JPH05260989A - Monoclonal antibody against indole-3-acetic acid and its production and use thereof - Google Patents

Abstract

PURPOSE: To obtain a new monoclonal antibody mAbI2G1F12 against a plant hormone IAA.

CONSTITUTION: This monoclonal antibody mAbI2G1F12 against a plant hormone indole-3-acetic acid is secreted from a hybridoma cell system I2G1F12 obtained by a hybridoma method.

COPYRIGHT: (C)1993,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel monoclonal antibody mAb I2G1F12 against the plant hormone indole-3-acetic acid (IAA) and its production and use, in particular produced by the hybridoma cell line I2G1F12. Monoclonal antibody mAb I2G1F12.

[0002]

IAA is an important plant hormone. We are trying to elucidate the role of phytohormones in the development and pathogenesis of plants based on our own research.

In 1985 Mertens et al., IAA.
Immunized with a conjugate of BSA and bovine serum albumin
Disclosed hybridomas from alb / c mice, the produced monoclonal antibodies showed high specificity for short IAA side chains, especially IAA methyl esters. This product, the monoclonal antibody mAb17-
II-A is Idetek. Inc. More commercially available under the trade name PHYTODETEK®-IAA
It is sold at. However, IAA had the disadvantage that it had to be treated with ethereal diazomethane to IAA-Me (IAA methyl ester) before it could be detected in the sample.

[0004]

DISCLOSURE OF THE INVENTION The present invention provides a novel monoclonal antibody mAbI2 against the plant hormone IAA.
G1F12 is provided.

Another object of the present invention is to produce an immunogenic IAA antigen, to produce a hybridoma from an immunized animal by the hybridoma method, to IAA.
It is intended to provide a method for producing a monoclonal antibody, which comprises a step of screening a monoclonal hybridoma cell line secreting an antibody having a high specificity for and a step of isolating such cell line.

Another object of the present invention is to provide a method for producing a hybridoma cell line for a monoclonal antibody.

Another object of the invention is to provide the use of the monoclonal antibody produced according to the invention for the direct detection of IAA and for the isolation of IAA.

The above and other objects, advantages and features of the invention will be more fully understood and appreciated by the following description.

[0009]

The present invention is directed to IA in plants.
In producing a monoclonal antibody that can be used to detect A directly, the plant hormone indole-
Novel monoclonal antibody mAb I2G1F12 against 3-acetic acid (IAA) and hybridoma cell line I2G
It relates to a method for producing the monoclonal antibody mAbI2G1F12 using the hybridoma method for producing 1F12.

The present invention relates to the hybridoma cell line I2G1.
F12 and its monoclonal antibody mA against IAA
and bI2G1F12. The feature of the monoclonal antibody in the present invention is that the free IAA molecule can be directly analyzed by a simple procedure without treatment with ethereal diazomethane and methylation, and without using a device such as a hood, nitrogen, HPLC, and a spectrophotometer. It is something that can be detected. Furthermore, the monoclonal antibody from the hybridoma cell line I2G1F12 shows a high degree of specificity after purification and qualitative analysis.

The method of the present invention comprises the following steps.

(A) a step of binding IAA and BSA (bovine serum albumin) to produce an IAA-BSA conjugate as an immunogenic antigen; (b) Balb / c mice using the IAA-BSA antigen. Immunizing step,
(C) a step of fusing the spleen cells of the immunized mouse and the myeloma cell NS-1 by incubating with a PEG 1500 solution by the hybridoma method,
(D) screening the hybridoma cell line by HAT (hypoxanthine aminopterin thymidine) culture and IELISA (indirect enzyme immunoassay) method,
Obtaining a hybridoma polyclone having a high EIA (enzyme immunoassay) value for IAA-BSA and a low EIA value for BSA, (e) IAA-BSA
Cloning a hybridoma cell line having specificity for, (f) isolating the monoclonal antibody secreted by the cloned hybridoma cell line, (g) characterizing the resulting antibody.

The invention will be further described by way of examples.

The obtained monoclonal antibody can also be used for the following development.

1. ELI for direct detection of IAA amount
Establishment of SA system: It is well known that the binding between an antigen and an antibody shows a positive correlation under an appropriate concentration. In plant tissue extracts or pure cultures of plant pathogens, if non-IA
If the A component does not affect the antigen-antibody binding, then IAA
The abundance of can be detected directly without a purification step.

2. Use of Affinity Chromatographic Method to Separate IAA Molecules: To capture IAA molecules in a complex composition, a monoclonal antibody against IAA is immobilized on a ligand and its pH value is varied to
Elute the AA molecule. The separation method according to the invention simplifies the conventional complicated purification procedure and allows the purification to be carried out in a single step. Thus, this method used to separate trace amounts of IAA in plant extracts is the fastest and most effective.

3. Use of immunoelectron microscopy to measure and trace the physiologically active site of IAA: The amount and distribution of phytohormones differ during development of the plant. Immunoelectron microscopy can be used to measure and track the amount and active site of IAA in plants, which is useful in studying plant growth and physiology during plant development. ..

4. Studies on pathogenic mechanisms in plant pathology:
It is known that under certain circumstances and culture substrates, certain pathogens can be used to synthesize IAA, and that an imbalance in the amount of phytohormones results in the appearance of symptoms. Therefore, the developed monoclonal antibody described above is
In combination with ELISA and immunoelectron microscopy, pathogens,
It can be used to study the pathogenic mechanism relationship between host and IAA.

From the above description, it is clear that the monoclonal antibody mAbI2G1F12 obtained according to the present invention is effective for plant studies.

Accordingly, the present invention provides a novel monoclonal antibody against IAA produced by the hybridoma method. The production method according to the present invention has immunogenicity I.
Producing AA antigens, producing hybridomas by the hybridoma method, screening hybridoma cell lines that secrete specific antibodies against IAA, and isolating monoclonal antibodies.

In accordance with the present invention, we have found that free IAA
M that can directly detect the molecule and shows high specificity
The hybridoma cell line I2G1F12 secreting AbI2G1F12 (IgG1) was screened.

[0022]

The above and other objects, features and advantages will become more apparent by the following examples. These are meant to represent some aspects of the invention and are not meant to limit its scope.

Example 1: Monoclonal antibody mAbI2
Method for producing G1F12 (1) Production of antigen: IAA was combined with BSA to form an IAA-BSA conjugate as an immunogenic antigen.

(2) Production of monoclonal antibody: a. B
Immunization of alb / c mice: Equal amounts of IAA-BSA antigen and Freund's complete adjuvant were mixed well, then 4
Balb / c mice, ~ 5 weeks old, were immunized by intraperitoneal injection. Three to four weeks later, the mice were immunized twice more by intraperitoneal injection with a mixture of the IAA-BSA conjugate and incomplete Freund's adjuvant. After an additional 3-4 weeks, 3 days prior to cell fusion, booster immunization with a complete mixture of saline and IAA-BSA conjugate was given by intraperitoneal injection. The amount of protein for each dose was 200-250 mg.

B. Cell Culture of Myeloma Cell NS-1: NS
-1 cells were cultured in CRPMI-1640 medium and maintained in exponential growth phase.

C. Hybridoma production: Balb / c mouse splenocytes 3 days after the third immunization under sterile conditions were fused with NS-1 cells in the presence of PEG1500 and then hybridomas were screened using HAT medium.

D. Screening hybridomas secreting antibodies: Hybridoma polyclones with high EIA values for IAA-BSA and low EIA values for BSA are screened using indirect enzyme immunoassay (IELISA). Obtained. A second cloning was performed on this hybridoma polyclone and a hybridoma cell line secreting an antibody specific for IAA-BSA was screened using IELISA.

E. Isolation of secreted antibody.

Example 2: Monoclonal antibody mAbI2
Characterization of G1F12 (1) Subclass (isotype) of monoclonal antibody
Determination: Determined by ELISA using MAB-isotyping kit from PMarMingen.

(2) Titration of monoclonal antibody: A series of dilutions of the IAA-BSA antigen from a protein amount of 1 ppm and dilutions of the culture supernatant of the hybridoma cell line I
It can be determined by ELISA.

(3) Measurement of Identification Ability of Monoclonal Antibody Against Free IAA Molecule: The binding site for a predetermined amount of monoclonal antibody after absorption of the IAA-BSA antigen was allowed to compete with the free IAA molecule.

(4) Effect of pH value on binding of IAA-BSA to monoclonal antibody: 1N HCl and 1
PH = 3 of hybridoma cell line with N NaOH
Culture supernatants of 10 were made and then determined by IELISA.

(5) Effect of pH value after binding of IAA-BSA to antibody: IAA-B according to IELISA procedure
After the SA was bound to the monoclonal antibody and treated with 1N HCl for 30 minutes, the effect of pH value on the antigen-antibody binding was observed.

(6) Cross-reaction of monoclonal antibody against antigen analog: The following IAA according to competitive ELISA
The binding reaction of the monoclonal antibody to the analog was measured.

Indole-3-acetic acid (Sigma), indole-2-carboxylic acid (Sigma), indole-3-acetone (Sigma), indole-3-acetamide (Sigma), indole-3-propionic acid (Sigma), Tryptohol (Sigma), indole-3-pyruvate (Sigma), β-indolylacetonitrile (Sigma), (indole-3-acetonitrile) indole-3-carboxylic acid (Sigma), indole-3-aldehyde (Sigma), Indole-3-
Acetaldehyde (Sigma), indole-3-acetic acid ethyl ester (Sigma), indole (Sig)
ma), tryptophan (Sigma), tryptamine (Sigma), IAA-L-Aspartnie, I
AA-glycine, 1-O- (IAA) -β-D-glucopyranose, IAA-Myo-inositol, IAA-
Me, indole-3-butyric acid.

Example 3: Monoclonal antibody mAbI2
Specificity test of G1F12 A competition ELISA was used to detect the recognition of plant hormone molecules by monoclonal antibodies.

(1) Preparation of IAA solution: appropriate amount of IA
Crystals A were dissolved in 95% alcohol and then diluted with distilled water to 1 ppm.

(2) 1 ppm of IAA solution and antibody
The reaction was carried out for 1 minute, and then the mixture was added to an ELISA plate having 1 ppm of IAA-BSA adsorbed thereon at 37 ° C. If the monoclonal antibody can recognize free molecules of phytohormones, the monoclonal antibody is washed out. That is, the amount of antibody bound to the originally adsorbed IAA-BSA molecule is decreased, and the OD405nm value is also decreased. The operating procedure is shown below: a. Adsorption: 1 ppm of IAA-BSA was distributed at 50 μl / well, and left at 37 ° C. for 2 hours or at 4 ° C. overnight.

B. Wash: Washed 6 times with PBST.

C. Competitive antibody: 50 μl of IAA at 50 μl
react with 1 monoclonal antibody for 30 minutes at 20 ° C,
Add 100 μl / well to the ELISA plate and add 3
It was allowed to stand at 7 ° C for 2 hours.

D. Wash: Washed 6 times with PBST.

E. Enzyme-labeled goat antibody: 50 μl / well ELI of enzyme-labeled goat anti-mouse IgG antibody
It was added to the SA plate and left at 37 ° C. for 2 hours.

F. Reaction substrate: 100 μl / well of enzyme reaction substrate having a density of 1 mg / ml was added to the ELISA plate and left standing at 37 ° C. for 2 hours.

Example 4 Cross-Reaction of Monoclonal Antibody Against Antigen Analogues (1) Separation Step of IAA and Methylation (Cohe
n, 1984): The appropriate amount of IAA crystals was placed in a vial and then processed in the following steps.

A. It was dissolved in 100 μl of methanol.

B. 1 ml of ethereal diazomethane was added and reacted at room temperature for 5 minutes.

C. The vial was purged with nitrogen, leaving IAA and IAA-Me on the viral wall (note that the entire reaction process should proceed in an airtight hood and gloves should be worn).

D. 50% methanol was used to elute the remaining IAA and IAA-Me on the wall.

E. A microsyringe was used to take 10 μl of the solution in the vial, which was then injected into the HPLC (High Performance Liquid Chromatography) for the assay: Pump: TRI ROTAR II (JASCO, Japan), Injection device: GP-A40 gradient. Programmer, Column: Lichrosorb C (10 μm particles,
4.6 mm × 25 cm), detector: ABI analytical Kratos
Division, Spectroflow 78
3. Wavelength: IAA: 280 nm, flow rate: 1 ml / min, solvent: 50% methanol.

F. Harvest: Me showed high peak elution with a retention time of 12 minutes.

G. IAA-Me was analyzed by spectrophotometer and confirmed by absorption spectrum.

(2) Determination of cross-reactivity of monoclonal antibodies against some analogs according to competitive ELISA: 1
ppm of IAA-BSA was adsorbed on the ELISA plate, and the culture supernatant of I2G1F12 was diluted 250 times. A solution of the following substances with an OD405 nm = 0.1 was prepared.

Indole-3-acetic acid (Sigma), indole-2-carboxylic acid (Sigma), indole-3-acetone (Sigma), indole-3-acetamide (Sigma), indole-3-propionic acid (Sigma), Tryptohol (Sigma), indole-3-pyruvate (Sigma), β-indolylacetonitrile (Sigma), (indole-3-acetonitrile) indole-3-carboxylic acid (Sigma), indole-3-aldehyde (Sigma), Indole-3-
Acetaldehyde (Sigma), indole-3-acetic acid ethyl ester (Sigma), indole (Sig)
ma), tryptophan (Sigma), tryptamine (Sigma), IAA-L-Aspartnie, I
AA-glycine, 1-O- (IAA) -β-D-glucopyranose, IAA-Myo-inositol, IAA-
Me, indole-3-butyric acid.

(3) I for all 21 kinds of IAA analogs and precursors synthesized in plants and their metabolites
The cross-reactivity of 2G1F12 was measured. From the results of the cross-reactivity test, the IAA and IAA-Myo-inositol tests showed reactivity of about 78-87% and about 17-20%, respectively. The specificity of I2G1F12 is quite high, the antibody obtained in the present invention does not need to be pre-treated by methylation, and Idetek. Inc. It is clearly noted that it is very different from the commercially available PHYTODETEK®-IAA. The results are shown in Table 1 below.

[0055]

[Table 1]

The monoclonal antibody secreted by the hybridoma cell line I2G1F12 against the plant hormone IAA is IgG1. The amount of IAA contained in the plant detected by a 900-fold dilution of the culture supernatant was 46 pg
And pH 6-10 is suitable for binding free IAA molecules to the monoclonal antibody. After binding the monoclonal antibody to the absorbed antigen, using 1N HCl,
Bound antibody can be separated from the antigen at a rate of 80%. Regarding IAA analog cross-reactivity to monoclonal antibodies, only IAA-Myo-inositol showed 17-20% reactivity, while others showed 0 to 1 reactivity.
It showed less than% reactivity.

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Claims (12)

[Claims] 1. A monoclonal antibody against indole-3-acetic acid (IAA), which recognizes free IAA molecules and shows high specificity. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is obtained from the hybridoma cell line I2G1F12. 3. A. Producing an immunogenic indole-3-acetic acid antigen and a hybridoma, b. Screening a hybridoma cell line secreting an antibody with specificity for indole-3-acetic acid, c. Isolating the monoclonal antibody produced in the above step, and a method for producing a monoclonal antibody. 4. The method of claim 3, wherein the immunogenic indole-3-acetic acid antigen is produced by coupling indole-3-acetic acid with bovine serum albumin. 5. A. Producing an immunogenic indole-3-acetic acid antigen, b. Producing a hybridoma by the hybridoma method, c. Screening a hybridoma cell line secreting an antibody having specificity for indole-3-acetic acid, and a method for producing a hybridoma cell line secreting a monoclonal antibody. 6. The hybridoma method is an indole-based method.
The method according to claim 5, wherein the splenocytes of an animal preimmunized with 3-acetic acid-bovine serum albumin antigen are used to fuse with myeloma cells. 7. The method according to claim 6, wherein the immunized animal is a Balb / c mouse. 8. The method of claim 6, wherein the myeloma cells are NS-1 cells. 9. A monoclonal antibody used to establish an ELISA system for the direct detection of the amount of indole-3-acetic acid. 10. A monoclonal antibody used in affinity chromatography for the isolation of indole-3-acetic acid. 11. A monoclonal antibody used in immunoelectron microscopy for qualitative analysis and tracking of the physiologically active site of indole-3-acetic acid. 12. A monoclonal antibody used in the study of plant pathogenic mechanisms.