incidentally, the composition for detecting a specific antibody of rainbow trout against fish antigens is produced by a fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a monoclonal antibody specific for immunoglobin M of rainbow trout. It may contain a monoclonal antibody.

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

1 is an SDS-PAGE photograph of purified rainbow trout IgM according to an embodiment of the present invention.
Figure 2 shows the results of Western blotting showing the rainbow trout IgM recognition site of the monoclonal antibody according to an embodiment of the present invention.
3 is an ELISA result showing the response of a monoclonal antibody to MBP IgM according to an embodiment of the present invention.
4 is a graph showing the detection result of a specific rainbow trout antibody against bovine serum albumin (BSA) using a monoclonal antibody according to an embodiment of the present invention.
5 is a table showing the isotyping results of a monoclonal antibody (44H10) against rainbow trout IgM (MBP IgM) purified using an MBP affinity column according to an embodiment of the present invention.

The present invention has excellent sensitivity and specificity to immunoglobin M (Ig M) of rainbow trout and is specific to immunoglobin M (Ig M) of rainbow trout. Therefore, it is preferable to detect a specific antibody of rainbow trout against fish antigens. The present invention relates to a monoclonal antibody specific for immunoglobin M, a fusion cell line producing the same, and a use thereof.

Hereinafter, the present invention will be described in detail with reference to specific examples.

Example

<Experiment method>

1. Serum separation of rainbow trout

Rainbow trout serum used in the experiment was collected from the microarrhythmic veins of rainbow trout (weight: 900-1,200 g) reared at the Namhae Specialty Test Center, Jeollanam-do Institute of Marine Science and Technology, left at room temperature for at least 30 minutes, 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. Rainbow trout IgM purification

IgM contained in rainbow trout serum was purified using Mannan binding protein (MBP) affinity column (ImmunoPure IgM Purification Kit, Pierce, USA). After washing the ImmunoPure MBP column with 5 ml of preparation buffer, the column was equilibrated with 20 ml of ImmunoPure IgM binding buffer. After mixing the rainbow trout serum and ImmunoPure IgM binding buffer 1:1, put the column and react at 4℃ for 30 minutes. To remove the unbinding protein, 42 ml of ImmunoPure IgM binding buffer was put into a column and washed with water. 3 ml of ImmunoPure IgM elution buffer was added to the column and reacted at room temperature for 12 hours, and then IgM of rainbow trout was collected. Purified rainbow trout IgM (MBP IgM) was stored at 80°C until use in the experiment.

3. Structural protein analysis of IgM of rainbow trout

SDS-PAGE was performed to analyze the structural protein of the purified rainbow trout IgM. The same amount of SDS-sample buffer was added to the purified rainbow trout IgM and heat-treated at 100° C. for 5 minutes, followed by SDS-PAGE (10% acrylamide separating gel, 4% acrylamide stacking gel). After electrophoresis, the gel was stained with coomassie brilliant blue.

4. Rainbow trout IgM immunity and hybridoma production

For immunity, purified rainbow trout IgM (MBP IgM, 100 ug) and complete Freund's adjuvant (Gibco, USA) were first inoculated into the abdominal cavity of 2 BALB/c mice at 100 uL by mixing equal amounts. Rainbow trout was second inoculated with IgM. One week later, after the third inoculation with purified rainbow trout 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. After suspension with HAT medium (0.1 mM hypoxanthine, 4×10 ^-4 mM aminoprotein, 1.6×10 ^-2 mM thymidinein Dulbecco's modified eagle medium), 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 rainbow trout IgM. For antigen, purified rainbow trout IgM was dispensed into a 96 well plate by 50 µl (250 ng/well) per well 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. Using Western blot Monoclonal Antibody response investigation

Western immunoblot analysis was performed to confirm the recognition site of the monoclonal antibody against IgM in rainbow trout. SDS-PAGE was performed in the same manner as above. After electrophoresis of purified rainbow trout IgM, the protein in the gel was blotting on a nitrocellurose membrane (advantec, Japan) for 1 hour at 144 ㎃ using a transblot device (ATTO, Japan). After blotting, it was blocked with 2% skim milk and reacted for 1 hour, followed by washing with buffer 1 (0.1M maleic acid, 0.15M NaCl, pH 7.5) for 15 minutes. As the primary antibody, the hybridoma culture supernatant prepared in this study was diluted twice with 2% skim milk, reacted for 1 hour, and washed in the same manner as above. As a secondary antibody, alkaline phosphatase (AP) labeled goat polyclonal anti-mouse IgG antibody (novus, USA) was diluted 1,000 times with 2% skim milk, reacted for 1 hour, washed 5 times, and then washed 5 times. -HCl, 100 mM NaCl, 50 mM MgCl ₂ solution (pH 9.5) 20 ml, NBT (75 mg/ml 4-nitrotetrazolium blue chloride) 90 μl, BCIP (50 mg/ml 5-Bromo-4-chloro-3- Indolyl phosphate p-toluidine salt/dimethyl-formamide) (70 µl) was used and visually confirmed. After the color development was completed, a color development stop solution (1mM EDTA, 10mM Tris-HCl) was added.

7. Using ELISA Monoclonal Investigation of antibody reaction

ELISA was performed to confirm the response of the monoclonal antibody to MBP IgM. About 250 ng of MBP 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, 50 µl of ELISA color reagents (TMB, color reagents) were dispensed per well to develop color. 50 µl of 1N H ₂ SO ₄ was added to each well to stop the color development, and the absorbance was measured at 450 nm.

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

Inject 1 mg/200 µl of BSA into the rainbow trout (9001,200 g) raised in the Namhae special test site of Jeollanam-do Institute of Oceans and Fisheries, and then put it in a 2 ton water tank (water temperature: 13-16℃) for 61 days. I did. On day 12 during breeding, 1 mg/200 μl of BSA was injected again. After BSA inoculation, about 1 ml of blood was collected from the caudal vein of rainbow trout on days 12, 24, 40, and 61, and then centrifuged (6,000 rpm, 4° C., 20 minutes) to separate the serum. The separated serum was stored at -80°C until used in the experiment.

In order to detect the specific rainbow trout antibody 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, the rainbow trout serum collected from the stomach was diluted 40 times with 5% skim milk and reacted for 1 hour, and 50 µl per sample was dispensed into two wells. As an antibody, 50 µl of the monoclonal antibody produced in this study was used, and as a tertiary antibody, horseradish peroxidase (HRP) diluted 1,000 times in 5% skim milk was labeled goat anti-mouse IgG (Youngin, Korea) was dispensed 50 μl each. Each antibody reaction was reacted at 25° C. for 1 hour. After washing with T-PBS 5 times, 50 µl of ELISA color reagents (TMB, color reagents) were dispensed into each well, followed by color development at 25°C. 50 µl of 2N H ₂ SO ₄ was added to each well to stop the color development, and the absorbance was measured at 450 nm.

9. Of monoclonal 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.

<Experiment result>

1. Rainbow trout IgM refine

1 is an SDS-PAGE photograph of purified rainbow trout IgM. As a result of SDS-PAGE using rainbow trout IgM (MBP IgM) purified using MBP affinity column, heavy chain (79 kDa) and light chain (28 kDa) were observed.

2. Hybridoma Production and Monoclonal Antibody screening

Hybridomas were prepared by fusion of the spleen tissue of a mouse immunized with MBP IgM and Sp2/0Ag14 myeloma cells with PEG. As a result of screening antibodies generated from hybridomas by ELISA and western blot, 47 samples showed positive reactions. As a result of cloning four times by ELISA for 47 samples, one clone (44H10) was finally selected.

The clone 44H10 (hybridoma) was deposited with the Korea Cell Line Research Foundation with 00000 letters and was given the deposit number 000000 (fusion cell line, rainbow trout IgM 44H10, rainbow trout IgM 44H10).

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

3. Rainbow Trout IgM for Monoclonal Investigation of the recognition site of antibodies

Figure 2 is a Western blotting result showing the recognition site of the monoclonal antibody for rainbow trout IgM. The produced antibody was confirmed to recognize the heavy chain (79 kDa) of rainbow trout IgM.

4. Monoclonal Antibody Rainbow Trout IgM reaction

3 is an ELISA result showing the response of a monoclonal antibody to MBP IgM. 44H10 had an OD value of about 1.4 for MBP IgM. No OD value was observed in the control (antigen: skim milk).

5. Detection of specific rainbow trout antibodies to BSA

4 is a graph showing the detection result of a specific rainbow trout antibody against BSA using a monoclonal antibody. In order to investigate the specificity of the monoclonal antibody produced in the present invention, after immunizing rainbow trout with BSA, serum was isolated on days 12, 24, 40, and 61 to measure antibody titer against BSA. The serum of BSA-immunized rainbow trout was found to increase in antibody titer with time. No increase in antibody titer was observed in the control rainbow trout (control 1, control 2). That is, in rainbow trout (BSA 1, BSA2) immunized with BSA, antibody titers were measured on days 40 and 61.

6. Monoclonal Of antibodies isotype

5 is a table showing the isotyping results of a monoclonal antibody (44H10) against MBP IgM. As a result of investigating subtypes of heavy and light chains of IgG using a rapid ELISA mouse mAb isotyping kit for the monoclonal antibody produced by MBP IgM, H-chain was identified as IgG1 and L-chain as κ.

Korea Cell Line Research Foundation KCLRFBP00438P 20180614

Claims (8)

Rainbow Trout ( Oncorhynchus mykiss ) immunoglobin M (Immunoglobulin M, Ig M)-specific monoclonal immunoglobin M of rainbow trout produced by the fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a specific monoclonal antibody A kit for diagnosing infectious diseases of rainbow trout containing antibodies. The method of claim 1,
The diagnostic kit is a kit for diagnosing an infectious disease of rainbow trout 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, 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 infectious diseases of rainbow trout, which is a strip type using immunochromatography. The method of claim 4,
The strip-type diagnostic kit is a conjugate of a monoclonal antibody specific to immunoglobin M of rainbow trout and gold produced by a fusion cell line that produces a monoclonal antibody specific to immunoglobin M of rainbow trout, and is specific for mouse immunoglobulin. A kit for diagnosing infectious diseases of rainbow trout by rapid immunochromatography, comprising a membrane coated with an antibody and a strip with a moisture absorbing pad attached to the subject. Rainbow Trout ( Oncorhynchus mykiss ) Rainbow trout against fish antigens containing monoclonal antibodies produced from the fusion cell line (RT IgM 44H10) of KCLRF-BP-00438, which produces a monoclonal antibody specific for immunoglobin M (Immunoglobulin M, Ig M) of mykiss A composition for detecting a specific antibody of. Rainbow Trout ( Oncorhynchus mykiss ) monoclonal antibody produced in the fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 that produces a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M). Rainbow Trout ( Oncorhynchus mykiss ) fusion cell line (RT IgM 44H10) of accession number KCLRF-BP-00438 producing a monoclonal antibody specific to immunoglobin M (Immunoglobulin M, Ig M).

Images