JP2002112767A - Anti-aristolochic acid monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a simple means for analyzing aristolochic acid. SOLUTION: This monoclonal antibody belongs to a subclass of IgG2b, has κlight chain and specifically reacts with aristolochic acid. The anti-aristolochic acid monoclonal antibody is obtained by fusing an antibody-forming cell of an animal (preferably mouse) immunized against an aristolochic acid-serum albumin complex, which is obtained by reacting aristolochic acid with bovine or human serum albumin, with a myeloma cell to give a hydridoma, selectively culturing the hybridoma, screening and cloning the hydridoma for producing the monoclonal antibody against aristolochic acid. Aristolochic acid can be simply determined in high sensitivity by applying the monoclonal antibody to an immunoassay method such as ELISA method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention belongs to the technical field of monoclonal antibodies, and more particularly, to an anti-aristrochiic acid monoclonal antibody that specifically reacts with aristolochic acid.

[0002]

2. Description of the Related Art Aristolochic acid (Aristolochic acid: hereinafter may be abbreviated as Ari) is a component contained in plants of the family Echinacea. In Europe, people who consumed slimming tea caused severe nephritis. The causative components were examined, and this compound was isolated and identified. Also in Japan, patients who took Chinese medicine, especially Chinese-made Toki Shikakkashiyuyu-shokyo-to, had similar nephritis, which was a serious side effect. This is due to the fact that the combination of herbal medicines differs between Japanese and Chinese prescriptions, and that Chinese herbal medicines belonging to the family Echinacea are used. Under such circumstances, it has been pointed out that it is important to test for the presence or absence of aristolochic acid in herbal medicines and crude drugs in order to avoid side effects, and a simple and accurate method for analyzing the present compound has been eagerly desired.
In addition, since the physiological mechanism by which this compound causes nephritis is unknown, it is necessary to investigate the pharmacokinetics and metabolism, as well as the binding to proteins, in detail.
A simple and highly sensitive means for analyzing aristolochic acid meeting this requirement has not yet been established.

[0003] On the other hand, the cell fusion technology has been rapidly developed since the report of Koehler and Milstein (Nature, pp. 495-497, 1975). Hybrid cells (hybridomas) obtained by fusing mammalian spleen cells (splenocytes) immunized with a specific antigen and cancer cells, myeloma cells (myeloma cells), show the ability of the spleen cells to produce antibodies against the specific antigen. Therefore, antibodies specific to a large number of high molecular compounds such as proteins and peptides, that is, monoclonal antibodies (hereinafter, sometimes referred to as MAbs) are produced by using the MAbs. Attempts have been made to construct assay systems for such proteins and the like. However, since aristolochic acid is a small molecule, it cannot normally serve as an antigen, and no monoclonal antibody against aristolochic acid has been known.

[0004]

Means for Solving the Problems The present inventors have now immunized an animal with a specific treatment of aristolochic acid, obtained a hybridoma producing a monoclonal antibody against aristolochic acid by cell fusion, and obtained a hybridoma from the hybridoma. The present inventors have succeeded in producing a large amount of an anti-alistrochiaic acid monoclonal antibody which can be subjected to acid analysis, and have derived the present invention.

[0005] Thus, the present invention provides an aristolochic acid I
And / or an anti-aristrochiic acid monoclonal antibody that recognizes aristolochic acid II is provided for the first time. Further, the present invention provides, as a specific example of such an anti-aristrochiic acid monoclonal antibody, an anti-aristrochiic acid monoclonal antibody belonging to the subclass of IgG2b and having a κ light chain.

[0006] The present invention further provides a method for producing an anti-alistrochiaic acid monoclonal antibody as described above, which method comprises the steps of: producing a monoclonal antibody against aristolochic acid in the presence of N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide; Antibody production in animals immunized with a complex of aristolochic acid I and / or aristolochic acid II and serum albumin formed by reacting aristolochic acid I and / or aristolochic acid II with bovine or human serum albumin A step of selectively culturing a hybridoma obtained by fusing the cells with myeloma cells, and screening (selecting) and cloning a hybridoma producing a monoclonal antibody against aristolochic acid I and / or II. In a preferred embodiment of the method for producing an anti-alistrochiaic acid monoclonal antibody according to the present invention, mouse spleen cells are used as antibody-producing cells, and mouse myeloma cells are used as myeloma cells. Further, the present invention provides
Hybridomas producing these anti-alistrochiaic acid monoclonal antibodies are also provided.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The anti-alistrochiaic acid monoclonal antibody of the present invention can be produced by devising and applying the cell fusion technique and the monoclonal antibody production method devised so far. That is, (1) antibody-producing cells of an animal immunized with a complex of aristolochic acid and serum albumin as an antigen are prepared. (2) Myeloma cells (myeloma cells) are cultured and grown. (3) The above 2
The seed cells are fused using a fusion promoter such as polyethylene glycol as a medium. (4) The obtained hybridoma is cultured (proliferated) in a selection medium such as a HAT medium.
(5) Screening (selection) of anti-aristrochiic acid-producing hybridomas. (6) Cloning the selected hybridoma. (7) The obtained hybridoma is cultured (proliferated) in vitro or in the animal intraperitoneal cavity to obtain an anti-aristrochiaic acid monoclonal antibody. Hereinafter, each step will be described in detail.

(1) Preparation of antibody-producing cells Aristolochic acid includes aristolochic acid I (Ari
I) and aristolochic acid II (AriII) are known (see FIG. 1). In the present invention, these aristolochic acids and bovine serum albumin (BSA) are used.
Alternatively, the animal is immunized with a complex (conjugate: conjugate) with human serum albumin (HSA). That is, AriI and / or AriII and BS in a weakly acidic (pH 5) solution in the presence of EDC [N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide].
A (or HSA) is reacted with an aristolochic acid-BSA (or HSA) complex formed by peptide-bonding the carboxyl group of the aristolochic acid and a lysine residue of BSA (or HSA). Immunize. FIG. 1 schematically shows a reaction for obtaining such a complex. In the present invention, the MALDI-tof spectrum measurement
An aristolochic acid-BSA (or HSA) complex confirmed to have 5-40 molecules of aristolochic acid bound per BSA (or HSA) molecule is used.

According to the present invention, AriI and AriII
(Ie, a mixture of AriI and AriII) using B
Immunization as a complex with SA (or HSA) provides an anti-aristrochiic acid that recognizes AriI and AriII. Immunization with either AriI or AriII as a BSA (or HSA) complex yields anti-aristrochiic acid that recognizes only one of AriI and AriII, and recognizes both AriI and AriII. It has been found that anti-alistrochiaic acid monoclonal antibodies can also be obtained. As an immunization method, a method using Freund's complete adjuvant is used. Examples of animals to be immunized include mice, rats, rabbits, guinea pigs, sheep, and the like. Cells isolated from spleen, lymph nodes, peripheral blood and the like are used as antibody-producing cells.

(2) Preparation of myeloma cells Myeloma cells used for cell fusion are not particularly limited, and various mammalian cell lines can be used. It is preferred to use a cell line of A particularly preferred combination for producing anti-aristrochiic acid monoclonal antibodies of the invention is mouse splenocytes / mouse myeloma cells. Considering that after cell fusion, unfused myeloma cells cannot survive in the selection medium and only hybridomas can proliferate after cell fusion, thereby selecting unfused cells and fused cells. Those having a particular drug resistance are preferred. For example, 8-azaguanine-resistant cells are preferably used because they have a property that they cannot grow in a HAT medium. Specifically, mouse myeloma cell line, PAI, P3-X63-Ag
8, P3-X63-Ag8-UI, P3-NSI / 1-
Ag4-1, X63-Ag8-6.5.3. , SP2 /
0-Ag14, FO, S194 / 5XXO, BU. 1,
MPC11-45.6. , TG. 1.7 or the like is used.

(3) Fusion cells Cell fusion is usually performed in MEM medium, RMI1640 medium, IM
Myeloma cells and antibody-producing cells are mixed in an e-RDF medium such as a DM medium in the presence of a fusion promoter (mixing ratio is usually 1: 1:
4 to 1:10). As a preferred fusion promoter, polyethylene glycol (PEG) having an average molecular weight of 1,000 to 6,000 can be used. The working concentration of PEG is usually 30 to 50%.

(4) Selective culture of hybridomas The cells after fusion are appropriately diluted with e-RDF medium containing 10% FCS and centrifuged. The precipitate is suspended in a selection medium (for example, HAT medium), inoculated into a 96-well microplate, and then cultured in a 5% carbon dioxide gas culture apparatus. The cells growing on the selection medium are hybridomas.

(5) Screening of antibody-producing hybridomas Screening of antibody-producing hybridomas may be performed according to a conventional method, and is not particularly limited. For example, a culture in which a hybridoma has grown is collected, reacted with a complex of aristolochic acid (AriI and / or AriII) and BSA (or HSA), and reacted with a secondary antibody labeled with an enzyme, a fluorescent substance, a luminescent substance, or the like. Hybridomas having an anti-aristroic acid monoclonal antibody producing ability of interest are selected by the ELISA method.

(6) Cloning of Hybridoma Cells in culture wells confirmed to contain antibody-producing hybridomas are cloned by a limiting dilution method or the like to obtain anti-aristrochiic acid-producing hybridomas.

(7) Preparation of Anti-Ari MAb The hybridoma obtained as described above is cultured in vitro in a suitable medium such as a serum-free medium, and the desired anti-aristrochiaic acid monoclonal antibody is obtained from the culture supernatant. (Anti-Ari MAb) is obtained. MAb
When a large amount of is produced, an animal of the same species as the myeloma cell-derived animal is intraperitoneally administered with a mineral oil such as pristane and then inoculated with a hybridoma. After inoculation, ascites is collected, and an anti-Ari MAb is obtained by a usual antibody separation operation.

[0016]Characterization of anti-Ari MAb
® down  Of the immunoglobulin class of the obtained anti-Ari MAb
Properties and light chain properties can be determined by the usual methods (for example,
-Method). Also, the molecular weight of the anti-Ari MAb
Is, for example, a multi-
Know from MALDI-tof mass spectrum measurement
Can be

According to the present invention, aristolochic acid (AriI and / or AriII) is converted to B
Immunization of mice as a complex with SA (or HSA) produces a monoclonal antibody that specifically reacts with aristolochic acid (AriI and / or AriII) but does not show cross-reactivity with other related compounds. can get. The resulting monoclonal antibody is generally
Monoclonal antibodies belonging to the subclass G2b and having a κ light chain, but belonging to other immunoglobulin classes are also obtained. Examples are shown below to further clarify the features of the present invention, but the present invention is not limited by these examples.

[0018]

【Example】Example 1: Preparation of anti-Ari MAb  (1) Preparation of antigen: aristolochic acid (AriI and Ar
iII mixture: obtained from Sigma) 3 mg
3.8 mg of B dissolved in 1.2 ml of lofuran
SA (manufactured by Wako Pure Chemical Industries) in 20 mM phosphate buffer (p
It was added to the solution dissolved in H5) with stirring. Reaction
EDC 18 mg was added to the liquid, and the mixture was stirred and reacted for 15 hours. Anti
After the reaction, it was dialyzed for purification, lyophilized and treated with 3.5 mg of A
An ri-BSA complex was obtained.

(2) Examination of the number of haptens in the antigen: Take a trace amount of the obtained Ari-BSA complex, add excess sinapinic acid and mix, put a small amount of this mixture into the well of the cassette, It was measured by Mardi Tohumas. FIG. 2 shows the spectrum. From FIG. 2, it was confirmed that 20 molecules of Ari were bound to one BSA molecule.

(3) Preparation of immune cells: Ari-BSA
50 μg of the complex was emulsified in Freund's complete adjuvant and administered intraperitoneally to BALB / C mice. Thereafter, 50 μg of the Ari-BSA complex (incomplete Freund's adjuvant solution) was similarly administered twice at two-week intervals, and finally 100 μg of the Ari-BSA complex alone (incomplete Freund's adjuvant solution) was administered for immunization. Completed. Three days later, the mouse was sacrificed under anesthesia, and the spleen was removed. After chopping the spleen, the spleen was filtered through a 100 mesh nylon net to obtain isolated spleen cells.

(4) Preparation of hybridoma:
Add hypotonic solution (155 mM ammonium chloride) to the cells.
After red blood cells are eluted, the cells are cultured three times in e-RDF medium.
Washed. Mouse myeloma cells (cultured in the laboratory of the present invention)
Were also washed three times with e-RDF medium. Count both cell counts
Centrifuge cells and myeloma cells at a 10: 1 ratio
You. Discard the supernatant, dissolve and separate the precipitated cells.
Ethylene glycol (PEG) 4,000 diluted in medium
The fusion was performed by dropping 1.0 ml of a 50% solution. 37
After standing still at 1 ° C. for 1 minute, 5 ml of e-RDF medium was
And added. Centrifuged at 1,000 rpm for 10 minutes. Sinking
Is washed with IMDM containing 10% FCS and centrifuged
Abandoned Qing. Hypoxanthine 10⁻²M, Aminopteri
4 × 10 ⁻⁷M and thymidine 1.5 × 10⁻⁵Add M
(HAT) using 10% FCS-added e-RDF medium
And resuspend the precipitate in a 96-well microplate.
Each 100 μl was dispensed. 50 μl of the same medium every 3 days
Additional cell proliferation was confirmed.

(5) Screening of antibody-producing hybridoma: A liquid from the well in which the hybridoma has grown is collected, added to another well to which the Ari-BSA complex has been bound, and the MAb-producing hybridoma against aristolochic acid is detected by direct ELISA. Screened. That is, 0.5 μg / 100 μl / well of Ari-BSA complex was dispensed into a 96-well microplate and incubated at 37 ° C. for 1 hour to be adsorbed to the well. 100 μl of the culture supernatant was dispensed into each well to perform an antigen-antibody reaction. The plate was washed three times with a phosphate buffered saline solution containing 0.05% Tween 20 (T-PBS). Peroxidase-labeled goat anti-mouse IgG antibody (secondary antibody) 1000-fold diluted solution was added at 100 μl per well, and 1 hour later, T-PB
Washed with S. 0.003% hydrogen peroxide, ABTS
[2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] 0.3 mg / ml citrate buffer was added to develop color. After 20 minutes, the absorbance was measured at a wavelength of 405 nm using a plate reader. The cells of the colored wells were collected.

(6) Cloning of anti-Ari-MAb-producing hybridoma: Antibody-producing hybridoma was subjected to limiting dilution and dispensed into wells. Hybridomas having antibody-producing ability and proliferating were similarly cloned three times to obtain a desired clone, that is, a MAb against aristolochic acid.
Was obtained.

(7) Preparation of anti-Ari-MAb: The above antibody-producing hybridoma 1A8 was added to a serum-free medium (100 μg).
/ Ml insulin, 35 μg / ml transferrin,
20 μM ethanolamine, 25 nM selenium added
RDF medium) at 37 ° C. in a carbon dioxide incubator. The supernatant was adjusted to pH 7 with Tris buffer and purified using a protein G affinity column. 10 mM column
After washing with phosphate buffer, the adsorbed antibody is
Elution with citrate buffer. The obtained antibody solution is PBS
Against aristolochic acid monoclonal antibody (MAb-1A8) purified by freeze-drying
I got

(8) Immunoglobulin class and molecular weight of MAb: The immunoglobulin class of purified MAb-1A8 was examined by the Ouchterlony method.
It belongs to the subclass b and has a κ light chain.

Further, the purified MAb-1A8 was mixed with sinapinic acid, and the purity was confirmed and the molecular weight was examined with Maldeit Fumath (manufactured by JEOL Ltd.).
It was about 147,100. FIG. 3 shows the spectrum of Maldeit Fuma.

[0027]Example 2: Quantitative test by competitive ELISA
Trial  The monoclonal antibody (MAb-1A) prepared in Example 1
To check the performance of 8), competitive ELISA was performed as follows.
Quantitative test was performed by the method. Ari-BSA complex solution
(5 μg / ml) was added to each well at 100 μl
The reaction was allowed to take place for a while. To remove non-specific binding.
Add 300 μl of PBS containing Kinmilk and react for 1 hour
And blocked. 50 μl of various concentrations of AriI
Or 10% 10 mM sodium bicarbonate dissolved in AriII
Liquid and methanol (9: 1).
Lonal antibody (MAb-1A8) solution (2 μg / ml)
50 μl was added and incubated for 1 hour. TP
Washed 3 times with BS and diluted 1000 times
100 μl of ze-labeled anti-mouse antibody was added and reacted for 1 hour.
After 1 hour, the plate was washed with T-PBS. 0.003% peroxide
Hydrogen, citrate buffer containing ABTS 0.3 mg / ml
The color was developed by addition. After 15 minutes, the color development was measured at 405 nm.
And check from the absorbance of AriI or AriII at each concentration.
A dose line was created. FIG. 4 shows the results. As shown in FIG.
As such, the concentration of AriI or AriII is 10^-2
-10¹Absorbance and good phase in the concentration range of μg / ml
This monoclonal antibody is associated with aristolochic acid
Excellent Quantitative Measures for I and Aristolochic Acid II
It is understood to provide.

[0028]Example 3: Cross-reactivity test  The monoclonal antibody (MAb-1A) prepared in Example 1
To confirm the specificity of 8), aristolochic acid (A
riI and AriII) and various related compounds
Cross-reactivity was determined. The method of the cross-reactivity test is roughly as follows.
It is as follows: each compound (1 mg) is accurately weighed and 1
Mixture of 0 mM sodium bicarbonate solution and methanol
(9: 1) (pH 8.3) (1 ml) for 10 minutes
Sonicated. Dissolve the supernatant in 10 mM sodium bicarbonate
Diluted with a mixed solution of liquid and methanol (9: 1)
A liquid was prepared. AriI, a test solution and standard
The solution was dispensed at 50 μl / well into an immunoplate and
Incubated for hours. After blocking the plate,
50 μl of the primary antibody solution [MAb-1A8 (0.1 μg /
ml)] and incubated for 1 hour. Next,
After adding the secondary antibody solution and incubating for 1 hour,
The liquid was added and the coloring intensity was measured. The cross-reactive CR is
CR (%) = [(AriI concentration that satisfies A / Ao = 0.5)
Degree) / (concentration of test solution where A / Ao = 0.5)] × 10
0 where A is the absorbance of the sample and Ao is 10 mM carbonated water.
Absorption of mixed solution of sodium hydrogen solution and methanol (9: 1)
Indicates degree. Table 1 shows the results.

[0029]

[Table 1]

[0030]

As is apparent from the above description, the present invention provides a monoclonal antibody having a very high specificity to aristolochic acid (aristrochiic acid I and / or aristolochic acid II) contained in a plant of the family Echinacea. provide. If the anti-aristrochiic acid monoclonal antibody of the present invention is applied to an ordinary immunoassay such as ELISA, it is possible to analyze aristolochic acid with simple, high sensitivity and excellent reproducibility without the need for pretreatment. Quantification of aristolochic acid contained in natural medicines, plants, their various organs, etc., can contribute to the evaluation of their pharmacological quality and elucidation of the pharmacokinetics and metabolic mechanism of physiological actions involving aristolochic acid. it can.

[Brief description of the drawings]

FIG. 1: Aristolochic acid-BSA used to generate anti-aristrochiaic acid monoclonal antibodies of the invention
(Or HSA) schematically illustrates the reaction to form a complex.

FIG. 2 is an example of a spectrum of Malditohumas measured to confirm the formation of an aristolochic acid-BSA complex.

FIG. 3 shows an example of a spectrum of Malditohumas measured for confirming the purity of the monoclonal antibody of the present invention and examining the molecular weight.

FIG. 4 is an ELISA using the monoclonal antibody of the present invention.
1 is a graph showing the relationship between aristolochic acid concentration and absorbance in Method A.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/577 C12N 5/00 BF Term (Reference) 4B024 AA11 BA53 GA03 HA15 4B064 AG27 CA10 CA20 CC24 DA13 4B065 AA92X AB05 AC14 BA08 CA25 CA46 4H045 AA11 AA20 AA30 BA50 CA40 DA76 DA86 EA50 FA72 GA10 GA26 HA07

Claims (5)

[Claims] 1. An anti-aristrochiic acid monoclonal antibody, which recognizes aristolochic acid I and / or aristolochic acid II. 2. The anti-alistrochiaic acid monoclonal according to claim 1, which belongs to the IgG2 subclass and has a κ light chain. 3. A method for preparing the anti-aristrokiaic acid monoclonal antibody according to claim 1 or 2, wherein aristolochic acid I and / or aristolochic acid are present in the presence of N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide. Antibody-producing cells and myeloma cells of an animal immunized with a complex of aristolochic acid I and / or aristolochic acid II formed by reacting acid II with bovine or human serum albumin and the serum albumin; The hybridoma obtained by the fusion is selectively cultured,
Screening and cloning a hybridoma producing a monoclonal antibody against aristolochic acid I and / or II. 4. The method according to claim 3, wherein mouse spleen cells are used as the antibody-producing cells and mouse myeloma cells are used as the myeloma cells. 5. A hybridoma that produces the anti-aristrochiaic acid monoclonal antibody according to claim 1 or 2.