JPH05130888A - Monoclonal antibody - Google Patents

Abstract

PURPOSE:To provide a new monoclonal antibody effective for easily detecting soybean infected with soybean mosaic virus in a short time owing to the property to specifically bind with soybean mosaic virus. CONSTITUTION:A monoclonal antibody recognizing soybean mosaic virus (SMV), having antibody class (subclass) of IgG1(kappa), IgG2(kappa) or IgG3(kappa) and exhibiting positive reaction to SMV. The antibody can be prepared by immunizing an animal with SMV separated and purified from soybean infected with SMV, fusing the splenocyte of the immunized animal to a myeloma cell, cloning the obtained hybridoma, selecting a clone capable of producing an antibody exhibiting specificity to SMV and letting the strain to produce the objective antibody.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody capable of recognizing soybean mosaic virus (SMV) and a hybridoma capable of producing the monoclonal antibody produced by using soybean mosaic virus (SMV) as an immunogen.

[0002]

2. Description of the Related Art In soybean, the presence or absence of a disease often affects the yield and quality of beans. Examples of viruses that affect soybean include soybean chlorotic spot virus, soybean dwarf virus, soybean micro mosaic virus, soybean atrophy virus and soybean mosaic virus (hereinafter sometimes abbreviated as SMV). .. Of these, SMV is the most common.

Generally, once a plant is infected with a virus, it does not heal naturally, so it is necessary to remove the virus by shoot apical culture or the like in order to obtain a plant that is not infected with the virus (virus-free plant). Furthermore, in growing this shoot apical seedling, it is necessary to test whether or not the parent plant is virus-free. In the conventional method, the local sickness plant of the sap to be tested (Top Crop: kidney bean in the case of SMV) is rubbed and inoculated, and the presence or absence of the local sickness is tested. However, in order to carry out this method, at least 1 g or more of the plant to be tested is required, so it is necessary to raise the shoot apical culture seedlings and wait for about one month for growth. The local diseased plant of No.1 had to be sown and cultivated about one month before the test, and it took about two weeks after the inoculation for the actual test result to be known.
Further, there are various problems such that the assay sensitivity varies depending on the physiological condition of the assay plant.

[0004] The virus assay of plants consists in creating breeding that prevents the disease, promotes proper growth of breeding, and enhances the marketability.

[0005] Monoclonal antibodies such as rice virus and citrus mosaic virus have been known as previous reports on virus assays. However, these monoclonal antibodies are not used for SMV considering their specificity.

Monoclonal Antibody-based Biotin-Avi for the soybean mottle virus monoclonal antibody
din ELISA for the Detection of Soybean Mosaic Viru
s inSoybean Seeds. J. gen. Virol. (1985). 6
Although already reported in 6.2809-2094, it is not clear whether it is an antibody specific to the strain of soybean mosaic virus and strongly bound.

[0007]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
It is to provide a monoclonal antibody that specifically and strongly reacts with MV. Further, it is intended to simplify the virus diagnosis and shorten the diagnosis period by producing an antigen detection reagent for the specific virus.

[0008]

The present invention provides a monoclonal antibody capable of recognizing soybean mosaic virus (SMV).

The monoclonal antibody of the present invention is a monoclonal antibody produced by a hybridoma produced using soybean mosaic virus (SMV) as an immunogen and having the following properties.

(A) The antibody class (subclass) is Ig
G ₁ (κ), IgG _2a (κ) or IgG ₃ (κ), and shows a positive reaction against (b) soybean mosaic virus (SMV).

The monoclonal antibody of the present invention can be produced by a cell fusion technique known per se. For example, a fusion hybrid is formed between an antibody-producing cell and a myeloma cell by an electrofusion method or a polyethylene glycol fusion method, the hybrid is cloned, and then SM is prepared.
It can be produced by selecting a clone producing an antibody showing specificity for V.

Immunogen (antigen) for producing antibody-producing cells
As the SMV, SMV isolated from soybean infected with SMV and purified can be used.

Examples of animals that can be immunized using the above-mentioned immunogens include mice, rats, horses, goats, rabbits, guinea pigs, and the like. The immunogens are, for example, Freund Complete Adjuvant. After mixing, these animals can be immunized. Immunization can be performed subcutaneously, intramuscularly or intraperitoneally in animals (mouse),
The amount of protein is 10 to 100 μg / injection, the immunization is performed 2 to 4 times every 1 to 7 weeks after the initial immunization, and the final immunization is further performed about 1 to 2 months later. About 3 to 5 days after the final immunization, antibody-producing cells such as splenocytes, lymph node cells, lymphocytes, etc., preferably splenocytes, are collected from the immunized animal.

On the other hand, as the myeloma cells, for example, those derived from mouse, rat, human and the like can be used.
It is desirable that the antibody-producing cells and the myeloma cells are of the same kind.

The fusion of antibody-producing cells with myeloma cells is a method known per se, for example, the method of Kohler and Milstein.
(Methods Enzymol., 73, 3-46 (1981)), polyethylene glycol and Sendai virus (HV
J) or the like as a fusion promoter, a method of performing cell fusion,
Electrofusion method [Kotaro Noda, Yoshishi Togawa (1989), Review of Shimadzu, Vol. 46, No. 4, 167-178] and the like.

The cells obtained by cell fusion are subjected to screening for hybridoma clones which produce the desired monoclonal antibody. For example, the fused cells are cultured in a microplate, and the antibody titer in the culture supernatant of the well in which proliferation is observed is measured by the ELISA method, the plaque method, the spot method, the Ouchterlony method, the RIA method, etc.,
Obtain the wells producing the desired antibody. Such a well is further cloned by, for example, the limiting dilution method to obtain a hybridoma clone producing the desired antibody.

The hybridoma producing the desired monoclonal antibody thus obtained is, for example,
SMV-D11, SMV-E06, SMV-06 and SMV-D10 are mentioned, and these hybridomas are respectively Micromachine Research Institute No. 12375 and No. 12373.
No. 12539 and No. 12374 are registered and stored in the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology.

The monoclonal antibody of the present invention can be collected from these hybridomas by, for example, culturing the hybridoma according to a conventional method, separating and purifying from the culture supernatant, or administering the hybridoma to a compatible mammal. It can be carried out by a method of separating and purifying from the ascites, and the antibody can be separated and purified by a suitable combination of salting out, gel filtration, affinity chromatography and the like. You can

Specific examples of the monoclonal antibody of the present invention thus produced include hybridoma SMV.
-D11, SMV-E06, SMV-D06 and SMV
-SMV-D11-Ig produced by D10
_{G 1, SMV-E06-IgG} 2a, SMV-D06-I
gG _2a and SMV-D10-IgG ₃ and the like.

[0020]

The monoclonal antibody of the present invention is SMV
It has a property of specifically binding to. Regarding the reaction specificity, an antibody group that reacts with all of virus strains A, B, C, D, and E (SMV-D11-Ig
G ₁ , SMV-D06-IgG _2a ), A excluding C, B,
D, antibody group which reacts with _{E (SMV-D10-IgG 3} ,
SMV-E06-IgG _2a) has been created. If this monoclonal antibody is used, soybean infected with SMV can be detected simply and in a short time by an ELISA method or an enzyme antibody method using membrane adsorption. The monoclonal antibody of the present invention can also be applied to assay the inoculation efficiency of viruses in SMV research.

[0021]

EXAMPLES Next, the present invention will be described more specifically by way of examples.

Example 1 (1) Preparation of immunogen: Soybean mosaic virus (SM
V) Proliferation and Purification Soybeans (cultivar Hyuga) 2 weeks after sowing were inoculated with SMV by the carborundum method, and after 2 to 4 weeks, leaves exhibiting viral symptoms were harvested.

2 volumes of 0.01M NaDI for the diseased leaf volume
A 0.3M phosphate buffer solution (PH7) containing ECA, 0.2% meltcaptoethanol, and 8% butanol was added, and the mixture was ground and filtered with a tetoron goose.

The filtrate is a high-speed centrifuge (8500 g × 10 minutes).
After that, the supernatant is separated and its volume is designated as (A). TX100, PEG and NaCl were added to this supernatant at 1%, 6%,
Add 0.1M to room temperature for 20 minutes or 4 ℃
Left for 1 hour.

Thereafter, further high speed centrifugation (8500 g × 1
(0 minutes), and the obtained precipitate has a volume (A) of 1/2 to 1
0.05M Phosphate buffer (PH) containing 0.5M urea
7) was added and resuspended and left overnight. The next day, high-speed centrifugation (8500 g × 10 minutes) was performed, the supernatant was recovered, and this supernatant was subjected to ultracentrifugation (78000 g × 90 minutes) to recover the precipitate, which was used as a crudely purified virus.

This crude virus was added with 0.005M ED
After adding 0.05M phosphate buffer (PH7) containing TA and dissolving, sucrose density gradient centrifugation (sucrose 10% to 50%), 47
After collecting 000 g x 3 hours and collecting the virus fraction,
0.05M phosphate buffer containing 0.005M EDTA (P
H7) was dialyzed. Subsequently, CsCl density gradient centrifugation (CsCl 3.7 g / 10 ml) was performed, and then the virus fraction was collected and dialyzed against 0.05 M borate buffer to give an SMV-purified virus.

The virus can be confirmed by scanning electron microscopy, molecular engineering and SDS-PAG.
The method according to E was used.

(2) Immunization 10 purified from soybean (cultivar Hyuga) in the above experiment
mg / ml SMV was diluted with physiological saline to 2 μg / μl, and then sterilized by filtration with a 0.2 μm filter to obtain an antigen solution. To this antigen solution, an equal amount of Freund's azimuth band was added, mixed and emulsified, and 100 μl of the emulsion was Balb / c.
Mice were injected subcutaneously. Repeat the same operation 4 weeks later 2
This was the second antigen stimulation. Five weeks after the second antigen stimulation, the same operation was repeated to give the final immunization. Confirmation of antibody production in antigen-stimulated mice was performed by immunoprecipitation method.
Four days after the final immunization, the mouse spleen was excised to give splenocytes, which were then used for the following cell fusion.

(3) Fused cells and cloning: The spleen cells prepared in (2) above were converted into mouse myeloma cells (P
3U1) and mixed with Kohler & Miristin (Kohler & Mi
lstein) method was partially modified, and cell fusion was performed by reacting with a 50% polyethylene glycol DF solution for 1 minute. 50% polyethylene glycol DF
Solution is polyethylene glycol (average molecular weight 4000)
4g 121 ℃, after autoclaving for 20 minutes 37 ℃
After cooling, the mixture was prepared by adding 2.8 ml of DF basal medium and mixing. The treated cells were seeded in a 96-well microplate, cultured in HAT medium for 14-21 days, transferred to HT medium, and further cultured in 10 ml.
The cells were cultured in F medium (containing 10% FBS). The antibody titer in the culture supernatant of the well in which proliferation was observed was measured by the enzyme antibody method, and the desired hybridoma was cloned from the appropriate well by the limiting dilution method. Cloning results SMV-D10 hybridoma and SMV-D06
Hybridomas were obtained.

(4) Recovery and Purification of Monoclonal Antibody 1 cloned strain obtained by the above screening
Cultured in DF medium containing 0% FBS, about 10-20μ
A culture supernatant containing g / ml of the monoclonal antibody was obtained. For subclass classification of this monoclonal antibody, SMV-D10 was IgG ₃ and SMV-D by using an isotyping kit RPN 29 (manufactured by Amersham).
06 was determined to be IgG _2a .

70% ammonium sulfate was added to this culture supernatant.
The mixture was added so as to have a concentration, and the precipitate fraction was collected. The precipitate fraction was dissolved in a small amount of 10 mM PBS solution and then dialyzed with 10 mM PBS solution as an external solution. After the dialysis was completed, an immunoglobulin G fraction was obtained using a protein A affinity column and used as a purified monoclonal antibody.

(5) Characteristics of monoclonal antibody Hybridoma cells (SMV-
The mouse monoclonal antibody produced by D10 and SMV-D06) was soybean SMV by Western blotting.
It became positive for the antigen.

Example 2 (1) Preparation of immunogen: SMV purified from the same soybean (cultivar Hyuga) used in Example 1 is used as a virus solution for immunization.

(2) Immunization SMV purified from soybean (Hyuga) was diluted with physiological saline to 2 μg / μl and then sterilized by filtration with a 0.2 μm filter to obtain an antigen solution. An equal amount of Freund's Ajyuband was added to this antigen solution, mixed and emulsified, and 100 μl of the emulsion was subcutaneously injected into Balb / c mice. The same operation was repeated 1 week, 7 weeks, and 12 weeks later, and was used as the second, third, and fourth antigen stimulation. Five weeks after the fourth antigen stimulation, the same operation was repeated to give the final immunization. The antibody production in antigen-stimulated mice was confirmed by immunoprecipitation method. Four days after the final immunization, the mouse spleen was excised and used as splenocytes for the following cell fusion.

(3) Fused cells and cloning: The above mouse spleen cells and mouse myeloma cells P3U1 were mixed, and the method of Kohler & Milstein was partially modified to prepare a 50% polyethylene glycol DF solution. Cell fusion was performed by reacting for 1 minute with. A 50% polyethylene glycol DF solution contains 121 g of polyethylene glycol (average molecular weight 4000).
After autoclaving for 20 minutes at ℃, cool to 37 ℃ 2.
8 ml of DF basal medium was added and mixed to prepare. The treated cells were seeded in a 96-well microplate and HAT
After culturing in the medium for 14-21 days, transfer to HT medium, and after being able to further culture to 10 ml, DF medium (10%
(Including FBS). The antibody titer in the culture supernatant of the wells in which proliferation was observed was measured by the enzyme antibody method, and the desired hybridoma was cloned from the appropriate well by the limiting dilution method to obtain several clones. As a result of this screening, each clone
The MV-D06 SMV-E06, SMV-D10 and SMV-D11 hybridomas were used.

(4) Recovery and Purification of Monoclonal Antibody One cloned strain obtained by the above screening was used.
Cultured in DF medium containing 0% FBS, about 10-20μ
A culture supernatant containing g / ml of the monoclonal antibody was obtained. The subclass classification of these monoclonal antibodies is described in Example 1.
SMV-D06 was used for IgG _2a (κ) SM
V-E06 is IgG _2a (κ), SMV-D10 is Ig
G ₃ (κ) and SMV-D11 were determined to be IgG ₁ (κ).

70% ammonium sulfate was added to this culture supernatant.
The mixture was added so as to have a concentration, and the precipitate fraction was collected. After dissolving the precipitate fraction with a small amount of 10 mM PBS,
It was dialyzed as an external solution of 10 mM PBS solution. After dialysis,
An immunoglobulin G fraction was obtained using a protein A affinity column and used as a purified monoclonal antibody.

(5) Characteristics of monoclonal antibody Hybridoma cells obtained by cell fusion (SMV-
D06 SMV-E06, SMV-D10 or SM
The monoclonal antibody produced by V-D11) had an SMV-derived molecular weight of about 30,000 by Western blotting.
It was positive for ~ 32,000 antigenic coat proteins (see Figure 1).

Example 3 Antibody Dilution Curve Using a covalent bond type 96-well ELISA plate (manufactured by Nunc), purified SMV was added to each well at 50 μl and 10 μg in 10 mM PBS solution, and the mixture was added at 37 ° C. for 2 hours.
Reacted for hours.

After washing three times with a gelatin buffer (10 mM PBS solution containing 0.3% gelatin), 1% B was added.
200 μl of 10 mM PBS solution containing SA (bovine serum albumin) and 0.05% sodium azide was added 37
The reaction was carried out at ℃ for 1 hour.

After washing three times with a gelatin buffer, the hybridoma culture supernatant was diluted with 10 mM PBS, 50 μl was added to each well, and 37 ° C.
Was reacted for 1 hour.

After washing three times with gelatin buffer,
Peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Katspel) was diluted 1000 times with 10 mM PBS to give 50.
μl was added and reacted at 37 ° C.

After washing three times with gelatin buffer,
Color developing solution (12 mg o-PDA, 6 μL 31% H
50 μl of ₂ O ₂ and 30 ml citrate buffer solution (PH5) were added, and the mixture was reacted at 37 ° C. for 30 minutes under dark conditions. After completion of the reaction, 50 μl of 5N sulfuric acid was added to stop the reaction, and the absorbance at 491 nm was measured. A plot of the concentration of mouse antibody and the absorbance at 491 nm after the enzyme antibody reaction is shown in FIG.

Example 4 Reaction of Monoclonal Antibody by Dot Immunobainding (DIBA) Method to Inoculated Leaf of Each SMV Line Inoculated leaf (Hyuga), 100 mg of TBS containing 1 ml of TTBS (0.05% Tween 20) in a microtube. )
Was ground, centrifuged for 5 minutes, and the supernatant was used as a sample.

Separately, a nitrocellulose sheet (manufactured by Amasyamu Co., Ltd.) was cut into a required area and a 1 × 1 cm square was drawn with a pencil. (Hereafter referred to as a sheet) This sheet is 2
TBS containing 20% methanol (20 mM Tris, 500
Immersed in mM NaCl aqueous solution, PH 7.5) for several minutes,
It was immersed in TBS without methanol for about 15 minutes and dried on filter paper for about 5 minutes.

Next, 1 .mu.l of the above-prepared sample was spotted on the center of the squares of the sheet and dried for about 5 minutes.

The sheet was dipped in a block solution (TTBS containing 2% polyvinylidone (PVP, Mw 40,000) 2% bovine serum albumin (BSA)) and allowed to stand at room temperature for 30 minutes. Put the sheet on the parafilm and TTBSPB
(2% polyvinylidone (PVP, Mw 40,000) 0.
TTBS containing 2% bovine serum albumin (BSA))
Then, 30 μl of the hybridoma culture supernatant (anti-SMV antibody) diluted about 100 times was dropped on a sheet per sample.

After left at room temperature for 1 hour, the sheet is TTBSP.
B was shaken twice for 10 minutes and washed. Alkaline phosphatase-labeled anti-mouse globulin antibody (manufactured by Kuppel) diluted 500 times with TTBSPB was dropped on the sheet in the same manner as above. After leaving at room temperature for 1 hour, TTB
After washing with SPB, further AP impact solution (100 mM
Tris, 100 mM NaCl, 5 mM MgCl ₂
It was washed twice with an aqueous solution, pH 9.5) for 10 minutes.

Next, a coloring solution (A: AP buffer containing 0.33 mg / ml nitroblue tetrazolium (NBT), B)
N, N containing 5.0 mg / ml 5-bromo-4-chloro-3-indoxylphosphoric acid-P-toluidine salt (BclP)
-Dimethylformamide, 10 μl of solution B was added to 3 ml of solution A immediately before the specification and mixed) was dropped on the sheet, and the sheet was allowed to stand for about 15 minutes in the dark. After confirming the color development,
Stop the sheet (10 mM Tris, 5 mM MgCl ₂
After immersing in an aqueous solution, pH 9.5) to stop the reaction, it was dried on filter paper and the result was measured.

The reaction of the monoclonal antibody against the inoculated leaves of each SMV strain is shown in FIG. 3 and Table 1 below.

[0051]

[Table 1] Table 1 Binding properties of anti-SMV antibody with antigen (SMV) in ELISA activity ━━━━━━━━━━━━━━━━━━━━━━━━━━━ Antibody titer ──────────── No. ¹⁾ EP ²⁾ ED ₅₀ ³⁾ ────────────────────── ───── 1-2-D06 2.80 3 × 10 ^-4 1.4 × 10 ^-2 1-4-D10 2.65 3 × 10 ^-3 9.6 × 10 ^-2 2-1-E06 2.64 3 × 10 ^-3 4.0 × 10 ^-2 2-3-D11 2.54 1 × 10 ^-3 7.1 × 10 ^-2 ─────────────────────────── Note 1) Antibody It shows the maximum absorbance at 410 nm in the dilution curve.

Note 2) Limiting concentration of antibody dilution by endpoint method Note 3) Half-saturation concentration of antibody against antigen

[Brief description of drawings]

FIG. 1 is a Western blot diagram showing SDS-PAGE of SMV protein and culture supernatant of each hybridoma, 1 is a molecular weight marker, 2 is SDS-PAGE, and 3 is
-6 are Western blots.

FIG. 2 is a dilution curve of the reaction of the culture supernatant of each hybridoma with an antigen (SMV).

FIG. 3 is a reaction diagram in the DIBA method using the culture supernatant of each SMV strain and each hybridoma.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location G01N 33/577 B 9015-2J // C12N 15/06 (C12P 21/08 C12R 1:91)

Claims (8)

[Claims] 1. A monoclonal antibody capable of recognizing soybean mosaic virus (SMV), wherein (a) the antibody class (subclass) is IgG ₁ (κ),
A monoclonal antibody which is IgG _2a (κ) or IgG ₃ (κ), and (b) shows a positive reaction against soybean mosaic virus (SMV). 2. The monoclonal antibody is SMV-D11-
_{IgG 1, SMV-E06-IgG} 2a, SMV-D06
-IgG _2a or SMV-D10-IgG ₃ a is claim 1, wherein the monoclonal antibody. 3. Monoclonal antibodies SMV-D11-IgG ₁ and S showing a positive reaction to all of tobacco mosaic virus (SMV) strains A, B, C, D and E.
MV-D06-IgG _2a. 4. A monoclonal antibody SMV-D1 which shows a positive reaction to strains A, B, D and E of tobacco mosaic virus (SMV) and a negative reaction to strain C.
0-IgG ₃ and SMV-E06-IgG _2a. 5. A hybridoma capable of producing the monoclonal antibody according to claim 1, which is produced by using soybean mosaic virus (SMV) as an immunogen. 6. The hybridoma is SMV-D11, SM
The hybridoma according to claim 5, which is V-E06, SMV-D06 or SMV-D10. 7. A monoclonal antibody SMV-D11-I
Hybridomas SMV-D11 and S capable of producing gG ₁ and SMV-D06-IgG _2a , respectively
MV-D06. 8. A monoclonal antibody SMV-D10-I
gG ₃ and hybridoma having SMV-E06-IgG _2a each capable of producing SMV-D10 and S
MV-E06.