KR20190138610A - Monoclonal antibody against immunoglobulin m of rainbow trout and use thereof - Google Patents

Abstract

The present invention relates to a monoclonal antibody that specifically binds to immunoglobulin M of rainbow trout, and a hybridoma cell line producing the same. The monoclonal antibody of the present invention specifically binds to immunoglobulin M of rainbow trout, and thus can be preferably used for detecting a specific antibody of rainbow trout against fish antigens.

Description

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

The present invention rainbow trout (rainbow trout, Oncorhynchus mykiss ) monoclonal antibody specific for immunoglobulin M (Ig M) and its use, and more specifically, a fusion cell line producing monoclonal antibody specific for immunoglobin M of rainbow trout and monoclonal produced therein The present invention relates to a method for detecting an antibody in rainbow trout serum using an antibody.

With the activation of the fish farming industry worldwide, the damage caused by fish disease has increased considerably, which is a major problem for the aquaculture industry. In particular, the mortality rate from viral diseases is estimated to account for more than 30% of fish production. However, the rapid diagnosis and prevention of disease remains a major challenge because there is no effective treatment to date.

In Korea, in order to systematically manage aquatic biological diseases and to minimize the damages caused by diseases, the Aquatic Disease Control Act has been implemented since 2008 to carry out quarantine and quarantine targeting 21 infectious diseases. The list of prevention measures for each situation of disease occurrence is divided to carry out staged prevention measures of killing, quarantine, and restriction of movement.

Among the marine aquatic diseases that are the target of quarantine and quarantine, 67% of viral diseases are viral diseases. In Korea, the diagnosis of viral infectious diseases used for quarantine and quarantine is mainly carried out by PCR targeting the viral genome. . In addition, isolation cultures and pathological methods using fish chemotaxis cells are used in parallel. In the case of fish, PCR is mainly used, and shellfish and crustaceans use both PCR and pathological methods. However, immunological methods are rarely used because they do not yet have specific antibodies against the pathogenic virus.

The diagnostic method by PCR is widely used as a virus detection method because it can be quickly diagnosed.However, quarantine of marine products from foreign countries, epidemiological investigations in aquaculture farms, pathological assessment, and periodical identification of virus infection in fish There are many inadequacies for use in quarantine protection, so there is a limit to application in the field. In other words, since PCR is a method of directly detecting pathogens, even aquatic products, such as koi carp, can be tested only by killing them. In addition, it is difficult to process a plurality of samples at one time, and has a disadvantage in that a large number of samples are expensive to inspect. In addition, since PCR tests target genes, genes are detected even when pathogens are inactivated. When two or more viruses are mixed and infected in cultured fish, accurate diagnosis is difficult.

In contrast, immunological diagnostic methods are methods for diagnosing pathogen infections 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.

The immunological diagnostic method by direct method is a method of directly detecting a pathogen, and has the promptness to test a disease within 6 hours. In addition, the sensitivity and specificity is very high, it is easy to trace the pathogen in the body, it is also possible to check the infected cells and the degree of infection. Once the stable production of antibodies specific for the pathogen is possible, the diagnosis kit can be easily diagnosed at the aquaculture site by producing a diagnostic kit.

Indirect immunological diagnosis is a method of detecting specific antibodies present in the blood. It is possible to process a large number of samples at low cost, and has high detection sensitivity and high reliability. It is widely used for monitoring and verification of vaccine efficacy.

Indirect immunological diagnosis does not require killing the organism to be tested and can be diagnosed with only a small amount of serum. In addition, a large number of samples can be quickly diagnosed at low cost. By using the antibody detection ELISA method, more than 300 samples can be processed at one time, and 15 times more samples can be processed at about 100 times lower cost than PCR. In addition, it is possible to diagnose the non-existent infected fish and grasp the virus infection history, and is useful for determining the efficacy of the vaccine after determining the efficacy of the vaccine and performing the prevention of the infection.

In order to use such indirect immunological diagnosis, a specific antibody to IgM of fish is required. However, fish antibodies are known to be composed of IgM with low affinity unlike mammalian IgG. When antibody detection ELISA is applied to fish, high background is caused by nonspecific adsorption of fish IgM and blocking agent. Problems have not been reported in mammals. For this reason, OIE does not accept antibody detection ELISA as a diagnostic method because of its low reproducibility.

However, recent studies have suggested methods for inhibiting nonspecific adsorption of fish IgM with blocking agents. Therefore, there is an urgent need for a low specific non-specific adsorption with a blocking agent on the ELISA and to produce and isolate specific antibodies against fish IgM and use them for diagnosis of aquatic pathogen infection.

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

The present invention specifically binds to rainbow trout immunoglobulin M and provides a monoclonal antibody, a fusion cell line producing the monoclonal antibody, and a method of using the same, which can be preferably used for detecting specific antibodies of rainbow trout against fish antigens. It aims to do it.

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

Specifically, the present invention rainbow trout ( Oncorhynchus mykiss ) is directed to a fusion cell line producing monoclonal antibodies specific for immunoglobulin M (Ig M). In this aspect, the present invention specifically the fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438 for producing monoclonal antibodies specific for immunoglobin M (Ig M) of rainbow trout ( Oncorhynchus mykiss ) Can be.

In another aspect, the present invention is a rainbow trout ( Oncorhynchus mykiss ) to a monoclonal antibody produced in a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438, which produces monoclonal antibodies specific for immunoglobin M (Ig M). Specifically, the present invention rainbow trout ( Oncorhynchus mykiss ) may be a monoclonal antibody produced in a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438 that produces a monoclonal antibody specific for immunoglobin M (Ig M).

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

In detail, the kit for diagnosing an infectious disease of rainbow trout may include a monoclonal antibody specific for immunoglobin M of rainbow trout produced by a fusion cell line producing monoclonal antibodies specific for immunoglobin M of rainbow trout. . The fusion cell line may be a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438.

In this aspect, the present invention preferably the kit for diagnosing the infectious disease of rainbow trout is a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438 for producing monoclonal antibodies specific for immunoglobin M of rainbow trout. It may be a kit for diagnosing an infectious disease of rainbow trout comprising a monoclonal antibody specific for immunoglobin M of rainbow trout produced by.

The diagnostic kit may include detecting an antigen-antibody complex by color particle binding, preferably an antibody condensed with a label that reacts with a substrate and develops color; And a chromogenic substrate, wherein the label 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 monoclonal specific for the rainbow trout immunoglobin M produced by the fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438, which produces monoclonal antibodies specific for immunoglobin M of rainbow trout. It may be by rapid immunochromatography including a conjugate of an antibody and a gold, a membrane coated with an antibody specific for mouse immunoglobulin, and a strip to which a test pad of a subject is attached.

In another aspect, the present invention may be directed to a composition for detecting rainbow trout specific antibodies to fish antigens.

Preferably, the composition for detecting a specific antibody of rainbow trout against the fish antigen is produced by a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438 which produces monoclonal antibodies specific for immunoglobin M of rainbow trout. It may be to include a monoclonal antibody.

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

1 is an SDS-PAGE photograph of purified rainbow trout IgM according to one embodiment of the present invention.
Figure 2 shows the Western blotting results showing the rainbow trout IgM recognition site of the monoclonal antibody, according to an embodiment of the present invention.
Figure 3 is an ELISA result showing the response of the monoclonal antibody to MBP IgM, according to an embodiment of the present invention.
Figure 4 is a graph showing the specific rainbow trout antibody detection results for bovine serum albumin (BSA) using a monoclonal antibody, according to an embodiment of the present invention.
5 is a table showing isotyping results of 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 is excellent in the sensitivity and specificity of the rainbow trout to immunoglobin M (Ig M) and specific to the immunoglobin M (Ig M) of the rainbow trout, rainbow rainbow trout that is preferable for the specific antibody detection of rainbow trout against fish antigen It relates to a monoclonal antibody specific for immunoglobin M, a fusion cell line producing the same and its use.

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

Example

Experimental Method

1.Isolation of Serum from Rainbow Trout

The rainbow trout serum used in the experiment was collected from the tail vein of the rainbow trout (weight: 900-1,200 g) in the Namhae Specialty Research Center, Jeollanam-do Marine Fisheries Research Institute. Serum was isolated. Serum was stored at −80 ° C. until used in the experiment.

2. Rainbow Trout IgM Tablets

IgM in rainbow trout serum was purified using a 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 1: 1 of rainbow trout serum and ImmunoPure IgM binding Buffer, add column and react at 4 ℃ for 30 minutes. To remove the unbind protein, 42 ml of ImmunoPure IgM binding buffer was added to the column and washed. 3 ml of ImmunoPure IgM elution buffer was added to the column and reacted at room temperature for 12 hours to collect rainbow trout IgM. Purified rainbow trout IgM (MBP IgM) was stored at 80 ° C. until used in the experiment.

3. Structural Protein Analysis of Rainbow Trout IgM

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

4. Rainbow Trout IgM Immunization and Hybridoma Production

Immunization was first inoculated into the abdominal cavity of two BALB / c mice in 100 uL of purified rainbow trout IgM (MBP IgM, 100 ug) and complete Freund's adjuvant (Gibco, USA), followed by two weeks of purification. Second inoculation with rainbow trout IgM. One week later, after inoculation with purified rainbow trout IgM, the spleen was isolated from the mouse and fused with SP2 / 0 myeloma cells using polyethylene glycol (Roche, Germany), followed by the addition of 10% fetal bovine serum. HAT medium (0.1 mM hypoxanthine, 4 × 10 ^-4 mMaminoprotein, 1.6 × 10 ^-2 mMthymidineinDulbecco's modified eagle medium) was suspended and dispensed into 96 well plates and incubated in a CO ₂ incubator set at 37 ° C.

5. Hybridoma  Screening

ELISA was performed to screen hybridomas that produce antibodies that specifically react to rainbow trout IgM. Antigen was dispensed with purified rainbow trout IgM 50 96 (250 ng / well) per well in 96 well plate and coated overnight at 4 ℃ or 2 hours at 37 ℃. After blocking by blocking 200 μl of 2% skim milk, washing was performed three times with TBST, and 50 μl of the hybridoma culture supernatant as a primary antibody was dispensed per well for 2 hours at 37 ° C. After washing three times with TBST, goat anti-mouse IgG labeled with HRP was diluted 5,000-fold with 2% skim milk as a secondary antibody, and 50 µl of each well was reacted at 37 ° C for 1 hour. After washing 5 times with TBST, 50 μl of color reagents (TMB, color reagents) were dispensed per well. 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. Western blot Monoclonal  Antibody Response Investigation

Western immunoblot analysis was performed to confirm the recognition site of monoclonal antibody against rainbow trout IgM. SDS-PAGE was carried out in the same manner as above. After electrophoresis of purified rainbow trout IgM, the protein in the gel was blotting on the nitrocellurose membrane (advantec, Japan) for 1 hour at 144 하여 using a transblot apparatus (ATTO, Japan). After blotting, the reaction was blocked with 2% skim milk for 1 hour, and then washed 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 for 1 hour and then washed in the same manner as above. As a secondary antibody, goat polyclonal anti-mouse IgG antibody (novus, USA) labeled with alkaline phosphatase (AP) was diluted 1,000-fold with 2% skim milk, reacted for 1 hour, washed five times, and developed as a coloring agent (100 mM Tris). -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- 70 μl of indolyl phosphate p-toluidine salt / dimethyl-formamide) was visually confirmed. After completion of color development, color stop solution (1 mM EDTA, 10 mM Tris-HCl) was added.

7. Using ELISA Monoclonal  Investigation of antibody response

ELISA was performed to confirm the response of the monoclonal antibody to MBP IgM. About 250 ng of MBP IgM was dispensed into 50 well each of 96 well ELISA microplates (Greiner-bio-one, Germany) and then coated overnight at 37 ° C. Washed three times with T-PBS (0.05% Tween-20 / PBS (v / v)) and 5% skim milk was dispensed by 380 μl and blocked at 25 ° C. for 1 hour. 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, horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (Youngin, Korea) was diluted 1,000-fold with 5% skim milk, and 50 μl were dispensed per well. Reaction was time. After washing 5 times with T-PBS, 50 μl of ELISA coloring solution (TMB, color reagents) was dispensed per well for color development. 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.

8. Detection of specific rainbow trout antibodies against Bovine serum albumin (BSA)

Rainbow trout (9001,200 g), bred at the Namhae Specialty Laboratory, Jeollanam-do Marine Fisheries Research Institute, injected 1 mg / 200 μl of BSA into the abdominal cavity, and then housed in a 2 ton tank (water temperature: 13-16 ° C.) for 61 days. It was. On day 12 of breeding, 1 mg / 200 μl of BSA was injected again. After BSA inoculation, blood was collected from the arrhythmia of rainbow trout at 12, 24, 40, and 61 days by about 1 ml of blood, and then centrifuged (6,000 rpm, 4 ° C., 20 minutes) to separate serum. The separated serum was stored at -80 ° C until used in the experiment.

To detect specific rainbow trout antibodies to BSA, ELSIA was performed using the serum obtained above. 5 μg BSA / 50 μl was dispensed into each well on ELISA plates, and then antigen coated at 37 ° C. overnight. Washed three times with T-PBS (0.05% Tween-20 / PBS (v / v)) and 380 μl of 5% skim milk were blocked at 25 ° C. for 1 hour. After washing three times with T-PBS, the rainbow trout serum collected from the stomach was diluted 40-fold with 5% skim milk for 1 hour, and then 50 μl was dispensed into two wells per sample. 50 μl of the monoclonal antibody was used as an antibody and goat anti-mouse IgG (Youngin, labeled with horseradish peroxidase (HRP) diluted 1,000-fold in 5% skim milk was used as a tertiary antibody. Korea) was dispensed 50 μl each. Each antibody reaction was reacted at 25 ° C. for 1 hour. After washing with T-PBS five times, 50 μl of ELISA coloring solution (TMB, color reagents) was 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 reaction, and the absorbance was measured at 450 nm.

9. Monoclonal antibody Isotyping

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

<Experiment Result>

Rainbow Trout IgM  refine

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

2. Hybridoma  Production and Monoclonal  Screening of Antibodies

Hybridomas were prepared by fusion of Sp2 / 0Ag14 myeloma cells with Sp2 tissue from mice immunized with MBP IgM with PEG. Antibodies produced from hybridomas were screened by ELISA and western blot. As a result of cloning four times with ELISA of 47 samples, one clone (44H10) was finally selected.

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

In subsequent experiments, specificity studies were performed using antibodies produced from one clone (the name of the antibody: identically to the name of the clone).

3. Rainbow Trout IgM For Monoclonal  Investigation of recognition site of antibody

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

4. Monoclonal  Rainbow Trout of Antibodies IgM  reaction

Figure 3 is an ELISA result showing the response of the monoclonal antibody to MBP IgM. 44H10 showed 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 against BSA

Figure 4 is a graph showing the results of detection of specific rainbow trout antibody for BSA using a monoclonal antibody. In order to investigate the specificity of the monoclonal antibody prepared in the present invention, after immunizing BSA to rainbow trout, serum values were isolated at 12, 24, 40 and 61 days to determine the antibody titer against BSA. Serum from rainbow trout immunized with BSA showed an increase in antibody titer over time. No increase in antibody titer was observed in the rainbow trout (control 1, control 2) of the control. In other words, antibody titers were measured at days 40 and 61 in rainbow trout (BSA 1 and BSA2) immunized with BSA.

6. Monoclonal  Antibody isotype

5 is a table showing isotyping results of monoclonal antibody (44H10) against MBP IgM. Using the rapid ELISA mouse mAb isotyping kit, the monoclonal antibodies prepared with MBP IgM were examined for subtypes of heavy and light chains of IgG. H-chain was identified as IgG1 and L-chain as κ.

Korea Cell Line Research Foundation KCLRFBP00438P 20180614

Claims (8)

Rainbow Trout ( Oncorhynchus Immune globin of mykiss) M (Immunoglobulin M, Ig M) in specific monoclonal the immune globin M of rainbow trout are produced by the fusion cell line (RT IgM 44H10) of deposition No. KCLRF-BP-00438 which produces specific monoclonal antibody Kit for diagnosing infectious diseases of rainbow trout containing an antibody. The method of claim 1,
The diagnostic kit is a diagnostic kit for infectious diseases of rainbow trout that detects the antigen-antibody complex by color particle binding. The method of claim 2,
The diagnostic kit includes an antibody condensed with a label that reacts with a substrate and develops color; And a chromogenic substrate, wherein the label 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 diagnostic kit for infectious diseases of rainbow trout of the strip type using immunochromatography. The method of claim 4, wherein
The strip-type diagnostic kit is specific for monoclonal antibodies and gold conjugates specific for immunoglobin M of rainbow trout, produced by a fusion cell line producing monoclonal antibodies specific for immunoglobin M of rainbow trout, and mouse immunoglobulins. Kit for diagnosing infectious diseases of rainbow trout by rapid immunochromatography comprising a membrane coated with an antibody and a strip with a subject absorbent pad. Rainbow Trout ( Oncorhynchus Immune globin M (Immunoglobulin M, Ig M) rainbow trout with fish antigen comprising a monoclonal antibody produced from accession number of fused cell lines (RT IgM 44H10 of KCLRF-BP-00438) for producing a specific monoclonal antibody to the mykiss) Specific antibody detection composition. Rainbow Trout ( Oncorhynchus monoclonal antibody produced in a fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438, which produces monoclonal antibodies specific for immunoglobin M (Ig M) of mykiss ). Rainbow Trout ( Oncorhynchus mykiss ) fusion cell line (RT IgM 44H10) of Accession No. KCLRF-BP-00438 which produces monoclonal antibodies specific for immunoglobulin M (Ig M).

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