KR20010051314A - Monoclonal antibody for Hog cholera virus vaccine strain protein, hybridoma cell line producing the same and the methods of detecting Hog cholera virus antibody using the monoclonal antibody - Google Patents

Abstract

PURPOSE: Provided are a monoclonal antibody of Hog cholera virus(HCV) vaccine strain (LOM strain) proteins, a hybridoma strain producing the monoclonal antibody and a Hog cholera antibody detecting method using the monoclonal antibody. Therefore, the monoclonal antibody peroxidase conjugate can be used in detecting antibody for Hog cholera and its activity rapidly and exactly. CONSTITUTION: The monoclonal antibody specifically binds with Hog cholera virus vaccine strain proteins. The hybridoma strain producing the monoclonal antibody JENO HCV-3 Hybridoma cell(BALB/c Mice Myeloma)(KCTC 0878BP) is produced by the steps of: preparing hybridoma strains by fusion of inguinal lymphnode and myelocyte of mouse immunized by an immunogen of Hog cholera virus; adsorbing the immunogen of attenuated Hog cholera virus and Hog kidney cell protein as a control antigen onto a microplate for ELISA; reacting the microplate with the fermented culture of the hybridoma strain then with antimouse HRP conjugate, and adding ABTS pigments into the reaction mixture; and measuring the optical density of the reaction mixture to select the hybridoma strain producing monoclonal antibody specifically binding with Hog cholera.

Description

Monoclonal antibody against hog cholera vaccine strain virus protein, hybridoma cell line producing the same, and detection method of pig cholera virus vaccine using the monoclonal antibody (monoclonal antibody for Hog cholera virus vaccine strain protein, hybridoma cell line producing the same and the methods of detecting Hog cholera virus antibody using the monoclonal antibody}

The present invention relates to a monoclonal antibody against a swine cholera vaccine strain virus protein, a cell line producing the same, and a method for detecting swine cholera antibody using the monoclonal antibody. More specifically, the present invention relates to a monoclonal antibody that specifically reacts to the swine cholera vaccine strain virus E2 protein, a cell line producing the same, and the presence of antibody formation and antibody titers in pigs to which the pig cholera vaccine is administered using the monoclonal antibody. It relates to a method for quickly and accurately detecting.

Hog cholera (HC; Classical swine fever) is caused by hog cholera virus (HCV; Classical swine fever virus) and is one of the infectious diseases that cause economic loss to pig industry in domestic pig disease. Despite being vaccinated against live poison, the situation occurs sporadically. Swine cholera is highly contagious, mortality and morbidity and high susceptibility to pigs of all ages. Infected pigs usually involve high fever, skin flare, loss of appetite, constipation, diarrhea, leukocyte reduction, posterior palsy, and propagation disorders such as pseudoacids.

Porcine cholera has been reported worldwide since it was first introduced in the United States in 1830, but recently, it has not been reported in the United States, Canada, the United Kingdom, Iceland, Ireland, Scandinavia, New Zealand, and Australia. In Korea, it was officially reported in 1947 in a farm near Seoul. Since then, the vaccine has been immunized with LOM strain, a swine cholera purified live poison virus, but it is sporadically distributed every year in Korea. Eradication is needed.

Swine cholera is the only natural host of pigs and is a major source of the virus. Direct contact between infected pigs and susceptible pigs is the main route of transmission. Infected pigs begin to release the virus prior to the onset of disease and continue to release the virus throughout the entire disease period. HCV is mainly released into the oral nasal cavity, tear urine, feces, etc. Pigs recovered from HC disease continue to release the virus until the formation of specific antibodies. Thus, pigs infected with virulent HCV excrete large amounts of virus for 10-20 days. On the other hand, infection of low virulence virus after birth has a very short virus ejection period. Thus virulent HCV spreads rapidly in one herd and shows high morbidity. Chronic infected pigs release the virus continuously or intermittently until death. Pregnancy pigs infected with low virulence virus are initially inconsistent, but the virus spreads to the fetus in the womb and causes symptoms such as stillbirth or weak piglets, and dies shortly after birth. Since HCV occurs continuously in the fetus, a large amount of virus is transmitted at delivery. However, if congenital infected piglets are healthy, they are viable for several months, but the infection is difficult to recognize, and because of the ongoing virus transmission, HCV spreads the disease to piglets for months before eradication is eliminated. Infections may be transmitted from pig farms or to pigs on new farms through places where many pigs gather or through contamination media. Therefore, the best way to prevent swine cholera is to prepare a virus vaccine and administer it to pigs in advance to prevent infection. However, if the pig cholera virus vaccine is a weaning pig, the first dose must be inoculated twice at the age of 40 days and the second at 60 days, and the sow should be inoculated once a year. After confirming the degree of administration, the effect can be obtained. However, the conventional ELISA method or virus neutralization method for confirming this tends to take a long time and is less reliable.

In view of the above, the present inventors have studied to prepare a kit for rapidly and accurately diagnosing antibody formation and antibody titers in pigs after administering a prophylactic agent for porcine cholera virus. As a result, pig cholera virus vaccine strain (LOM strain) E2 Peroxidase was conjugated to the monoclonal antibody against the protein to detect antibody formation and titer within 3 hours when reacted with sera of vaccinated pigs. In other words, the antibody titer can be detected faster than the virus neutralization test, which takes 3 to 5 days. Thus, if there is an antibody with high titer before or after vaccination, it can be quickly detected and judged.

Accordingly, an object of the present invention is to provide a monoclonal antibody against a pig cholera vaccine strain viral protein and a hybridoma cell line producing the same.

Another object of the present invention is to provide a kit for measuring antibody formation and titer after porcine cholera virus vaccination by conjugating a peroxidase to a monoclonal antibody against a swine cholera virus vaccine protein.

Still another object of the present invention is to provide a method for rapidly and accurately detecting porcine cholera virus antibodies and antibody titers using the kit.

The object of the present invention is to inject an immunogen antigenic protein into a mouse with an antigen adjuvant to extract inguinal lymph nodes and to fused it with myeloma cells to produce hybridoma cell lines, and then to the immunoplates and control antigens on a microplate. After adsorbing and reacting the fusion cell culture, a cell line was produced which produced monoclonal antibodies specific for porcine cholera virus by reacting and developing anti-mouse horse radish peroxidase. Subsequently, the selected hybridoma cell line was injected intraperitoneally into a mouse to generate ascites, and a monoclonal antibody was obtained therefrom, conjugated with peroxidase, an antigen was distributed on a microplate, and the test serum was diluted and transferred to an ELISA plate. It was then achieved by reacting and developing the anti-porcine cholera monoclonal antibody peroxidase conjugate to detect porcine cholera antibody and its titer.

Hereinafter, the configuration of the present invention will be described in detail.

1 is a photograph showing the detection of E2 protein for porcine cholera virus by Western blotting using a monoclonal antibody against porcine cholera virus.

Figure 2 is a photograph showing the detection of porcine cholera vaccine strain virus (LOM strain) using a fluorescence antibody immunoassay using a monoclonal antibody against porcine cholera virus.

3 is a graph showing the optimal concentration of E2 protein adsorption for detecting porcine cholera virus antibody.

Figure 4 is a graph showing the determination of the optimal serum dilution concentration for the antibody detection for porcine cholera virus.

Figure 5 is a graph showing the determination of the optimal anti-piglet cholera virus peroxyday conjugate concentration for porcine cholera virus antibody detection

6 is a flowchart illustrating a method of detecting an antibody against porcine cholera virus using an anti-porcine cholera virus monoclonal antibody.

The present invention is inoculated with porcine cholera virus vaccine in pig kidney cells as a mouse immunization antigen and cultured by measuring the virus titer, inactivated the culture supernatant and again measured the virus titer to observe the presence of virus proliferation antigen protein Preparing a; Admixing the antigenic protein with an adjuvant to inject the mouse and harvesting inguinal lymph nodes; Preparing a bone marrow cell line by culturing myeloma cell SP2 / O-Ag14 in Dulbecco's modified eagle media (D-MEM) medium containing fetal bovine serum; Preparing a hybridoma cell line by fusing the inguinal lymph nodes obtained from the immunized mice with a bone marrow cell line prepared by culturing; The immunogen inactivated porcine cholera virus and the control antigen were adsorbed onto an ELISA microplate, reacted with the hybridoma cell culture supernatant, and then reacted with an anti-mouse HRP (horse reddish peroxidase (HRP)) conjugate. , 2'-azino-di [3-ethylbenzthia-

zolin sulfonate] diaminium salt) selecting a cell line producing monoclonal antibodies specific for porcine cholera virus by adding a colorant and measuring absorbance; Injecting the selected monoclonal antibody-producing cell line into a mouse intraperitoneally to generate ascites, purifying monoclonal antibody from the same, concentrating and conjugating it by reacting with peroxidase; Synthesizing cDNA from RNA of porcine cholera vaccine strain LOM strain and cloning the protein gene LOME2 obtained by pGemT and cloning the gene into a baculovirus expression vector to produce a recombinant protein; The recombinant protein LOME2 produced above was dispensed on microplates, diluted with serum and transferred to an ELISA plate, and then reacted with an anti-porcine cholera monoclonal antibody peroxidase conjugate, and a colorant ABTS was added, followed by measurement of absorbance. Detecting antibody titers.

The present invention provides an ELISA plate prepared by conjugating a monoclonal antibody produced from a cell line producing a specific monoclonal antibody against swine cholera virus with a peroxidase and dispensing a recombinant protein antigen with an ELISA plate, and diluting the test serum. After transfer to the anti-porcine cholera monoclonal antibody peroxidase conjugate and the color was developed a kit for detecting porcine cholera virus antibody using the principle of detecting the porcine cholera antibody and its titer.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Hybridoma Cell Line Producing Monoclonal Antibody to Porcine Cholera Vaccine Virus Protein

Step 1: Preparation of Antigen Protein for Immunization

Porcine cholera vaccine strain (LOM strain) was inoculated into pig kidney cells as a mouse immunization antigen and cultured in a cutlet carbonate incubator for 4 days. Cells and the supernatant were harvested, repeated freeze-thawing three times, and centrifuged at 6000 rpm for 30 minutes to harvest the supernatant. The harvested supernatants were inoculated into pig kidney cells by 10-fold step dilution, and cultured in a carbonated cutlet incubator for 4 days and stained for viral titers. In addition, the harvested supernatant was inactivated overnight at 37 ℃ by adding 0.001% of Binary ethyleneimine (BEI, sigma). To confirm inactivation, the supernatant was inoculated into pig kidney cells by 10-fold step dilution, incubated in a carbonated cutlet culturer for 4 days, and stained to confirm the proliferation of the virus.

Second Step: Mouse Immunization for Monoclonal Antibody Production Cell Line Preparation

The antigen protein prepared in the first step was well mixed 1: 1 with incomplete adjuvant (Gibco BRL Co., Ltd.), and then injected into the foot of the hind feet by 50-100 μl of Balb / C mice. Inguinal lymph nodes were collected between 10-15 days post-immunization and used for cell fusion.

Third Step: Bone Marrow Cell Line Culture

Four to five days before cell fusion, myeloma cells, SP2 / 0-Ag14, were removed from the nitrogen tube and suspended in DMEM medium containing 10% fetal calf serum and separated by 500 x g for 5 minutes. The supernatant was discarded and the precipitated cells were carefully suspended in DMEM with 10% fetal calf serum and incubated in a 37 ° C. incubator fed with 5% cutlet.

Fourth Process: Cell Fusion

Cell fusion was carried out as follows using a general method using polyethylene glycol. Lymph nodes were extracted from the inguinal lymph nodes of immunized mice. Lymphocytes were isolated from inguinal lymph nodes extracted with DMEM, mixed with myeloma cells to be fused, and mixed with PEG1500. After completion of the fusion, the cells were properly diluted in HAT medium, and the cells fused to 96well tissue culture plates were dispensed.

Step 5: Selection of Cell Lines Producing Monoclonal Antibodies Specific to Porcine Cholera Virus Proteins

Inactivated porcine choleravirus supernatant, which is the immunogen used in the first step, and porcine kidney cell protein treated with 1% Triton-X 100 (bio-rad) as a control antigen. Each cell culture supernatant was reacted for 1 hour. After washing four times with phosphate buffer, the anti-mouse HRP conjugate (kpl) was reacted. After washing four times with phosphate buffer, ABTS color development was performed, followed by color development for 10 minutes. A spectrometer was used to measure 405 nm and cloned by selecting fusion cells with an absorbance ratio of 2 or more between porcine cholera virus antigen and control antigen, and repeating the selection method three times to produce a specific monoclonal antibody against swine cholera virus. Cell lines were selected. This shows a monoclonal antibody that specifically reacts with porcine cholera virus protein by Western blotting method, and used as a control the normal pig kidney cells. In addition, in FIG. 2, the porcine cholera virus-infected porcine kidney cells and the uninfected porcine kidney cells were identified by the fluorescence antibody immunoassay to confirm that they are monoclonal antibodies that specifically react with the porcine cholera virus protein. This cell line, which produces monoclonal antibodies that specifically react with porcine cholera virus proteins, is named JENO HCV-3 Hybridoma cell (BALB / c Mice Myeloma). It was.

Example 2: Detection of Porcine Cholera Virus Antibodies and Antibody Titers

First Step: Multiple Production, Purification and Monoclonal Antibody Conjugation of Jose Radish Peroxidase

Cell lines producing the monoclonal antibody selected in the fifth step of Example 1 were cultured 1 × 10 ⁶ and injected into the abdominal cavity of Balb / c mice priming with Pristen (Sigma, Inc.). The harvested ascites was centrifuged at 10,000 X g for 10 minutes, and the supernatant was harvested and used for purification. Purification of monoclonal antibodies from ascites was performed using immunoaffinity chromato-garphy method. Agarose beads conjugated with Protein A (Pharmacia Co., Ltd.) were charged to the column and then reacted with a monoclonal antibody treated with ammonium sulfate. Monoclonal antibodies attached to Protein A agarose beads were separated from pure monoclonal antibodies using elution buffer and dialyzed in distilled water. Dialysed monoclonal antibodies were concentrated using a lyophilizer and used for peroxidase conjugation. Conjugation was performed by dissolving 10 mg of peroxidase in phosphate buffer (0.1 M, pH 6.8), adding glutaraldehyde to 1.25%, and reacting at room temperature overnight. Activated peroxidase and glutaraldhyde were distinguished by gel-filtration. 10 mg of the purified monoclonal antibody was dissolved in carbonate buffer (0.5M, pH9.5), mixed with an appropriate amount of activated peroxidase, and reacted at 4 ° C. overnight. 50 μl of sodium borohydride (10 mg / ml) was added, followed by reaction at 4 ° C. for 1 hour, and then dialyzed into a phosphate buffer solution.

Process 2: Production of Porcine Cholera Recombinant Protein for ELISA Antigen

Extract RNA from porcine cholera virus vaccine LOM strain and primer (5'-CGCGGATCCT-

After synthesis of 1st cDNA using TCTGCGAAGTAATCTGA-3), 5'-CGCGGATCCCGGCTAGCCTGCAAGGAAGAT-3 'and reverse primer 5'-CGCGGATCCTTCTGCGAAG as forward primer

TAATCTGA-3 'was used to amplify the thermocycler (PerlkinElmer) for 35 cycles by polymerase chain reaction (PCR) conditions for 1 cycle at 92 ° C, 1 minute at 45 ° C, and 2 minutes at 72 ° C. The amplified gene was named LOME2-1, which was purified and cloned into pGemT (promega) and named as pGemLOME2-1. Also, the gene LOME2-1 was cloned into the baculovirus expression vector and called pBacLOME2-1. Used. Recombinant protein was found to react with the monoclonal antibody that specifically reacts with porcine cholera virus as shown in FIG.

Third Step: Antibody and Antibody Titer Detection Using Anti-Swine Cholera Virus Monoclonal Antibody Conjugate

The recombinant LOM-E2 protein produced in the second process was diluted 1:50 and dissolved in 0.05M carbonate buffer, and then, 100 µl was aliquoted into an enzyme immunoassay microplate and adsorbed at 4 ° C. overnight. At this time, the optimal concentration of the antigen adsorbed on the plate is shown in FIG. The adsorbed antigen was discarded from the plate and washed once with washing solution (Tween20 0.05%, 10 mM phosphate buffer pH 7.2). 100 μl of the antigen-adsorbed plate was added to 10 mM phosphate buffer (pH 7.2) containing 1% bovine serum albumin and 3% gelatin, followed by reaction at 37 ° C. for 2 hours. Diluted the test serum with serum dilution buffer (Tween 20 at 1%, 10 mM phosphate buffer pH 7.2) twice in the dilution plate (4-fold step dilution in potency test). After transfer to the finished ELISA plate and reacted at 37 ℃ for 1 hour. At this time, the optimal serum dilution concentration for the antibody detection for porcine cholera virus is shown in FIG. After the reaction, the cells were washed three times or more with the washing solution, and 1/500 dilution of the anti-pork cholera monoclonal antibody peroxydays conjugate with 1% Tween 20-phosphate buffer was added thereto, and 100 μl of each was added. Reacted. At this time, the optimum concentration of the anti-pigletola virus peroxidase conjugate for porcine cholera virus antibody detection is shown in FIG. After the reaction, the remaining solution after washing with the phosphate buffer solution added 1% Tween 20 at least three times as a washing solution was removed. 100 μl of ABTS (KPL) solution as a colorant was added for 10 minutes, and the absorbance was measured at 405 nm using an ELISA reader. Judgment of results should maintain the standard negative serum absorbance above 0.9 and the standard positive serum absorbance below 0.35. The percentage of test serum / standard negative serum absorbance (%) was positive if less than 60%, positive if it was 60% to 70%, and negative if more than 70%. Antibody titer was positive in the dilution factor of serum. The inverse of the last dilution factor was determined as the titer. The procedure is shown schematically in FIG.

As described above, the anti-piglet choleravirus monoclonal antibody peroxidase conjugate obtained by conjugating peroxidase to a monoclonal antibody that specifically reacts with the porcine cholera virus vaccine protein as described above is used for the porcine cholera virus. It is a very useful invention in the hog industry because it has an excellent effect of detecting antibody presence and antibody titer quickly and accurately.

Claims (5)

Monoclonal antibody that specifically reacts with porcine cholera virus vaccine protein. Preparing a hybridoma cell line by fusing inguinal lymph nodes and bone marrow cell lines of mice immunized with porcine cholera virus immunogens; Adsorbing each of the inactivated porcine choleravirus immunogens and a control antigen, porcine kidney cell protein treated with 1% TritonX, to a microplate for ELISA; Reacting the microplates to which the immunogens and the control antigens are adsorbed with the hybridoma cell line culture medium, and then reacting the anti-mouse HRP conjugates and applying color development by ABTS color development; Hybridoma cell line JENO HCV-3 Hybridoma cell (BALB / c Mice Myeloma) (KCTC) comprising the steps of selecting a hybridoma cell line producing monoclonal antibodies specific for porcine cholera virus by measuring absorbance 0878BP). A hybridoma cell line JENO HCV-3 Hybridoma cell (BALB / c Mice Myeloma) (KCTC 0878BP), which is obtained by the method of claim 2 and produces a monoclonal antibody that specifically reacts to porcine choleravirus vaccine strain proteins. An anti-swine choleravirus monoclonal antibody peroxidase conjugate, which is obtained by concentrating and purifying monoclonal antibodies that specifically react to the porcine cholera vaccine strain protein according to claim 1, followed by conjugation with peroxidase. A method of using the anti-pigmentary choleravirus monoclonal antibody peroxidase conjugate according to claim 4 in a kit for antibody presence and antibody titer test against porcine cholera virus.