KR100478435B1 - Monoclonal antibody for Classical Swine Fever Virus (CSFV), hybridoma cell line producing the Kit for detecting Classical Swine Fever Virus (CSFV) using the monoclonal antibody for CSFV - Google Patents

Abstract

The present invention relates to monoclonal antibodies against Swine Fever Virus (CSFV), cell lines producing the same, and swine cholera virus detection and diagnostic kit using the monoclonal antibodies. More specifically, a coating monoclonal antibody that specifically reacts with porcine cholera virus is coated on an immunoplate and attached to the plate, and the monoclonal antibody prepared by conjugating the enzyme to the monoclonal antibody prepared above is reacted with a pretreated sample. The present invention relates to a method for quickly and accurately detecting the presence or absence of porcine cholera virus in a sample.

Description

Monoclonal antibody for Classical Swine Fever Virus (CSFV), hybridoma cell line producing the Kit for detecting Classical Swine Fever Virus (CSFV) using the monoclonal antibody for CSFV}

The present invention relates to a swine cholera virus diagnostic kit using a monoclonal antibody against the classical swine fever virus (CSFV). In particular, in the swine cholera virus detection method according to the present invention, a monoclonal antibody for detection, which is prepared by attaching a monoclonal antibody for a specific reaction to the swine cholera virus to an immunoplate and conjugating an enzyme to the monoclonal antibody, with a sample. By reacting, the presence or absence of porcine cholera virus in the sample is quickly and accurately diagnosed.

Swine cholera virus is a causative agent of swine cholera disease and is highly contagious, mortality and morbidity, and high susceptibility to infect 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. Swine cholera virus is the only natural host for pigs and is an important 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 before the disease occurs and continue to release the virus throughout the course of the disease. In addition, the virus is mainly released into oral, nasal, tear, urine, and feces. Pigs recovered from swine cholera disease continue to excrete the virus until a specific antibody titer is formed. Therefore, pigs infected with strong poisoning virus release large amounts of virus for 10 to 20 days, and low virulence virus infections have a very short virus ejection period. As a result, strong poisoning CSFV is rapidly spread and high morbidity in a group of pigs. Chronic infected pigs continue to release the virus continuously or intermittently until death, and pregnant pigs infected with low virulent viruses are initially invisible, but are spread to the fetus in the womb and deliver stillbirths or weak piglets. The piglets die soon after birth. Since CSFV is constantly secreted in the fetus, a large amount of virus is transmitted at delivery. If innately infected piglets are healthy, they are viable for several months, but the infection is difficult to recognize and also causes CSFV to spread the disease to piglets for months before eradication is eliminated as a cause of ongoing viral transmission. Infections can be transmitted from species to pigs or spread to pigs on new farms through contamination media in places where many pigs are gathered.

Pig cholera has occurred 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 near Seoul. Since then, the vaccine has been vaccinated with LOM strain, a pure cholera virulence virus, and there has been no recurrence of swine cholera disease in the past two years. According to OIE standards, a country that develops swine cholera may proclaim it after six months of discontinuation if the cholera does not occur for another year.

Accordingly, the present inventors conducted a continuous study to provide a method for quickly and accurately diagnosing the presence of porcine cholera virus causing a serious situation as described above, as a result of the monoclonal antibody for coating having a trapping ability against porcine cholera virus Using a detection monoclonal antibody prepared by conjugating an enzyme to a monoclonal antibody, a method for diagnosing the presence of a virus within 3 hours was examined by examining a sample taken from a pig suspected of swine cholera disease. In other words, the virus cultivation method or gene amplification method that requires 3 to 5 days is used as a method for detecting swine cholera virus, but the method according to the present invention can be diagnosed within a short time without any special technology. In addition, the presence of a large amount of antigens can be determined quickly and accurately.

It is therefore an object of the present invention to provide a monoclonal antibody for the coating of porcine cholera virus and a hybridoma cell line producing the same.

Another object of the present invention is to provide a monoclonal antibody for detecting porcine cholera virus and a hybridoma cell line producing the same.

Still another object of the present invention is to provide a method for diagnosing the presence of porcine cholera virus by conjugating an enzyme to a detection monoclonal antibody and a diagnostic kit for use in this purpose.

In order to achieve the object according to the present invention, the present inventors produce a monoclonal antibody for coating and a monoclonal antibody for detection and detection monoclonal antibody having a capturing ability against porcine cholera virus, as described in detail in the following Examples Conjugates of cloned antibodies and peroxidase were prepared, and a method for detecting porcine cholera virus in a sample was developed by enzyme-linked immunoassay using them. This method can be used to quickly and accurately diagnose the presence of antigen in a sample.

That is, the pig cholera virus diagnostic method using the enzyme-linked immunoassay according to the present invention,

(1) diluting the coating monoclonal antibody capable of capturing porcine cholera virus with a coating buffer, aliquoting the ELISA plate and adsorbing;

(2) washing and removing monoclonal antibodies not adsorbed on the plate;

(3) reacting the specimen to be tested on the plate;

(4) washing and removing specimens not captured in the monoclonal antibody for coating;

(5) reacting the detection monoclonal antibody with the conjugate of the enzyme;

(6) washing and removing unbound conjugates; And

(7) characterized in that it comprises the step of measuring the absorbance by the color reaction by adding a substrate and confirming the presence of porcine cholera virus. The detection method of porcine cholera antigens according to the present invention is shown schematically in FIG.

In addition, the kit for diagnosing porcine cholera virus according to the detection method according to the present invention includes (i) a monoclonal antibody for coating according to the present invention, and (ii) a monoclonal antibody for detecting according to the present invention and peroxidas. It comprises a conjugate, and (iii) a substrate for the peroxidase enzyme, the present invention is another object to provide such a diagnostic kit.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to help the understanding of the present invention, the scope of the present invention in any sense is not limited to these examples.

Example 1 Preparation of Coating Monoclonal Antibody

Step 1: Preparation of Immunogenic Protein

Porcine cholera virus 1 × 10 ⁴ TCID ₅₀ / ml (LOM strain, a vaccine strain in Korea) was inoculated into 4 × 10 ⁵ Cells / ml of pig kidney cells (PK-15, ATCC CRL-1542) Cultured for 4 days in a cutlet carbonate incubator (37 ℃). Cells and supernatant were harvested, repeated freeze thawing three times, and centrifuged at 6000 rpm for 30 minutes to harvest supernatant. The harvested supernatant was diluted 10-fold step by step, and 50 µl of each was added to a 96 well plate, 50 µl of medium without serum was added, and finally 50 µl of porcine kidney cells was added. After culturing for 4 days, virus titer was measured by staining with indirect immunoassay (Neutralizing Peroxidase Linked Assay (NPLA)) (result: 10 ⁷ TCID ₅₀ / ml). In addition, 0.001% of Binaryethyleneimine (BEI, sigma) was added to the harvested supernatant and incubated overnight at 37 ° C to inactivate the virus. Confirmation of inactivation is centrifuged inactivated culture medium at 6,000 rpm for 30 minutes, the supernatant is diluted 10 times step by step in the same manner as the pig cholera virus titer method and inoculated in pig kidney cells and cultured in a carbonated cutlet culturer for 4 days. Afterwards, it was confirmed that the virus was not inactivated at all when stained by indirect immunoassay (NPLA) and stained at all.

Step 2: Mouse Immunization for Monoclonal Antibody Production Cell Line Construction

The antigenic protein prepared in step 1 was mixed with incomplete adjuvant (Gibco BRL Co., Ltd.) in the same amount (1: 1 by volume) and then injected into the sole of the hind paw of BALB / C (female, 5-6 week old) mice. Inguinal lymph nodes were collected 14 days after immunization (between 10-15 days) and used for cell fusion.

Stage 3: Bone Marrow Cell Line Culture

4-5 days before cell fusion, remove myeloma cells SP2 / O-Ag14 (ACTC CRL-1581) from nitrogen bottle and suspend in DMEM (Gibco BRL) medium with 10% fetal calf serum and centrifuge at 500xg for 5 minutes. Separated. The supernatant was discarded and the precipitated cells were carefully suspended in DMEM with 10% fetal calf serum and cultured in a 37 ° C. incubator fed with 5% cutlet.

Step 4: Cell Fusion

Cell fusion was performed according to the general method using polyethylene glycol (Ed Harlow, David Lane: Antibodis, A laboratory manual. Cold Springs Harbor press, 1988 P139-244). On day 14 after the mice were immunized with the virus, lymph nodes were extracted from the inguinal lymph nodes of the mice. Lymphocytes from the inguinal lymph nodes were isolated using a cell strainer (Falcon) and diluted to a concentration of 10 ⁸ cells / ml. 10 ⁷ cells / ml of myeloma cells and 1 ml of PEG1500 (Sigma) were mixed in equal amounts and fused. After completion of the fusion, the cells were diluted in 200 ml of HAT (Sigma) medium, and the cells fused to 96 well tissue culture plates were dispensed.

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

The selection of monoclonal antibodies that specifically respond to porcine cholera virus was confirmed by the indirect immunofluorescent antibody technique (IFA). As in step 1, porcine cholera virus was inoculated into porcine kidney cells and incubated for 4 days in a cutlet carbonate incubator (37 ° C.), and the cells were washed once with phosphate buffered saline (PBS: pH 7.2). The washed cells were fixed for 5 minutes at room temperature with acetone / distilled water (80:20). After collecting the supernatant secreted from the fusion cells prepared in step 4 to the immobilized cells, the reaction mixture was raised to the reaction solution and allowed to react at room temperature for 1 hour, followed by anti-mouse IgG FITC (fluorosein isothiocyanate; KPL). G) was added at a concentration of 10 µg / ml and reacted at room temperature for 1 hour. After the reaction, washed three times with phosphate buffered saline (PBS: pH 7.2) three times for 5 minutes and fluorescence microscopy to examine the monoclonal antibody that specifically reacts with porcine cholera virus (see Figure 2). By repeating the selection method three times, cell lines producing monoclonal antibodies specific for porcine cholera virus were selected, and the cell lines were deposited with the Korea Cell Line Bank on October 9, 2002 (deposit name: CSFV-LOM 1; accession number KCLRF-BP-00069).

Step 6: Multiple Production of Monoclonal Antibodies

1 × 10 ⁶ cells / intraperitoneally in BALB / C (female, 10-12 week old) mice cultured with monoclonal antibody-producing cell line (CSFV-LOM 1) selected in step 5 and primed with pristen (sigma) 0.5 ml of the solution was injected at the concentration of ㎖, and ascites was produced in the abdominal cavity of the mouse. The harvested ascites was centrifuged at 10,000xg for 10 minutes and the supernatant was harvested to determine the optimal dilution concentration using check-borad titration (DM Kemeny and SJ Challacomble; ELISA and others solid phase immunoassay: Theroretical and Practical Aspects. Wiley Medical Publication. 1988) (see FIG. 6) was used as a monoclonal antibody for capturing coatings.

In FIG. 6, the virus is LOM strain of CSFV, and RK-15 cells are porcine kidney cells (acquired from ATCC CRL 1542).

Example 2 Preparation of Detection Monoclonal Antibody

Step 1: Preparation of Immunogenic Protein

Porcine cholera virus 1 × 10 ⁴ TCID ₅₀ / ml (LOM strain, a vaccine strain in Korea) was inoculated into 4 × 10 ⁵ Cells / ml of pig kidney cells (PK-15, ATCC CRL-1542) Cultured for 4 days in a cutlet carbonate incubator (37 ℃). Cells and supernatant were harvested, repeated freeze thawing three times, and centrifuged at 6000 rpm for 30 minutes to harvest supernatant. After diluting the harvested supernatant step by step 10 times, and finally added to the pig kidney cells 50μL in the same manner as in the swine cholera virus titer method of Example 1 step 1 and cultured in cutlet carbonate incubator (37 ℃) for 4 days The virus titer was then stained by indirect immunoassay chemistry (Neutralizing Peroxidase Linked Assay, NPLA) (titer results: 10 ⁷ TCID ₅₀ / ml). In addition, 0.001% of Binaryethyleneimine (BEI, sigma) was added to the harvested supernatant and incubated overnight at 37 ° C to inactivate the virus. Confirmation of inactivation is centrifuged inactivated culture medium at 6,000 rpm for 30 minutes, the supernatant is diluted 10 times step by step in the same manner as in the swine cholera virus titer of Example 1 step 1 inoculated in pig kidney cells and carbonated After incubation for 4 days in a cut incubator, it was confirmed that the virus was not inactivated at all when the dye was not stained at all by indirect immunoassay chemistry (NPLA).

Step 2: Mouse Immunization for Monoclonal Antibody Production Cell Line Construction

The antigenic protein prepared in step 1 was mixed in the same amount (1: 1 Volume) with incomplete adjuvant (Gibco BRL Co., Ltd.), and then injected into the sole of the leg of the BALB / C (female, 5-6 week old) mice. Inguinal lymph nodes were collected 14 days after immunization and used for cell fusion.

Stage 3: Bone Marrow Cell Line Culture

4-5 days before cell fusion, remove myeloma cells SP2 / O-Ag14 (ATCC CRL-1581) from nitrogen bottle and suspend in DMEM (Gibco BRL) medium with 10% fetal calf serum and centrifuge at 500xg for 5 minutes. Separated. The supernatant was discarded and the precipitated cells were carefully suspended in DMEM with 10% fetal calf serum and cultured in a 37 ° C. incubator fed with 5% cutlet.

Step 4: Cell Fusion

Cell fusion was performed according to the general method using polyethylene glycol (Ed Harlow, David Lane .: Antibodis, A laboratory manual. Cold Springs Harbor Laboratory press, 1988. P139-244). On day 14 after the mice were immunized with the virus, lymph nodes were extracted from the inguinal lymph nodes of the mice. Lymphocytes from the inguinal lymph nodes were isolated using a cell strainer (Falcon) and diluted to a concentration of 10 ⁸ cells / ml. 10 ⁷ cells / ml of myeloma cells and 1 ml of PEG1500 (Sigma) were mixed in equal amounts and fused. After completion of the fusion, the cells were diluted in 200 ml of HAT (Sigma) medium, and the cells fused to 96 well tissue culture plates were dispensed.

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

Selection of monoclonal antibodies that specifically respond to porcine cholera virus was confirmed by indirect immunoenzyme chemistry (NPLA). Porcine cholera virus was inoculated into porcine kidney cells as in step 1, incubated for 4 days in a cutlet carbonate incubator (37 ° C.), and the cells were washed once with phosphate buffered saline (PBS: pH 7.2). The washed cells were fixed for 5 minutes at room temperature with acetone / distilled water (80:20). The fixed cells were raised using the cell supernatant secreted from the fusion cells prepared in step 4 as a reaction solution, and then reacted at room temperature for 1 hour, and then washed three times with phosphate buffered saline (PBS: pH 7.2). Anti-mouse IgG HRP (horseradish peroxidase: KPL) diluted at a concentration of 1 μg / ml was added thereto, reacted at room temperature for 1 hour, and washed three times with phosphate buffered saline (PBS: pH 7.2). A monoclonal antibody that specifically reacts with porcine cholera virus by coloring with diaminobenzidine (DAB, Pierce) and 0.03% H ₂ O ₂ dissolved in 0.01 M PBS (pH 7.2) at a concentration of 2 μg / ml. Selected. By repeating the selection method three times, cell lines producing monoclonal antibodies specific for porcine cholera virus were selected (see FIG. 3). The cell lines were deposited with the Korea Cell Line Bank on October 9, 2002 (deposit name: CSFV- LOM 2; Accession No. KCLRF-BP-00070)

Step 6: Multiple Production, Purification and Peroxidase Conjugation of Monoclonal Antibodies

1x10 ⁶ intraperitoneally of BALB / C (female, 10-12 week old) mice cultured with the monoclonal antibody producing cell line (CSFV-LOM 2) selected in step 5 and primed with pristen (sigma) 0.5 ml of the cells / ml was injected and collected when ascites formed in the abdominal cavity of mice. The harvested ascites was centrifuged at 10,000 × g for 10 minutes and the supernatant was harvested and used as a monoclonal antibody for detection. Monoclonal antibodies were purified using immunoaffinity chromatography. That is, agarose beads (Bio-Rad) conjugated with Protein A were filled in a column, and monoclonal antibodies collected from the plurality were added. Pure monoclonal antibodies were isolated by eluting the monoclonal antibody attached to the protein A-agarose beads using an elution buffer (100 mM glycine PH 3.0) and then dialyzed in distilled water. The dialysis monoclonal antibody was concentrated in a lyophilizer and used for peroxidase conjugation.

In the conjugation step, 2 mg of peroxidase (Promega) was dissolved in distilled water, and then 0.1M sodium periodate (sodium periodate) was added and reacted at room temperature for 20 minutes. It was dialyzed in 1 mM sodium acetate buffer (pH 4.4). 4 mg of the purified monoclonal antibody was dissolved in carbonate buffer (pH 9.5), and then mixed in a 1: 1 equivalent ratio with the peroxidase solution dialyzed in the same amount of acetate buffer and reacted at room temperature for 2 hours. 5 μl of sodium borohydride (4 mg / ml) was added, followed by reaction at 4 ° C. for 2 hours, and dialyzed in phosphate buffer (pH). Optimal dilution concentrations were determined (see FIG. 7) and used.

In FIG. 7, the virus is LOM strain of CSFV, and PK-15 Cell is porcine kidney cell (acquired from ATCC CRL 1542).

Example 3 Detection of Porcine Cholera Virus Using a Monoclonal Antibody for Coating with Porcine Cholera Virus Capture Capability and a Detection Monoclonal Antibody Conjugated with Peroxidase

The monoclonal antibody for coating prepared in Example 1 was dissolved in 0.05M carbonate coating buffer at a concentration of 0.15µg / ml, and then dissolved in 100µl of ELISA plate and adsorbed at 4 ° C overnight. After the adsorption reaction, non-adsorbed monoclonal antibodies were removed by washing three times with washing solution (Tween20 0.05%, 10 mM phosphate buffer pH 7.2). In order to suppress the nonspecific reaction, 10 mM phosphate buffer (pH 7.2) containing 1% bovine serum albumin and 5% gelatin was dispensed in 300 μl onto a plate to which the coating antibody was adsorbed and reacted at 37 ° C. for 2 hours.

Prepare the test sample by reacting the sample to be tested with sample dilution 5% horse serum in TBST pH 7.3 for 1 hour, add 50 μl of the sample to the ELISA plate, and then dilute 5% horse serum in TBST pH 7.3 with the same amount. Was added. The plate to which the sample was added was reacted at 37 ° C for 90 minutes, and washed five times with a washing solution. The detection monoclonal antibody was diluted to a concentration of 1 µg / ml using a diluent, and 100 µl was added thereto, followed by reaction at 37 ° C for 1 hour. After the reaction, the solution was washed 5 times with a washing solution, and 100 μl of a colorant TMB substrate (1.2 mM 3,3; 5.5'-Tetramethylbenzidin.Moss) was added thereto for 10 minutes to react, followed by 50 μl of 0.5M sulfuric acid solution. Stopped. Within 10 minutes after stopping color development, absorbance at 450 nm was measured using an ELISA reader. In the result judgment, the strong positive control absorbance should be 0.8 or more, the weak positive control absorbance should be 0.35 or more, and the negative control absorbance should be 0.30 or less. The ratio between the difference between the sample absorbance and the negative control absorbance and the difference between the strongly positive control absorbance and the negative control absorbance was calculated, and a value of 0.20 or more was positive, and a value of 0.15 was negative. 8 and 9).

8 and 9 are diagrams for analyzing the positive sample and the negative sample of pig cholera infection by calculating the absorbance difference between the positive and negative control and the absorbance ratio of the sample. First, S / P-CP (%) is the ratio of the absorbance of the sample to the negative control. The ratio of the absorbance of the sample to the positive control can be explained easily. In other words, if the sample shows more than a certain absorbance ratio, it is a kind of formula for determining positive.

And, by calculating the absorbance ratio to analyze how accurately the sample is true positive (True Positive), true negative (False positive), false negative (False Negative) as a method for analyzing how accurately 9 method is used. 8 is a table showing the specificity and sensitivity numerically, Figure 9 is a graph showing the change in specificity and sensitivity values TG-ROC. In the present invention, the positive classification of more than 20%, negative less than 15%, and the value between the positive and false in the meanwhile to derive a judgment value that satisfies both the specificity (96.5%) and sensitivity (100%) highly. This is the result derived by 9.

As described in the above examples, using a monoclonal antibody for coating having a capturing ability to specifically react to porcine cholera virus, and a detection monoclonal antibody obtained by conjugating an enzyme to a detection monoclonal antibody, It can be detected quickly and accurately, making it extremely valuable for the hog industry.

1 is a diagram illustrating a method for detecting porcine cholera virus using an indirect enzyme-linked immunoassay developed using a monoclonal antibody according to the present invention;

Figure 2 is a result of the detection of porcine cholera virus by indirect fluorescent antibody chemistry using a monoclonal antibody according to the present invention;

3 is a table showing the reaction relationship between the monoclonal antibody and porcine cholera virus strain;

Figure 4 is a result of confirming the specificity for the pig cholera virus using a monoclonal antibody for the anti-swine cholera virus coating obtained in Example 1;

5 is a result of measuring the sensitivity of the tissue culture virus using a monoclonal antibody for anti-pigletera virus coating obtained in Example 1;

FIG. 6 is a determination table of optimal adsorption concentrations of a monoclonal antibody for coating having a capturing ability for detection of porcine cholera virus antigen obtained in Example 1; FIG.

FIG. 7 is a determinant table of optimal concentrations of anti-porcine cholera virus peroxidas conjugates for porcine cholera virus antigen detection; FIG.

8 is a table of criteria for detecting porcine cholera virus antigens;

9 is a TG-ROC assay table for detecting porcine cholera virus antigens.

Claims (7)

delete delete delete delete delete delete (Iii) a monoclonal antibody for coating that specifically reacts with porcine cholera virus, prepared from the hybridoma cell line CSFV-LOM1 (Accession No. KCLRF-BP-00069), (ii) the hybridoma cell line CSFV-LOM2 ( Prepared from Accession No. KCLRF-BP-00070), comprising a conjugate of a detection monoclonal antibody and peroxidase, which specifically reacts with porcine cholera virus, and (iii) a substrate for a peroxidase enzyme. Pig cholera virus diagnostic kit, characterized in that.