JPH08242883A - Monoclonal antibody against 2-mthylisoborneol, its production and measurement of 2-methylisoborneol with the same - Google Patents

Abstract

PURPOSE: To provide a method for measuring 2-methylisoborneol, capable of specifically, rapidly and simply measuring the concentration of the 2- methylborneol in high sensitivity, capable of measuring the concentration at a site, and thereby enabling the more flexible and accurate measurement. CONSTITUTION: A monoclonal antibody specifically reacting with 2- methylisoborneol (2MIB). A hybridoma producing the monoclonal antibody specifically reacting with the 2MIB. A method for producing the monoclonal antibody comprises selecting a hybridoma specifically reacting with 2MIB to produce the monoclonal antibody from hybridomas which are produced by cell-fusing myeloma cells with the lymphocytes of an animal preliminarily immunized with an antigen containing a 2MIB-like compound as a hapten, and subsequently culturing the selected hybridoma. A method for measuring the 2MIB with the monoclonal antibody.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to 2-methylisoborneol (hereinafter referred to as 2) which is one of the substances causing off-flavor in tap water.
(Referred to as MIB), a method for producing the same, and a method for measuring 2MIB using the monoclonal antibody.

[0002]

2. Description of the Related Art In recent years, various algae and microorganisms have abnormally proliferated due to eutrophication of rivers and lakes, and the odor generated thereby has a great influence on waterworks using these water bodies as a water source, which has become a problem. There is. Among them, 2MIB has become a problem as one of the substances that cause musty odor, and it is also listed in the comfortable water quality item in the Waterworks Law. As a method for measuring 2MIB, an expensive measuring device such as gas chromatography or gas chromatography / mass spectrometry is generally used. In addition, since a concentration method called the purge trap method is used, it is necessary to equip the measuring device with a special concentration device. Also, the measurement time for one sample is about 2 hours. Therefore, it is difficult to build a detailed monitoring system.

On the other hand, the immunoassay method is rapidly and easily operated by anyone, and simultaneous measurement of multiple specimens is possible. Therefore, the immunoassay method is widely used in medical and clinical examinations. Recently, it has also been applied to environmental analysis of agricultural chemicals. 2 MI
If the immunoassay method was developed in B as well, it would be possible to easily and quickly perform multi-specimen measurement, so it was considered to be extremely effective for monitoring in water sources throughout Japan.

As such an attempt, a method using a rabbit polyclonal antibody (J. Agric. Food Ch
em. 38 , 410-415 (1990)). When producing an antibody against a small molecule such as 2MIB, since it does not have immunogenicity by itself, it is generally necessary to bind it to a high molecular substance such as a protein and immunize an animal with this as an immunizing antigen. . In the case of a polyclonal antibody, antibodies with different properties such as antibody titer, affinity, specificity and antibody subclass are obtained each time depending on the lot difference of the immunizing antigen, the individual difference of the immunized animal, and the immunization method. Therefore, various effects such as instability occurred on the measurement result of 2MIB.

[0005]

As described above, an effective antibody is required for immunologically measuring 2MIB, but as described above, a sufficient antibody has not been obtained. An object of the present invention is to provide a novel monoclonal antibody that specifically immunoreacts with 2MIB, and a method capable of sensitively and rapidly measuring 2MIB in water using the monoclonal antibody.

[0006]

In order to solve the above problems, the present invention provides a monoclonal antibody which specifically immunoreacts with 2MIB, a method for producing the same, a hybridoma producing the monoclonal antibody, a method for producing the hybridoma, It also provides a method capable of sensitively and rapidly measuring 2MIB in water using the monoclonal antibody.

The present invention will be described in detail below.

The first aspect of the present invention is a monoclonal antibody characterized by specifically immunoreacting with 2MIB. To be exact, the monoclonal antibody is a compound similar to 2MIB, that is, in addition to 2MIB, isoborneol (hereinafter referred to as IMIB).
B), borneol, camphor,
It is a monoclonal antibody that does not substantially react with norborneol and norcamphor, and particularly has a measurement sensitivity of 2 MIB at a level of 5 ng / assay. The second aspect of the present invention is
A specific immune reaction with 2-methylisoborneol was made from hybridomas prepared by cell fusion of lymph cells and myeloma cells of an animal previously immunized with an antigen having 2-methylisoborneol or a similar compound as a hapten. The method for producing a monoclonal antibody is characterized in that a hybridoma producing the indicated monoclonal antibody is selected and the hybridoma is cultured. The third aspect of the present invention is the method for producing the above monoclonal antibody, wherein the 2MIB analog is IB. The fourth aspect of the present invention is a hybridoma producing the above-mentioned monoclonal antibody. The fifth aspect of the present invention is to specifically immunize 2MIB among hybridomas prepared by cell fusion of lymphocytes and myeloma cells of an animal previously immunized with an antigen having 2MIB or a similar compound as a hapten. The method for producing a hybridoma is characterized in that a hybridoma producing a monoclonal antibody showing a reaction is selected. A sixth aspect of the present invention is that the compound similar to 2MIB is IB
The method for producing the hybridoma is A seventh aspect of the present invention is a method for measuring 2-methylisoborneol, which comprises using the monoclonal antibody. This measuring method is suitable for measuring 2MIB in the environment, and by extension, for measuring odor and off taste, and is expected to be used particularly in the waterworks field.

The term "immune reaction" as used in the present invention has a broad meaning indicating the binding between an antigen or a hapten and an antibody regardless of whether it is inside or outside the living body.

Preparation of Immunizing Antigen When producing an antibody against a low molecular weight substance as described above,
Since it has no immunogenicity by itself, it binds to macromolecules such as proteins and immunizes animals with the conjugate. 2 MI
Similarly, when producing an antibody that recognizes B, 2 MIB
Is bound to a protein and the conjugate is used as an immunizing antigen. In addition, even if a similar compound such as camphor or isoborneol (IB), which is more easily available than 2MIB, is used,
An antibody that recognizes IB can be obtained. When camphor is used, for example, the method of Chung et al. (J. Ag.
ric. Food Chem. , 38 , 410-415
(1990)), an immunizing antigen can be prepared. 2 MI
When B or IB is used, the hydroxyl group is reacted with succinic anhydride or glutaric anhydride to introduce a carboxyl group, and the carboxyl group is used to bind to a protein by a carbodiimide method or an acid anhydride method. The body can be the antigen for immunization. The protein is not particularly limited, but bovine serum albumin (BS
A), ovalbumin (OVA), Jinkasai hemocyanin (KLH) and the like can be used.

Monoclonal Antibody Production Method The monoclonal antibody production method is basically based on Koehl.
er and Milstein et al. (Nature, 25
6 , 495, 1975) and its variants (J. Immuno)
l. Methods, 39 , 285, 1980).

Preparation of hybridoma Examples of animals to be immunized include mice and rats.
The method of inoculation is subcutaneous, intramuscular or intraperitoneal. Immunization is performed in 3 to 5 divided doses at an interval of 1 to 4 weeks, and it is preferable that the immunization be mixed with complete Freund's adjuvant or incomplete Freund's adjuvant for administration. Antibody-producing cells obtained from the spleen or lymph node of the immunized animal are fused with myeloma cells and isolated as hybridomas. As the myeloma cells, those derived from mice, rats, etc. are used, and preferably derived from the same species as the antibody-producing cells.

At the time of cell fusion, the mixing ratio of the antibody-producing cells and myeloma cells is about 1 to 10: 1, and polyethylene glycol (PEG) or Sendai virus (HV) is used.
J) and the like are used for fusion, but from the viewpoint of operation, 30% to 60% PEG (molecular weight 1000 to 8) is used.
000) solution is preferably used. Hybridomas are usually selected by a medium for animal cell culture (IMDM, DMEM, RPM) containing HAT (hypoxanthine, aminopterin, thymidine) and 10 to 20% fetal calf serum (FCS).
I1640, MEM, etc.).

The growth of the hybridoma is confirmed, and it is determined whether or not the desired antibody is produced in the culture supernatant. As the measuring method, RIA (radioimmunoassay) method, ELISA (enzyme immunoassay) method, FIA (fluorescent immunoassay) method, plaque measuring method, agglutination reaction method and the like can be used, and the following ELISA method is used. It is desirable to use.

Screening by ELISA The protein conjugate prepared by the same procedure as the immunizing antigen was subjected to ELI.
Immobilize on the surface of each well of the SA plate. Next, for the purpose of preventing non-specific adsorption, BSA, OVA, KLH
Alternatively, gelatin, skim milk, etc. are immobilized in each well. The hybridoma culture supernatant is added to each well and left for a certain period of time to carry out an immune reaction. Each well is washed with PBS (phosphate buffered saline) or the like as a washing solution. It is preferable to add a surfactant to this cleaning liquid. Add the enzyme-labeled secondary antibody and leave it for a certain period of time. As the labeling enzyme, β-galactosidase, alkaline phosphatase, peroxidase and the like can be used. After washing each well with the same washing solution, the substrate solution of the labeling enzyme used is added to carry out the enzyme reaction. When the desired antibody is present in the added hybridoma culture supernatant, the enzymatic reaction proceeds and the color of the substrate solution changes.

Cloning of hybridoma After confirming the well producing the desired antibody by the above method, cloning is performed to obtain a single clone.
As a cloning method, a limiting dilution method or the like in which hybridoma cells are diluted and cultured so that one colony is formed per well of a culture plate may be used.
For cloning by the limiting dilution method, use feeder cells to increase the colony formation rate, or use interleukin 6 (I
It is desirable to add cell growth factors such as L6).
The hybridoma cells obtained in this way are deposited with the deposit number [FERM P
-14789].

The single hybridoma thus obtained can be subjected to large-scale culture using a flask or a cell culture device, or can be cultured in the abdominal cavity of an animal to obtain a monoclonal antibody. When culturing in a flask, a cell culture medium containing 0 to 20% FCS (I
(MDM, DMEM, RPMI1640, MEM, etc.). When cultured in the abdominal cavity of an animal, it is preferable to use an animal of the same species and strain as the origin of the myeloma cells used for cell fusion,
The hybridoma is transplanted after pristane or the like is administered in advance. After 1-2 weeks, myeloma cells proliferate in the abdominal cavity, and ascites containing the monoclonal antibody can be obtained. The monoclonal antibody can be purified from the thus obtained cell culture supernatant and ascites by means such as salting out, gel filtration, ion exchange chromatography, affinity chromatography and the like.

According to the immunoassay method using the anti-2MIB monoclonal antibody thus obtained, qualitative and quantitative determination of 2MIB in environmental water such as rivers and lakes can be performed quickly and easily. Immunoassay methods include RIA and EL
Known methods such as ISA and FIA can be used.

[0019]

EXAMPLES The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples. Example 1 Preparation of antibody recognizing 2MIB 1. Method for Introducing Carboxyl Group of IB 50 mg of IB was added to 2 ml of pyridine that had been dehydrated in advance, 700 mg of glutaric anhydride (HG) was added after dissolution, and the mixture was sealed and reacted at 70 ° C. for 20 hours. After the reaction, add an equal amount of distilled water and stir, then centrifuge (15000 r
pm, 5 minutes) to remove the upper aqueous phase and extract with 20 ml of dichloromethane. The dichloromethane phase was washed 9 times with distilled water, dehydrated with anhydrous sodium sulfate, and then dried under a nitrogen gas stream. This residue was used as IB-HG for binding to carrier protein.

2. Method for Preparing IB-HG-Protein Conjugate 20 mg bovine serum albumin (BSA) was dissolved in 10 mM sodium phosphate buffer (pH 7.0) containing 0.9% sodium chloride, and 40 mg 1-ethyl-3- (3
-Dimethylaminopropyl) -carbodiimide hydrochloride was added. 2.4 mg of IB-HG 10
It was dissolved in 0 μl of dimethylformamide, and was added dropwise to the protein solution. BSA and IB-H at this time
The molar mixing ratio with G was 1:30. After stirring for 12 hours at room temperature, 3% against 0.9% sodium chloride solution
He dialyzed for a day. Ovalbumin (OVA) by the same operation
And IB-HG were added so that the molar mixing ratio was 1:20, and hemocyanin (KLH) and IB-HG were 1: 200. The prepared conjugate was centrifuged (12000 rpm, 30 minutes) after dialysis, and the supernatant was divided into small portions and stored at 4 ° C until use.

3. Immunization For the primary immunization, the antigen was diluted with physiological saline, Freund's complete adjuvant was mixed at 1: 1 to emulsify, and then Balb / c mice (female, 8 weeks old) were treated with 5 per mouse.
It was subcutaneously administered to the back at two sites so that the amount of protein became 0 μg. The booster was administered one week later, this time using Freund's incomplete adjuvant. Two weeks later, 50 ml of the antigen solution diluted with physiological saline was added to each mouse.
It was administered into the tail vein so that the dose became μg.

4. Cell fusion 3 days after the final immunization, the spleen of the mouse was aseptically removed and
Splenic lymphocytes were dispersed in EM medium. The lymphocyte suspension was collected by centrifugation (1600 rpm, 5 minutes),
It was washed three times by redispersion in MEM medium. The obtained 1 × 10 ⁸ splenic lymphocytes were mixed with 2 × 10 ⁷ myeloma cells (SP2 / 0-Ag14 strain), centrifuged (1600 rpm, 5 minutes), and the supernatant was completely removed by suction. The bottom of the centrifuge tube was tapped to loosen the precipitate.
Then, 1 ml of 50% PEG4000 solution was added dropwise over 1 minute. After shaking and stirring for another minute as it is,
2 ml of MEM was added dropwise over 2 minutes. 7 ml more
MEM was added over 3 minutes. Centrifuge (800r
pm, 6 minutes), the supernatant was removed by suction, the precipitate was loosened, and then HAT medium (1 × 10 ⁻⁴ M hypoxanthine, 4 ×) was used.
10 ⁻⁷ M azaguanine, 1.6 × 10 ⁻⁵ M thymidine, 2
30 ml of DMEM medium containing 0% FCS was added and resuspended. The total amount of the cell suspension was dispensed into each well of a 96-well plate in an amount of 0.1 ml and cultured in a CO ₂ incubator. The day after cell fusion, HAT medium was added to 2
It was added drop-wise. Further, on the third day, the sixth day, the eighth day, the tenth day, and the twelfth day after the fusion, half the amount of the medium was replaced with a new medium. As a result, proliferation of hybridoma was observed in almost all wells. On day 14, screening was performed by measuring the antibody titer in the culture supernatant of each well by the ELISA method.

5. Method for measuring antibody activity All antibody titers were determined by the ELISA method. First 9
100 μl of IB-HG-OVA (0.1 μg / ml) diluted with PBS was added to each well of a 6-well EIA plate and left overnight at 4 ° C. to immobilize IB-HG-OVA on the well surface. 0.05% to remove things
4 with PBS containing Tween 20 (PBS-Tween)
Washed twice. 125 μl of blocking solution (0.1% O
VA, PBS containing 0.01% sodium azide) was added, and the mixture was left at 4 ° C. overnight. PBS-Tween before use
100 μl of a diluted culture supernatant diluted with PBS-Tween was added and left at room temperature for 1 hour to allow the antigen-antibody reaction to proceed. To remove unreacted materials after leaving,
After washing 4 times with PBS-Tween, alkaline phosphatase (ALP) -labeled goat anti-mouse immunoglobulin G + M
Dilute antibody 5000 times with PBS-Tween and
0 μl was added. After leaving at room temperature for another hour, PB
After washing 4 times with S-Tween, 100 μl of ALP substrate solution was added. Immediately before use, the ALP substrate solution was prepared by dissolving disodium p-nitrophenylphosphate in diethanolamine buffer at a concentration of 1 mg / ml. And 3
After leaving it at room temperature for 0 minutes, the absorbance at 405 nm was measured by a microplate reader. As a result, 34
The culture supernatant of the well showed an immunoreactivity with IB-HG-OVA.

Further, a competitive ELISA method was carried out to identify hybridomas producing anti-2MIB antibody. Conflict E
The LISA method was performed by allowing free 2MIB to coexist during the reaction between the diluted culture supernatant of the ELISA method and the solid phase antigen. Here, since the anti-2MIB antibody competitively reacts with the free 2MIB and the immobilized antigen, it can be confirmed that the anti-2MIB antibody-producing hybridoma is present in the well where the inhibitory reaction against the immobilized antigen is observed. As a specific operation method, the same operation as in the ELISA method was performed except that 50 μl of 100 μl / ml 2MIB solution was added simultaneously with 50 μl of the culture supernatant diluted solution. As a result, as can be seen from Table 1, of the 32 positive wells showing an immunoreactivity with IB-HG-OVA, the wells in which the inhibition reaction by free 2MIB was remarkable in the culture supernatant of 9 wells, that is, the inhibition rate was high. That is, three wells containing a large amount of anti-2MIB antibody were confirmed.

[0025]

[Table 1]

6. Cloning The 3 strains of anti-2MIB antibody-producing hybridomas thus obtained were immediately cloned. Cloning was performed by the limiting dilution method. As a medium for cloning, HT medium (HAT medium without aminopterin) was used for the first time, 20% FCS-DMEM medium was used for the second time and thereafter, and 2 U / ml of IL6 was added.

The operation was carried out at a hybridoma cell concentration of 10 cells /
Dilute to 0.1 ml with the above medium and add 0.1
ml was dispensed into each well of a 96-well microplate. After culturing for 5 days, two drops of the same medium were added dropwise and culturing was carried out for 10 to 14 days. After confirming colony formation, the antibody activity of the supernatant was measured by the above method. Positive wells were purified by cloning three times, and as a result, a monoclonal antibody (hereinafter also simply referred to as anti-2MIB monoclonal antibody or anti-2MIB antibody) -producing hybridoma 2 that specifically immunoreacts with 2MIB was produced.
MIB-1, 2MIB-7, and 2MIB-8 were able to be obtained. The results of measuring these antibody titers are shown in Table 2.

[0028]

[Table 2]

From this table, those with particularly high antibody titers were selected and deposited. This anti-2MIB antibody producing hybridoma 2
MIB-1 (hereinafter, also simply referred to as hybridoma 2MIB-1 or 2MIB-1 etc.) was entrusted to the Institute of Biotechnology, Institute of Industrial Science and Technology as of February 24, 1995, and the accession number [FERM P-14789]. Is getting

7. Production of Monoclonal Antibody The hybridoma 2MIB-1 strain thus obtained was cultured using 20% FCS-DMEM.
The culture was performed until 80% or more of the cells were dead, and the culture supernatant was collected by centrifugation (3000 rpm, 15 minutes). The recovered culture supernatant was diluted with 0.02 M phosphate buffer (pH 7.0) (PBS) and then diluted with protein G.
The anti-2MIB monoclonal antibody was purified by affinity chromatography using a column. The chromatography conditions are as follows:
After passing through a Sepharose column to wash and remove components other than the antibody with 0.02 M phosphate buffer (pH 7.0), the antibody was eluted with 0.1 M glycine-hydrochloric acid buffer (pH 2.7). The eluate was immediately neutralized and then dialyzed against PBS.

Intraperitoneally into Balb / c mice (female, 6 weeks old) pre-administered 0.5 ml / mouse of pristane 1
X10 ⁷ hybridoma 2MIB-1 were transplanted and 1
After 2 weeks, ascites fluid containing anti-2MIB monoclonal antibody was collected. The ascites was diluted 5 times with PBS, ammonium sulfate was added to have a concentration of 40%, and the precipitate was collected by centrifugation (12000 rpm, 20 minutes). The precipitate was redissolved in PBS and dialyzed against PBS. After dialysis, the monoclonal antibody was purified by affinity chromatography using a protein G column.

8. Characteristics of Monoclonal Antibody Mouse Monoclonal Antibody
The class and subclass of the monoclonal antibody were determined using Isotyping Kit (Amersham).
As a result, the obtained anti-2MIB monoclonal antibody was
It was an antibody belonging to IgG ₁ (κ). In order to investigate the specificity of the anti-2MIB monoclonal antibody, the antibody and IB-H
It was determined whether the reaction with G-OVA was inhibited by free 2MIB or its analogues. As a result, anti-2MIB
The detection sensitivity of 2MIB using a monoclonal antibody is 5
ng / assay (see Figure 1). Anti-2 MIB
Monoclonal antibodies reacted equally to 2MIB, IB, borneol and camphor and did not react with norborneol, norcamphor below 5 μg / assay. In addition, it did not react with diosmin, which is another off-taste substance in tap water, at the same concentration.

9. Measurement of 2MIB amount in water (1) 0.05 ml of antibody diluent (20%) on an EIA plate on which 100 ng / ml IB-OVA was immobilized in advance.
2 times concentrated PBS-Tween containing methanol) was added. The culture solution of actinomycetes which produces 2MIB was filtered through a 0.2 μm filter, and the filtrate was diluted with 0.
05 ml was added. A culture solution containing no actinomycetes that produces 2MIB was similarly filtered and used as a control together with a standard solution containing a known concentration of 2MIB. The mixture was well stirred and reacted at room temperature for 2 hours. The method described in the method for measuring antibody activity in 5 of Example 1 was followed. As a result, 0.16 mg / liter of 2M was added to the main culture solution.
It was confirmed that IB was present.

In this measuring method, it took about 3 hours to pretreat and measure 20 samples. On the contrary,
In the conventional gas chromatography method, about 30 minutes are required for each sample, and it takes time to measure a large amount of samples.

(2) 2 liters of river water was applied to a Bond Elute C18 column (GL Sciences) for solid phase extraction.
Pressure injection was performed at 0 ml / min. 2 ml of MIB was eluted by adding 6 ml of dichloromethane and dehydrated with anhydrous sodium sulfate. It was re-concentrated in a stream of N ₂ gas and replaced with methanol to make the total volume 0.1 ml. This concentrate 20μ
1 was dispensed on an IB-OVA-immobilized EIA plate containing 80 μl of an antibody diluent (diluted with PBS-Tween), and the reaction was carried out at room temperature. The method described in the method for measuring antibody activity in 5 of Example 1 was followed. As a result, it was confirmed that 10 ng / liter of 2MIB was present in the main lake water.

In the case of a low concentration sample, this measuring method is
The pretreatment required about 6 hours and the measurement required about 3 hours. This does not change even if the number of specimens increases.

[0037]

The novel monoclonal antibody of the present invention is 2
Since it reacts specifically with MIBs, the use of this monoclonal antibody enables the
E with a polyclonal antibody specifically for the MIB concentration
Compared to the LISA method, it has become possible to measure with high sensitivity and quickly and easily. In addition, a simple absorbance meter is sufficient for this measurement, and therefore it can be carried out at the outdoor site.
Therefore, there is no need to transport the sample to the analysis room, and quick response is possible. Especially, the difference in sensitivity from the conventional ELISA method using a polyclonal antibody is clear. Furthermore, since it enables on-site measurement, it is more flexible and enables precise measurement. The present monoclonal antibody is produced by a novel hybridoma, which can be stored semipermanently in liquid nitrogen and can be thawed and cultured to obtain a single antibody having the same characteristics at any time. You can get as many antibodies as you need.

[Brief description of drawings]

FIG. 1 2 MIB. 3 is a graph showing the cross-reactivity of 1 monoclonal antibody with 2MIB and 2MIB-like compounds.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location // (C12P 21/08 C12R 1:91) (C12N 5/10 C12R 1:91) (C12N 15 / 02 C12R 1:91)

Claims (7)

[Claims] 1. A monoclonal antibody which specifically immunoreacts with 2-methylisoborneol. 2. A hybridoma produced by cell fusion of myeloma cells and lymph cells of an animal previously immunized with an antigen having 2-methylisoborneol or a similar compound as a hapten, and specific to 2-methylisoborneol. 2. The method for producing a monoclonal antibody according to claim 1, wherein a hybridoma producing a monoclonal antibody that shows an immunological reaction is selected and the hybridoma is cultured. 3. The method for producing a monoclonal antibody according to claim 2, wherein the 2-methylisoborneol analogue is isoborneol. 4. A hybridoma producing the monoclonal antibody according to claim 1. 5. Among the hybridomas produced by cell fusion of myeloma cells and lymph cells of an animal previously immunized with an antigen having 2-methylisoborneol or a similar compound as a hapten, 2-methylisoborneol and The method for producing a hybridoma according to claim 4, wherein a hybridoma that produces a monoclonal antibody that specifically shows an immune reaction is selected. 6. The method for producing a hybridoma according to claim 5, wherein the 2-methylisoborneol analog compound is isoborneol. 7. A method for measuring 2-methylisoborneol, which comprises using the monoclonal antibody according to claim 1.