KR100307669B1 - Monoclonal Antibody-secreting Hybridoma for Detection of Lily Symptomless Virus and Preparation Method thereof and Monoclonal Antibodies - Google Patents

Abstract

The present invention relates to a monoclonal antibody secreted by hybridomas and hybridomas, which are fusion cells secreting a monoclonal antibody for lily symptomless virus (LSV) assay. Lily potential viruses are common in all parts of the world where lilies are grown, and these infections are a limiting factor in the production of high-quality cut flowers, as well as the production of superior species for lily production. The present invention is to develop a monoclonal antibody that specifically reacts to the lily potential virus using cell fusion technology to test the infection of the virus to a large number of samples, and the economic effect of the import of conventional antibody reagent It can solve various problems such as deterioration and timely supply caused during transportation during performance import.

Description

Hybridoma secreting monoclonal antibody for lily potential virus assay and preparation method and monoclonal antibody {Monoclonal Antibody-secreting Hybridoma for Detection of Lily Symptomless Virus and Preparation Method about and Monoclonal Antibodies}

The present invention relates to hybridomas, fusion cells that secrete monoclonal antibodies for lily symptomless virus (LSV) assay, methods for their preparation, and monoclonal antibodies secreted by hybridomas.

In order to control the lily potential virus, it is necessary to accurately test whether the virus is infected. In particular, during the production of high quality seedlings, an assay reagent that can easily and economically test a large amount of samples is required. In the past, such assay antibody reagents are mostly imported at a high cost from foreign countries. In some cases, it may deteriorate or the timely supply may not be smooth. Therefore, the inventors of the plant pathology department of Agricultural Science and Technology Institute conducted a study for 2 years from 1996, to produce a monoclonal antibody for LSV assay.

Lily potential virus is common in all parts of the world where lily is grown. The import of lily cultivation area and species in Korea is increasing and its importance is increasing. However, virus infection has become a limiting factor for the production of high quality cut lilies as well as the production of high quality lily. In recent years, randomly harvested casablanca varieties from the field showed a high infection rate of more than 95%, and the tissue culture in the cabin showed an infection rate of about 30-60%. The high rate of infection of lily potential virus is because lily grows through tissue culture and bulb growth, so if the mother is infected with LSV, the infection continues until later and drug control is impossible due to the biological characteristics of the virus. In order to control the virus, it is necessary to accurately test whether the virus is infected. To this end, an object of the present invention is to produce an antibody against a lily potential virus, which is an antigen, by hybridomas, and to use the antigen antibody reaction in large quantities. It is to be able to check whether the infection is.

1 is an electron micrograph of lily potential virus isolated from lily

Figure 2 is a picture of Lili Potential virus by Western blot (Western-blot)

3 is a photograph of the binding reaction between the monoclonal antibody secreted by the hybridoma and lily potential virus

Figure 4 is a virus assay picture of lily juice solution using a monoclonal antibody

The present invention is to prepare a hybridoma cells having the genetic properties of lymphocytes and tumor cells by fusion of lymphocytes obtained by animal immunoassay with tumor cells using the step of purifying the lily potential virus. Selection and cloning step, purification of monoclonal antibody for lily potential virus assay and measuring isotype of antibody, antigen determinant recognition experiment step of monoclonal antibody, virus assay using monoclonal antibody It consists of hybridomas that secrete specific antibodies against lily potential virus (LSV) obtained by a series of steps and monoclonal antibodies secreted thereby. Hybridoma constituting the present invention is monoclonal 1G6-B1 line to secrete lily latent virus (LSV) test monoclonal antibody, and as a condition for restoring microorganisms (1) the composition of the recovery agent RPMI-1640 Culture medium (15% FBS, added 100μg / ml streptomycin and penicillin) and environment of pH 7.0-7.4, (2) Sterilization by passing through 0.22㎛ membrane, (3) Temperature at 37 ° C Is the same as the restoring agent in composition and pH.

The culture conditions of microorganisms were (1) temperature: 37 ° C, (2) pH 7.0-7.4, (3) CO ₂ : around 5%, and storage condition was (1) Culture medium: RPMI-1640 culture solution (20% FBS, 10% DMSO added), (2) pH: 7.0-7.4, (3) Temperature: -196 ℃ (Liquid communication). Precautions for preservation should be pretreated at -70 ℃ for 1 day before storage. do. The microorganisms were deposited on February 19, 1999 using the Cell Line Bank as a depositary institution. The accession number is KCLRF-BP-00021.

Hereinafter, more specific details will be described through Examples and Test Examples, but the content of the present invention is not limited to Examples and Test Examples.

Example 1 Preparation of Antigen

The antigen used in the present invention is lily potential virus (LSV) and the purification method is as follows.

1) 0.5 grams (g) of virus two lobes were crushed with 2 milliliters (ml) 0.5 mole phosphate buffer (pH 7.5).

2) Carbon tetrachloride was added to 25 percent of the ground solution volume.

3) After low speed centrifugation (3,000 times / min) for 10 minutes, only the upper liquid layer was separated.

4) 4% polyethylene glycol (PEG, molecular weight: 6,000) was added to the separated liquid layer, and sodium chloride (NaCl) was added to 0.1 mol and stirred at 4 ° C. for 1 hour.

5) After centrifugation (10,000 times / min) for 10 minutes, the precipitate was diluted with 0.02 mol of phosphate buffer (pH 7.5).

6) Repeated 4 and 5, the purified virus was confirmed as Western blot (LSV) (see Figure 2) was used as the antigen. (Note: Virus electrophoresis and Western blot is a general Method, and LSV antibody for virus identification was used Sanofi product)

Example 2 Animal Immunity

Animals used for immunization were female Bob seed (BALB / c) mice about 6 weeks old and were intraperitoneally injected with the antigen LSV three times. The injection interval was 4 weeks between 1 and 2 times, and 1 week between 2 and 3 times.

Example 3 Cell Fusion

After three immunizations, fusion of Bob's mouse lymphocytes and tumor cells, NS-1 (NS-1) cells, with 50% polyethylene glycol (PEG, molecular weight 1,500) was performed to simultaneously combine the genetic properties of lymphocytes and tumor cells. Cell hybridomas were prepared. Cell fusion followed the general method and is as follows.

1) Lymphocytes and tumor cells were mixed in 10 milliliters of RMP-1640 (RPMI-1640) culture at a ratio of about 5: 1.

2) The cells were precipitated by low speed centrifugation (1,000 times / minute) for 10 minutes.

3) One milliliter of 50% polyethylene glycol (PEG) was added to the precipitate in 45 seconds and added dropwise.

4) After 75 seconds at 37 degrees Celsius, 2 milliliter culture was added for 60 seconds.

5) After 60 seconds at 37 degrees Celsius, 2 milliliter culture was added for 60 seconds.

6) After 60 seconds at 37 degrees Celsius, 20 milliliter culture was added for 120 seconds.

7) After 20 ml of the culture solution was added, low-speed centrifugation (1,000 times / minute) was carried out for 10 minutes.

8) After removing the culture medium from the top, the precipitate was mixed with HAT culture medium and dispensed into 96-hole culture plate.

9) Aliquoted culture plates were incubated in a thermostat supplied with 5 percent carbon dioxide (temperature controlled at 37 degrees Celsius).

Example 4 Selection and Cloning of Fusion Cells

Five lines were selected from fusion cells selected first by enzyme-linked antibody assay (ELISA, ELISA) and cloned by limiting dilution.

1) Mix about 200 fusion cells with 40 milliliters of thymus cells of RBM mice aged 3-4 weeks old and RPMI-1640 culture medium.

2) Dispense the mixed solution into two 96-hole culture plates

3) The aliquoted cells were incubated in a 37 ° C thermostat supplied with 5% carbon dioxide gas.

4) From the 10th day of culture, cell growth was examined and reselected. The enzyme-linked antibody assay (ELISA) used for reselection was as follows.

① 100 microliters (μl) of lily potential virus and whole control were added to the plate.

② After about 2 hours (or about 3 hours at room temperature) at 37 degrees Celsius, it was washed six times with a phosphate buffer solution of 0.02 mol etch (pH) 7.4.

③ Dissolve 0.1 percent BSA in 1 / 2-strength carbonate-bicarbonate buffer and add 100 microliters to each well of the plate.

④ Repeat step ②.

⑤ 50 microliters of culture medium of the fusion cells to be tested for antibody secretion was added.

⑥ Repeated ②

(7) added a secondary antibody linked with alkaline phosphatase, which reacts with mouse immunoglobulin-ji (IgG)

⑧ Repeat step ②.

⑨ 4-nitrophenyl phosphate as a substrate was reacted

37 After about 1 hour (or about 2 hours at room temperature) reaction at 37 degrees Celsius, the color reaction was observed or the light absorption value was measured at a wavelength of 405 nanometers (nm).

⑪ As a colorless, cell line showing a yellow response to the antigen-free LSV was selected, or a cell line showing a light absorption value more than two times higher than that of the sound control at wavelength 405 nanometers (nm).

(Storage and storage of specific antibody secreting fusion cell line against LSV)

The fusion cell lines researched and developed by the National Institute of Agricultural Science and Technology and stored and stored in liquid nitrogen communication are as follows (see Table 1).

Table 1. Fusion Cells and Monoclonal Antibody Lines Secreting Antibodies Responding to Lily Potential Virus

Number Hybrid cell lines Monoclonal antibody lines One 1F2 1F2-B3, -B4, -E5 2 1G6 1G6-B1, -B3, -C8 3 3G5 3G5-B8, -H12 4 4G5 4G5-D12, -E12 5 1G7 1G7-D1 system 5 lines 11 lines

Example 5 Purification of Monoclonal Antibodies for Lily Potential Virus Assay and Isotype Measurement of Antibodies

Of the 11 monoclonal antibody-secreting cell lines preserved in liquid nitrogen communication, 1F2-B3 and 1G6-B1 lines were cultured and 50% ammonium sulphate [(NH ₄ ) ₂ SO ₄ ], which is commonly used from the culture supernatant, was used. The monoclonal antibody for LSV assay was purified using anion exchange resin, dialysis, and the like, and the isocyta leaf assay of the purified antibody was performed using the mouse monoclonal antibody isota leaf assay reagent from Sigma. The results measured by ELISA using antibody isotyping reagents are as follows (see Table 2).

Table 2. Isotypes of Monoclones 1F2-B3 and 1G6-B1

Monoclonal Antibody Line Iso type 1F2-B3 IgG 2a 1G6-B1 IgG 2a

IgG = immunoglobulin G

Test Example 1 Antigen Determinant Recognition Test of Monoclonal Antibodies

In order to determine whether the monoclonal antibody developed by the Institute of Agricultural Science and Technology recognizes the antigen LSV, it was investigated by DIBA (dot immunobinding assay). The method is as follows.

1) Nitrocellulose paper (NC paper) was cut to a suitable size, immersed in a 0.05 mol TBS buffer solution for 5 minutes, taken out and dried for about 30 minutes.

2) The purified LSV, which is an antigen, was added to the NC paper by micropipette twice, and then left at room temperature for 1 hour.

3) Antigen prepared by the same process as LSV purification was used as a healthy control.

4) Washed 3 times for 10 minutes with TBS.

5) Blocked for 1 hour at 37 ° C. with 2% BSA diluted in TBS.

6) washed three times with 10 minutes each TBS.

7) Monoclonal antibody (1G6-B1 line), that is, conjugated with the enzyme alkaline phosphatase (1G6-B1 line), the conjugate (conjugate) was diluted 100 times in TBS and treated for 1 hour at 37 ℃.

8) After washing three times with TBS for 10 minutes, the substrate was treated with BCIP / NBT (BCIP / NBT) to confirm the color reaction.

It did not react in the healthy control, but as shown in FIG.

<Test Example 2> Virus assay using monoclonal antibody

As shown in FIG. 3, it was confirmed that the monoclonal antibody binds to the purified LSV, that is, the antigen. However, the monoclonal antibody normally reacts with the purified antigen as well as the juice from which the impurities are not removed in order to easily assay a large amount of samples. saw.

1) The crude juice triturated with Tris buffer solution (0.05, pH7.5) corresponding to 10 times (w / v) of LSV Lee Byung-yeop and dry whole leaf weight was used directly at the final dilution concentration of 20 times to 640 times without further purification. .

2) The sample of 1) was examined whether the virus assay was possible by the DIBA method (see Example 4).

As shown in FIG. 4, nonspecific reactions due to impurities were slightly up to diluting folds of 20 to 40 folds, but nonspecific reactions were removed as dilution folds increased, and virus assay was possible even when diluting up to 640 folds. As a result, LSV assay can be efficiently performed even if the monoclonal antibody developed by the Institute of Agricultural Science and Technology is diluted and reacted with 640 times of the lily crude juice triturated with Tris buffer without purification.

As a result of testing the monoclonal antibody against the lily virus (LSV) prepared by the above procedures, the lily juice was normally reacted not only with the purified LSV but also with the juice from which impurities were not removed. It was confirmed that LSV can be efficiently assayed by diluting the reaction to 640 times. As a result, many problems of the test for LSV infection, which have been a problem in the production of superior seedlings (high cost due to import of antibody reagent, concerns about alteration during transportation and timely supply, etc.) due to the completion of the present invention, Samples can be easily and economically assayed.

Claims (5)

Hybridoma 1G6-B1 line (KCLRF-BP-00021), characterized by secreting specific antibodies against lily potential virus (LSV). Fusing Lymphocyte Cells of Bob's (BALB / c) Mouse Immunized with Lily Potential Virus (LSV) and NS-1 (NS-1) Tumor Cells and Enzymatic Linkage of Fused Cells (KCLRF-BP-00021) A method for producing a hybridoma 1G6-B1 line, characterized by selecting by antibody assay (ELISA) and cloning by limiting dilution. 3. The hybridoma according to claim 2, wherein the fusion of lymphocytes of BALB / c mice with NS-1 tumor cells uses 50 percent polyethylene glycol (PEG, molecular weight 1,500). Process for producing 1G6-B1 line. 3. The fusion of lymphocytes of BALB / c mice with NS-1 (NS-1) tumor cells is mixed in 10 ml of AlMPM-1640 (RPMI-1640) culture solution at a 5: 1 ratio. Method for producing a hybridoma 1G6-B1 line, characterized in that. The monoclonal antibody secreted by the hybridoma of claim 1 is an isotype (Isotype) IgG 2a monoclonal antibody for assay LSV.