JP2643040B2 - Monoclonal antibody - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody having specific properties produced by a hybridoma prepared using carnation mottle virus (CarMV) as an immunogen.

[0002]

2. Description of the Related Art In carnation, the presence or absence of disease is the yield,
It often affects the quality of cut flowers. Normally, carnations are propagated by cuttings and buds, so contamination by transmission of a virus through a vegetative body becomes a problem.

The following types of viruses affect carnation. CarMV (carnation mottle virus), CaVMV (carnation bain mottle virus), CaLV (carnation latent virus), CaFRV (carnation etched ring virus), CaRSV (carnation ring spot virus) and the like. Of these, the most common is CarM
V.

[0004] In general, once a plant is infected with a virus, it does not heal spontaneously. Therefore, in order to obtain a virus-free plant (virus-free plant), it is necessary to remove the virus by shoot tip 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, the sap of the plant to be assayed is rubbed against a local lesion plant (in the case of CarM, red mosaic) and inoculated to determine whether a local lesion has appeared. In order to carry out this method, at least 1 g of the plant to be tested is required. Therefore, the shoot-top culture seedlings must be potted and grown for about one month. In addition, the local diseased plant for the test must be sown and raised about one month before the test, and also wait about 2 weeks after the inoculation for the actual test result to become clear. Further, the detection sensitivity varies depending on the physiological conditions of the test plant.

[0005] The virus assay of plants consists in the prevention of the disease, the promotion of proper breeding, and the creation of breeds which increase the vividness of petals and improve their commercial value.

[0006] As a report on virus assay, monoclonal antibodies such as rice virus and citrus mosaic virus are known. However, these monoclonal antibodies are not used for carnation virus in view of specificity. For carnation virus monoclonal antibodies, see "SUCCESSSFULPR.
ODUCTION OF MONOCLONAL AN
TIBODIESTO THREE CARRNATIO
N VIRUSES USING ANAMIX TU
RE OF ONLY PARTIALLY PURI
FIEDVIRUS PREPARATIONS AS
IMMUNOGEN ", Ramon Jordan,
PHYTOPATHOLOGY, Vol 79 N
o. 10,1989,1213 to have already been reported, only there is a description that bound to several carnation viruses, bind to the strong solid specific for carnation mottle virus The present inventors have targeted It is not disclosed whether it is an antibody.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a monoclonal antibody which specifically and strongly reacts with a specific virus. Furthermore, by preparing an antigen detection reagent for the specific virus,
An object of the present invention is to simplify virus diagnosis and shorten the period of diagnosis.

[0008]

SUMMARY OF THE INVENTION The present invention provides a monoclonal antibody that can recognize carnation mottle virus (CarMV).

[0009] Monoclonal antibodies of the present invention, carnation mottle virus (CarMV) hybridoma made using as an immunogen is a monoclonal antibody having the following properties produced: (a) the antibody class (subclass) (B) a carnation mottle virus-derived reduced molecular weight of about 38,000 (36,000 to 4000);
It shows a positive reaction to the antigenic coat protein of 0).

The monoclonal antibody of the present invention is produced by so-called cell fusion. That is, 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 the hybrid is specific to the virus (ie, a specific antigen having the above properties). It is produced by selecting a clone that produces an antibody exhibiting sexuality.

As an immunogen (antigen) for producing antibody-producing cells, carnation mottle virus (hereinafter referred to as C)
CarMV isolated from carnation infected with arMV) or purified or purified from CarMV or treated with RNase can be used in whole or in part.

Animals immunized with these immunogens include, for example, mice, rats, horses, goats, rabbits, guinea pigs, and the like. The immunogen is, for example, complete Freund's adjuvant (Freund Complete).
Adjuvant), the animals can be immunized.

The immunization is performed by injecting 10 to 100 μg / time as a protein amount subcutaneously, intramuscularly or intraperitoneally into the animal. Immunization is performed 2 to 4 times every 1 to 7 weeks from the initial immunization. A final immunization is performed after 1-2 months. 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 myeloma cells, for example, those derived from mice, rats, humans and the like can be used.
Preferably, the antibody-producing cells and myeloma cells are derived from the same animal.

The fusion of antibody-producing cells with myeloma cells can be carried out in a manner known per se, for example by Kohler and Milst.
ein et al. [Methods Enzymol. ,
73, 3-46 (1981)], cell fusion using polyethylene glycol, Sendai virus (HVJ) or the like as a fusion promoter, electrofusion [Kotaro Noda, Yoshishi Togawa, Shimadzu Review, Vol. 46 No.
4, pages 167 to 178 (1989)].

The cells obtained by cell fusion are subjected to screening for a hybridoma clone producing 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 performed is measured by a FLISA method, a plaque method, a spot method, an octalony method, an RIA method, or the like.
A well producing the desired antibody is obtained. Such a well is further cloned, for example, by a limiting dilution method to obtain a hybridoma clone producing the desired antibody.

The hybridoma producing the desired monoclonal antibody thus obtained includes, for example, C
AV-15, CAV-21 and CAV-6,
Each of these hybridomas was
09 issue, has been entrusted stored in the Agency Fermentation Research Institute as the No. 12310 No. and the second 12,372.

The monoclonal antibody of the present invention can be collected from these hybridomas by, for example, culturing the hybridoma according to a conventional method and separating and purifying it from the culture supernatant, or administering the hybridoma to a compatible mammal. The antibody can be separated and purified from the ascites by a method such as separation and purification, and the antibody can be separated and purified by appropriately combining salting out, gel filtration, affinity chromatography and the like.

Specific examples of the monoclonal antibody of the present invention thus produced include hybridoma CAV
-15, CAV-15-IgM, CAV-21-IgM and CV produced by CAV-21 and CAV-6, respectively
AV-6-IgM.

[0020]

EFFECT OF THE INVENTION The monoclonal antibody of the present invention comprises Car
Since it has the property of binding specifically to MV,
If this monoclonal antibody is used, the enzyme immunoassay using solid-phase enzyme immunoassay or membrane adsorption can be used.
Carnations infected with MV can be detected simply and in a short time.

[0021]

Next, the present invention will be described more specifically with reference to examples.

Example 1 (1) Preparation of Immunogen: Propagation and Purification of Carnation Mottle Virus (referred to as CarMV) In a house, seedlings were raised in a garden pan containing soil mixed with peat moss and perlite in a ratio of 1: 1. When grown to 2-3 leaves, transplanted to a planter containing a 1: 1 mixture of soil and peat moss, a dianthus barb (Dianthus barb)
atus L. ) Or Shabo Giant (D. caryop)
hyllus L. ) Was cultivated. About 2 months after sowing, the largest leaf was inoculated with CarMV (Ministry of Agriculture, Forestry and Fisheries, MAFF01-04008) at a 250-fold dilution by the carborundum method. Six weeks after inoculation, leaves with relatively severe symptoms were collected. 0.005 M Na was added to 600 g of the infected leaves.
-0.1 M phosphate buffer (referred to as PBE buffer) 600 containing EDTA and 0.1% thioglycolic acid pH 7.4
ml and chloroform 480 ml, n-butanol 120
ml, and the mixture was ground in a blender, and then centrifuged (10
000 rpm, 10 min, 4 ° C.), and the upper aqueous layer separated into two layers was collected. To this is added 6% polyethylene glycol (containing 0.1 M NaCl), and the mixture is allowed to stand at room temperature for 30 minutes, and further at 4 ° C. for 1 hour. After centrifugation (10,000 rpm, 10 min, 4 ° C.), the supernatant was removed. 0.05M was added to the precipitate remaining after removing the supernatant.
Add PBE, suspend, and centrifuge (10000 rpm,
10 min, 4 ° C), and the supernatant was collected. After centrifugation (38000 rpm, 90 min, 4 ° C.), the supernatant was removed, and the precipitate was resuspended by adding 0.05 M PBE buffer. Further, centrifugation (38000 r
pm, 90 min, 4 ° C.) and added to the previous suspension. Discontinuous sucrose density gradient centrifugation (24000 rpm,
(1 hour, 4 ° C.), and collect the cloudy part where the virus exists. After centrifugation (38000 rpm, 90 min, 4 ° C.)
Remove the supernatant. 0.05M PBE (4ml) for precipitation
And resuspend. Furthermore, after quantifying the virus concentration by ultraviolet absorption, it is stored at -70 ° C. The virus was confirmed by a direct negative method using 1% phosphotungstic acid.

(2) Immunity: Dianthus barbat
us L. ) Was diluted with physiological saline to 2 μg / μl, and then 0.2 μm
The solution was sterilized by filtration through a filter to obtain an antigen solution. An equal volume of Freund's adjuvant was added to this antigen solution, mixed and emulsified, and then 100 μl of the emulsified solution was subcutaneously injected into Balb / c mice. One week later, the same operation was repeated to obtain a second antigen stimulation. Three weeks after the second antigen stimulation, the antigen solution prepared above was further diluted twice with physiological saline (1 μg /
μl) After sterilization by filtration through a 0.2 μm filter, 100 μl was intravenously injected. Confirmation of antibody production in antigen-stimulated mice was performed by immunoprecipitation. Four days after the last immunization, the mouse spleen was excised and used as spleen cells, and used for the following cell fusion.

(3) Fusion cells and cloning: The spleen cells prepared in (2) above were used for the mouse myeloma cells NS.
-1 after mixing with -1
2 (manufactured by Shimadzu Corporation) to perform a fusion operation by electric pulse. The fusion conditions were as follows: C-02 was used as the fusion chamber.
(Manufactured by Shimadzu Corporation) using an AC voltage of 4 before pulse application
00 V / cm, AC application frequency 1 MHZ, AC application time 10 seconds, pulse voltage 2.3 KV / cm, pulse width 40
μs, the number of pulses was 2 times, the pulse interval was 1 second, and the AC application time after the pulse application was 30 seconds. The cells subjected to the fusion treatment are seeded in a 96-well microplate, and HAT
After culturing for 14 to 21 days in the medium, the medium was transferred to the HT medium, and the medium was further cultured in 10 ml.
% 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 antigen-positive well by the limiting dilution method. The hybridoma cells thus obtained are named CAV-15.

(4) Recovery and Purification of Monoclonal Antibody: The clonal strain obtained by the above screening was
Cultured in DF medium containing 0% FBS, about 10 μg / m
A culture supernatant containing 1 of the monoclonal antibody was obtained. The classification of the subclass of the mouse monoclonal antibody secreted by the mouse hybridoma CAV- 15 contained in the culture supernatant was performed using a mouse monoclonal antibody isotyping kit RPN29 (manufactured by Amersham).
Was identified.

Ammonium sulfate was added to the culture supernatant to a concentration of 50%, and a precipitate fraction was collected. The precipitate fraction was dissolved in a small amount of a 10 mM PBS solution as much as possible.
Dialysis was performed using 0 mM PBS as an external solution. After completion of the dialysis, an immunoglobulin fraction was obtained using an anti-mouse IgM affinity column, and the purified monoclonal antibody CAV-15 - I was obtained.
gM.

(5) Confirmation of Antigen Protein and Characteristics of Monoclonal Antibody: An equal amount of a reducing buffer (0.125 M Tris HCl (p) was added to the CarMV solution purified in Example 1.
H6.8), 4% sodium dodecyl sulfate (SDS),
After adding 8M urea and 5% (V / V) mercaptoethanol), the mixture was heated on a boiling water bath for 3 minutes. Diluent (0.0685 M Tris HCl (pH 6.8),
After dilution with 2% SDS, 4M urea), a sample solution was obtained.

SDS-PA with 4-20% gel concentration gradient
100, 10, 1 μg per sample lane on a GE plate (84 mm × 90 mm, manufactured by Daiichi Pure Chemicals Co., Ltd.)
Was added, and SDS-PAGE electrophoresis was performed.

After the electrophoresis, the gel of 1-4 lanes was cut out, and a protein stain (0.25 W / V% Coomas) was used.
sic Brilliant Blue R-250,
45.5% Etanol, 9.2% Acetic A
Cid and Water) and stained for 2 hours at room temperature with gentle shaking. Subsequently, the gel was taken out, and the destaining solution (25.5% ethanol, 8.
2% Acidic acid and Water), and the decolorization was continued several times until the background was decolorized, while gently shaking for several hours while changing the solution.

When the protein band became clear, the gel was taken out, and the preservation solution (5% ethanol, 7.5%
After the transfer to the (Acetic Acid and Water), the gel was photographed.

After the electrophoresis, the gel plate of 5-12 lanes was treated with a semi-dry membrane transfer device (Polyb manufactured by ABN).
The protein sample was transferred from the polyacrylamide gel to a nitrocellulose membrane (HYBOND-C manufactured by Amersham) using the following method. Next, 20 mM TB containing 1% BSA
S (20 mM Tris-HCl (pH 7.5), 0.8% Na
Cl) at 4 ° C overnight to block the nitrocellulose membrane.

20 containing 0.1% Tween 20
Washing for 5 minutes with mMTBS (hereinafter referred to as washing solution) was repeated three times. Next, the culture supernatant of the mouse hybridoma cells (CAV-15) was diluted 10-fold with a washing solution, and then reacted at room temperature for 2 hours. After the reaction, 5
The three minute wash was repeated three times. Then use cleaning solution 50
The mixture was reacted with a 0-fold diluted peroxidase-labeled anti-mouse IgM antibody (CAPEL) for 2 hours at room temperature. Further, after washing with a washing solution for 10 minutes was repeated three times, TBS containing 0.025% diaminobenzidine tetrahydrochloride and 0.0062% H ₂ O ₂ was added, and color development was performed at room temperature. Color development was stopped by washing with purified water.

The mouse monoclonal antibody produced by the hybridoma cells (CAV-15) obtained by cell fusion became positive for the CarMV virus antigen by Western blot.

Example 2 (1) Preparation of immunogen: CarMV (10 mg / ml) purified from Bishoujo (D. barbatus L.) was added to 1 mM T
The mixture is diluted with a ris-HCl buffer (2 mg / ml), 0.5 ml of diethyl ether is added to 0.5 ml of the solution, and after stirring, the ether layer is removed. After decompression treatment (room temperature, 10 minutes), 5 μg of RNase (Sigma, EC 3.
After adding 1.27.5), the mixture is allowed to stand at room temperature for 1 hour to prepare a CarMV solution for immunization.

(2) Immunization: RNase-treated CarM
The V solution was sterilized by filtration with 0.2 μm, mixed and emulsified with an equal volume of Freund's adjuvant, and then injected subcutaneously at 100 μl / mouse. One week later, the same operation was repeated to obtain a second antigen stimulation. The antigen solution prepared three weeks after the second antigen stimulation was further diluted twice with physiological saline (1 μg / μ
l) After sterilization by filtration through a 0.2 μm filter, 100 μl of peritoneal injection was performed. Four days after the final immunization, mouse spleen cells were excised and collected, and used for the following cell fusion.

(3) Fusion cells and cloning: The above mouse splenocytes and mouse myeloma cells NS-1 were mixed, and the method of Kohler and Milstein (Kohler and Milstein) was partially modified to obtain 50% polyethylene glycol. Cell fusion was carried out by reacting with the DF solution for 1 minute. A 50% polyethylene glycol DF solution was prepared by autoclaving 4 g of polyethylene glycol (average molecular weight 4000) at 121 ° C. for 20 minutes, cooling to 37 ° C., and adding and mixing 2.8 ml of DF basal medium. The treated cells were inoculated in a 96-well microplate, cultured in HAT medium for 14 to 21 days, then transferred to HT medium, and further cultured in 10 ml, and then cultured in DF medium (containing 10% FBS). . The antibody titer in the culture supernatant of the well in which proliferation was observed was measured by an enzyme-linked immunosorbent assay, and the desired hybridoma was cloned from an appropriate well by the limiting dilution method.
As a result of cloning, hybridoma CAV-21 was obtained.

(4) Recovery and purification of monoclonal antibody:
The clonal strain obtained by the above screening was
Cultured in DF medium containing 0% FBS, about 10 μg / m
A culture supernatant containing 1 of the monoclonal antibody was obtained.

Ammonium sulfate was added to the culture supernatant at 50%.
It was added to the concentration, and the precipitated fraction was collected. The precipitate fraction was dissolved in a small amount of a 10 mM PBS solution as much as possible, and dialyzed using the 10 mM PBS solution as an external solution. After completion of the dialysis, an immunoglobulin fraction was obtained using an anti-mouse IgM affinity column and used as a purified monoclonal antibody.

(5) Characteristics of monoclonal antibody: Hybridoma cell CAV-2 obtained by cell fusion
The mouse monoclonal antibody produced by No.1 was subjected to reduction treatment derived from CarMV virus by Western blot.
Having a molecular weight of about 38,000 (36,000 to 40,000)
Against the antigen of coat protein was Tsu positive der (see Figure 1).

Example 3 (1) Preparation of immunogen: CarMV (10 mg / ml) purified from Bishoujo (D. barbatus L.) was diluted with physiological saline to 2 μg / μl and used as an antigen solution. Was. The SMV virus solution (2 μg / μl) was used as a negative antigen solution.

(2) Immunization: An equal amount of Freund's adjuvant was added to the antigen solution, mixed and emulsified, and then emulsified with 100 μm.
1 was injected subcutaneously into mice. One week later, the same operation was repeated to obtain a second antigen stimulation. Two weeks after the first immunization, a negative antigen solution was used as the antigen solution, and the same operation was performed as the first time. Three weeks after the third antigen stimulation, immunization was performed in the same manner as the third immunization, and the final immunization was performed. Four days after the final immunization, mouse spleen cells were excised and collected, and used for the following cell fusion.

(3) Fusion cells and cloning: The above mouse spleen cells and mouse myeloma cells P3U1 were mixed, and the mixture was treated in the same manner as in Example 2 by Koehler & Milstein (K
Ohler & Milstein) was partially modified, and cell fusion was carried out by reacting with a 50% polyethylene glycol DF solution for 1 minute. The treated cells are seeded in a 96-well microplate, and
After culturing for 14-21 days in AT medium, the medium was transferred to HT medium,
After culturing to a further 10 ml, the cells were cultured in a DF medium (containing 10% FBS). Whether the antibody in the culture supernatant of the well where proliferation was observed was measured by the enzyme antibody method,
The desired hybridoma was cloned from the antigen-positive wells by the ultradilution method. As a result, hybridoma CAV-6 was obtained.

(4) Recovery and Purification of Monoclonal Antibody: Mouse hybridoma cells were transformed into D containing 10% FBS.
After culturing in F medium at 37 ° C., the cells are collected and washed with DF basal medium. 2-5 × 100,000 cells /
ml, and a serum-free medium (0.1% BSA, 10 ^-6 M
Cell culture is started in 2-mercaptoethanol, ^10-8 M sodium selenite, DF basal medium) and cultured for 5 to 7 days. When the culture supernatant becomes acidic, the culture supernatant is collected. After centrifugation at 10,000 rpm for 20 minutes to remove cell debris, ammonium sulfate is added, and the stirred protein is precipitated using a stirrer at 4 ° C. for 4 to 18 hours. After collecting the precipitate by centrifugation at 10,000 rpm for 20 minutes, purified water of 1/20 to 1/25 of the initial culture solution was added to dissolve the precipitate, and the mixture was placed in a dialysis tube and closed tightly. Containing TBS solution (100 mM NaCl, 20 mM Tris-HCl
(PH 7.5)). 100 after dialysis
The supernatant was collected by centrifugation at 00 rpm for 10 minutes,
G200 after filtration using a μm membrane filter
Sephadex column (5.0 cmo, 38.0 cm
L, 250 ml volume, buffer TBS). The active fractions were collected and used for affinity using a 50 ml column of DEAE Sephacel (Pharmacia) (TBS for the adsorption buffer, 150-200 mM Na for the elution buffer).
(TBS containing Cl). The activity and the degree of purification of the antibody were confirmed by the enzyme antibody method, 280 nm absorbance measurement, Lowry method and SDS-PAGE.

Example 6 Antibody Dilution Curve Covalent 96-well ELISA plate (Nunc)
Using 50 μl of purified CarMV per well, 10 μl per well.
Make a 10 mM PBS solution to give
The reaction was performed at 7 ° C. for 2 hours.

After washing three times using a gelatin buffer (10 mM PBS solution containing 0.3% gelatin), 1% B
Add 200 μl of 10 mM PBS solution containing SA (bovine serum albumin) and 0.05% sodium azide, and add 37 ° C.
The reaction was performed for 1 hour.

Subsequently, after washing three times using a gelatin buffer, mouse hybridomas (CAV-21, CAV
-15, CAV-6) After diluting the culture supernatant with 10 mM PBS, 50 μl was added to each well and reacted at 37 ° C. for 1 hour.

After washing three times using a gelatin buffer, a peroxidase-conjugated anti-mouse IgM goat antibody (manufactured by Kappel) was diluted 1000-fold and 50 μl was added, followed by reaction at 37 ° C. for 1 hour.

After washing three times with a gelatin buffer, a color developing solution (12 mg o-PDA, 6 μl 31% H
50 μl of ₂ O ₂ and 30 ml of citrate buffer (pH 5) were added, and the mixture was reacted at 37 ° C. for 30 minutes in a dark room. After the completion of the reaction for 30 minutes, 50 μl of 5N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 491 nm was measured using the absorbance at 600 nm as a blank.

FIG. 2 shows a plot of the dilution ratio and the absorbance of the culture supernatant.

Hybridoma CAV-21, CAV-6
Indicates higher absorbance than CAV-15. CAV
-21, CAV-15 and CAV-6 were used for about 5
When diluted 1:00, the absorption was about 1/2.

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a Western blot diagram of SDS-PAGE of CarMV protein and culture supernatant of hybridoma CAV-21, where 1 is a molecular weight marker, and 2 to 4 are SDs.
S-PAGE, 5-7 are Western blots.

FIG. 2 shows hybridomas CAV-21 and CAV.
It is a dilution curve of the reaction of the culture supernatant of -15 with the antigen (CarWV).

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication location G01N 33/577 9282-4B C12N 15/00 C (C12P 21/08 C12R 1:91) (56) REFERENCES VIROLOGY, 90-2! (1978) P.S. 288-298 PHYTOPATHOLOGY, 79 ~ 10! (1989) p. 1213 PHYTOPATHOLOGY, 72 ~ 8! (1982) p. 1018-1022

Claims (2)

(57) [Claims] The present invention relates to a carnation mottle virus (Car).
The MV) a monoclonal antibody produced by the hybridoma can recognize carnation mottle virus produced using as an immunogen, a (a) an antibody of class (subclass) of IgM (K), (b) Carnation The molecular weight of the reduced protein derived from the mottle virus is about 38,000 (36000 to 4000).
A monoclonal antibody which shows a positive reaction to the antigenic coat protein of 0). 2. A hybridoma CAV-15 (microbial lab fungi)
No. 12309) , CAV-21 ( Microtechnical Research No. 12)
No. 310) and CAV-6 (Microtechnical Laboratory No. 12372)
No.) produced CAV-15-IgM and CAV, respectively.
21-IgM and CAV-6-IgM.
2. The monoclonal antibody according to 1 .