JP2561766B2 - Monoclonal antibody - Google Patents

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 using soybean mosaic virus (SMV) as an immunogen.

[0002]

2. Description of the Related Art In soybeans, 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.

In general, once a plant is infected with a virus, it does not heal naturally. Therefore, in order to obtain a plant which is not infected with a virus (virus-free plant), it is necessary to remove the virus by a shoot apex culture or the like. In addition, in growing the shoot tip culture seedlings, it is necessary to test whether or not the parent plant is virus-free. In the conventional method, a local diseased plant (Top Crop: kidney bean in the case of SMV) of the juice of the plant to be tested is rubbed and inoculated, and the presence or absence of the local diseased site is tested. However, in order to carry out this method, at least 1 g or more of the plant to be tested is required. Therefore, shoot apical culture seedlings must be potted and allowed to grow for about one month. 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 results 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 producing breeding which prevents the disease, promotes proper growth of breeding, and improves the commercialability.

[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.

[0006] 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 is a strongly binding antibody.

[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. Furthermore, it is intended to simplify the virus diagnosis and shorten the diagnosis period by producing a reagent for detecting an antigen for the above-mentioned 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 SM
It can be produced by selecting a clone that produces an antibody showing specificity for V.

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

Examples of animals that can be immunized using the above immunogen include mice, rats, horses, goats, rabbits, and guinea pigs. The immunogens include, 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, and the immunization is performed 2 to 4 times every 1 to 7 weeks after the initial immunization, and the final immunization is performed about 1 to 2 months later. About 3 to 5 days after the final immunization, antibody-producing cells, for example, spleen cells, lymph node cells, lymphocytes, etc., preferably spleen cells, are collected from the immunized animal.

On the other hand, as the myeloma cells, for example, those derived from mouse, rat, human or 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 known per se, for example, the method of Kohler and Milstein et al.
(Methods Enzymol., 73, 3-46 (1981)), polyethylene glycol and Sendai virus (HV
J) or the like as a fusion accelerator, 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 screened 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 wells in which proliferation is observed is measured by the ELISA method, the plaque method, the spot method, the Ouchterlony method, the RIA method, etc.,
A well producing the desired antibody is obtained. Such wells are further cloned by, for example, the ultradilution 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- D 06 and SMV-D10. These hybridomas FERM 12375 respectively, the first 12373
No. 12539 and No. 12374 are registered and stored in the Institute of Microbial Technology, Institute of 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 such as isolation and purification from the ascites, and the antibody can be separated and purified by appropriately combining salting out, gel filtration, affinity chromatography, etc. 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 are mentioned.

[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 reactive with all of virus strains A, B, C, D, and E (SMV-D11-Ig
G ₁ , SMV-D06-IgG _2a ), A excluding C, B,
Antibody group that reacts with D and E (SMV-D10-IgG ₃ ,
SMV-E06-IgG _2a) has been created. By using this monoclonal antibody, soybean infected with SMV can be detected easily 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 Soybean (cultivar Hyuga) two weeks after sowing was 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 0.3 M 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).
Then, 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.

After that, further high speed centrifugation (8500 g × 1
For 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 crudely purified virus was added with 0.005M ED.
A 0.05 M phosphate buffer solution (PH7) containing TA was added and dissolved, followed by sucrose density gradient centrifugation (sucrose 10% to 50%), 47.
After collecting the virus fraction by performing 000g x 3 hours,
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 of 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 Ajyu-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.
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, then transferred to HT medium, and after further culturing to 10 ml, D
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 One cloned strain obtained by the above screening was used.
Cultured in DF medium containing 0% FBS, about 10 to 20 μm
A culture supernatant containing g / ml of the monoclonal antibody was obtained. For subclass classification of this monoclonal antibody, SMV-D10 is IgG ₃ and SMV-D by using the 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 precipitated 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 obtained by cell fusion (SMV-
The mouse monoclonal antibody produced by D10 and SMV-D06) was soybean SMV by Western blot.
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 an immunizing virus solution.

(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 give an antigen solution. An equal amount of Freund's Ajyuband was added to this antigen solution, mixed and emulsified, and 100 μl of the emulsified liquid 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. Confirmation of antibody production in antigen-stimulated mice was performed by immunoprecipitation. Four days after the final immunization, the mouse spleen was extracted and used as splenocytes for the following cell fusion.

(3) Fused cells and cloning: The above mouse splenocytes 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 plated in a 96-well microplate and HAT
After culturing in the medium for 14-21 days, transfer to the HT medium, and after further culture to 10 ml, the 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 to 20 μm
A culture supernatant containing g / ml of the monoclonal antibody was obtained. The subclass classification of these monoclonal antibodies is shown 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 precipitated 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 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.
Allowed to react for hours.

After washing three times with a gelatin buffer (10 mM PBS solution containing 0.3% gelatin), 1% B was added.
Add 200 μl of 10 mM PBS solution containing SA (bovine serum albumin) and 0.05% sodium azide, and add 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.
And reacted for 1 hour.

After washing three times with gelatin buffer,
A peroxidase-conjugated anti-mouse IgG goat antibody (manufactured by Kuppel) 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 (PH5) were added, and the mixture was allowed to react at 37 ° C. for 30 minutes under dark conditions. After the reaction was completed, 50 μl of 5N sulfuric acid was added to stop the reaction, and the absorbance at 491 nm was measured. A plot of mouse antibody concentration and 491 nm absorbance after 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 (0.05% Tween20 in 1 ml of microtube) )
Was added, and the mixture was ground and 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 previously 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 the sheet per sample.

After standing 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 and mixed with 3 ml of solution A immediately before the specification) was dropped on the sheet, and the sheet was allowed to stand under shading conditions for about 15 minutes. 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 The maximum absorbance at 410 nm in the dilution curve of was shown.

Note 2) Dilution limit concentration of antibody 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 front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12R 1:91) (56) References J. GEN. VIROL. , 65-3! (1984) P. 525-532 PHYTOPATHOLOGY, 79-11! (1989) P. 1261-1265

Claims (5)

(57) [Claims] 1. A monoclonal antibody capable of recognizing soybean mosaic virus (SMV), wherein (a) the antibody class (subclass) is IgG.
₁ (κ), IgG _2a (κ) or IgG ₃ (κ), and (b) at least soybean mosaic virus (SMV) .
Even lines A, B, shows a positive response against D and E, and (c) soybean mosaic Ui by Western Bro bracts
The molecular weight derived from Rus (SMV) is about 30,000 to 32,
A monoclonal antibody characterized by showing a positive reaction to 000 antigenic coat proteins . 2. A hybridoma SMV-D11 (Microtech Lab.
Bacteria No. 12375) and SMV-D06 (Microtechnology Research Institute)
No. 12539) produced by each
Antibodies, which are monoclonal antibodies SMV-D11-IgG ₁ and S showing a positive reaction against all of soybean mosaic virus (SMV) strains A, B, C, D and E.
_{MV-D06-IgG 2a.} 3. Hybridoma SMV-E06 (Microtech Lab.
Bacteria No. 12373) and SMV-D10 (Microtechnology Research Institute)
No. 12374) produced by each
An antibody, system A of soybean mosaic virus (SMV), indicated B, D, a positive response against E, monoclonal antibodies SMV-D1 showing a negative reaction for system C
0-IgG ₃ and _{SMV-E06-IgG 2a.} 4. A hybridoma capable of producing the monoclonal antibody according to claim 1, which is produced by using soybean mosaic virus (SMV) as an immunogen. 5. A hybridoma S MV-D11 (Bikoken
Bacteria nearest No. 12375), the SMV-E06 (FERM
No. 12373) , SMV-D06 ( Ministry of Microbiology Research Institute 125
39) or SMV-D10 (Ministry of Industrial Science, Microbiology Research Institute No. 1237)
No. 4), which is a hybridoma according to claim 4 .