JPH0438400B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to anti-aflatoxin monoclonal antibodies. More specifically, it relates to an IgG type anti-aflatoxin monoclonal antibody. [Prior art] Aflatoxins are produced by the fungus genus Aspergillus, and among them, Aspergillus flavus
It is a toxic secondary metabolite mainly produced by Aspergillus parasiticus and Aspergillus parasiticus. Aflatoxin B ₁ (hereinafter sometimes referred to as AFB ₁ ) is one of the most potent carcinogenic substances among aflatoxins, and is thought to be a substance that induces liver cancer when administered orally in excess of a certain amount. Examples of food contamination caused by aflatoxin include the yellowing rice incident that occurred in Japan in 1953 and the turkey incident that occurred in the United Kingdom in 1960, and recently there have been newspaper reports of food shipments being stopped due to aflatoxin contamination. As described above, aflatoxin is one of the most toxic mycotoxins, and contamination of grains, feed, meat, etc. is currently a problem. Thin layer chromatography, high performance liquid chromatography, and the like are used as methods for detecting aflatoxin from grains or their processed products, and recently, immunoassay methods have also been attempted. Now, it goes without saying that highly specific antibodies are required to specifically measure aflatoxin by immunoassay. When producing antibodies against low-molecular-weight compounds such as aflatoxin B ₁ , the low-molecular-weight compounds themselves are not immunogenic, so it is best to combine them with a polymeric carrier such as a protein and use them as hapten antigens to immunize animals with. This is the normal method. However, when there are many related compounds such as aflatoxins, it is difficult to create an antibody that is specific only for one of them, and it is usually difficult to create an antibody that is specific for only one type of compound. Cross-reactions are common. Antibodies against aflatoxin B ₁ have traditionally been produced by immunizing rabbits with a hapten antigen in which aflatoxin B ₁ is bound to bovine serum albumin (BSA), but the specificity of the antibodies obtained is not sufficient, and aflatoxin It showed cross-reactivity across all classes [e.g. JJPestka, PK
Gaur, FSChu, Appl.Environ.Microbiol., 40 ,
1027 (1980), see]. Also, JDGroopman et al. [Proc. Natl.
Acad.Sci.USA, 81 , 7728 (1984)] Monoclonal antibodies against aflatoxin _B1 also have cross-reactivity with other aflatoxins, and the type of monoclonal antibodies is IgM, making them difficult to use practically. . Despite the efforts of many researchers, the production of highly specific antibodies against aflatoxin has not yet been achieved, and there is a need to develop a specific method for measuring aflatoxin in grains. Since then, the production of specific antibodies has been eagerly awaited. [Problems to be Solved by the Invention] An object of the present invention is to provide a novel IgM type aflatoxin monoclonal antibody. Another object of the present invention is to provide an IgM type aflatoxin monoclonal antibody that is highly specific for aflatoxins. Further objects and advantages of the invention will become apparent from the description below. [Means and Effects for Solving the Problems] The present inventor immunizes with a hapten antigen, selects a clone that produces an antibody that specifically acts on the target antigen, and makes it a monoclone, thereby improving specificity. The present invention was completed based on the findings of the present invention. In general, in the conventional method of producing a new hapten antigen each time an animal is immunized for the purpose of producing antiserum, the titer, titer, etc. Antibodies with different specificities and antibody subclasses are obtained, so when these antibodies are applied to measurement reagents, they have a subtle effect on the measurement results, but if antibodies are produced using this antibody-producing hybridoma, there is no need to make antigens. Moreover, antibodies of stable quality can always be obtained regardless of individual differences between animals. This is another advantage of the invention. The above objects and advantages of the present invention are that, according to the present invention, IgG is produced from a hybridoma formed by cell fusion of mouse myeloma and lymphocytes of a mouse previously immunized with an antigen containing aflatoxin B ₁ as a hapten. They are anti-aflatoxin monoclonal antibodies, and when the reactivity ratio with aflatoxin B ₁ is taken as 100%, the reactivity ratio with aflatoxin B ₂ is 1.4 to 2.3%, and the reactivity ratio with aflatoxin G ₁ is 1.4% to 2.3%. 1.2-3.1%,
The reactivity ratio with aflatoxin M ₁ is 1.7 to 4.5% and the reactivity ratio with aflatoxin B _2a is 0.8 to 4.5%.
This is achieved by a monoclonal antibody characterized by 1.3%. The first step in achieving the objectives of the present invention is to establish new monoclonal hybridomas that produce antibodies. The specific details of the method for establishing this hybridoma will be shown in Examples, but it simply consists of the following three steps. 1. Immunization 2. Cell fusion 3. Selection and monocloning of hybridomas Aflatoxin B ₁ alone cannot serve as an antigen, so aflatoxin B ₁ is combined with a protein and used as an immunizing antigen. There is no need to select a protein that is generally available, but bovine serum albumin (BSA), which is generally easily available, is advantageously used. For the binding of aflatoxin B ₁ to a protein itself, a known method, such as the method of JJPestka described above, can be effectively employed. As a protein binding method, for example, aflatoxin B ₁ is converted into oxime to form aflatoxin B ₁ -O
-carboxymethyl oxime, which is then combined with 1
- Ethyl-3,3-dimethylamino-propyl-carbodiimide is used to combine with bovine serum albumin (BSA) and ovalbumin (OVA). Immunized animals are determined by the tumor cell line used for cell fusion. Generally, rats and mice are often used. Tumor cell lines that do not produce immunoglobulins have been established among mouse types.
BALB/C strain is often used. Hapten antigens are used after being dissolved in an isotonic buffer solution or physiological saline, but only once per mouse.
Preferably, doses of 10 μg to 300 μg are administered. Immunization is carried out in several doses, but the first immunization is often administered with an adjuvant. As the adjuvant, alum, killed tuberculosis bacteria, nucleic acid, etc. are used. Immunization is carried out at intervals of 2 to 4 weeks, and the final immunization is administered intraperitoneally or intravenously after dissolving in physiological saline or the like without using an adjuvant. Two to four days after the final immunization, the lymph nodes or spleen are removed, and the obtained lymphocytes are subjected to cell fusion. On the other hand, tumor cell lines used for cell fusion were initially MPC-11, P3-X63-Ag8, etc., but since these cells produce their own immunoglobulin, recently P3-X63-Ag8-U1, P3 -NS-1,SP2/
O-Ag14 (SP2/0), P3-X63-Ag8・653
(653) etc. are commonly used. During cell fusion, 5 to 5 lymphocytes compared to tumor cells
Use 20 times more. DMEM medium, McCOy medium,
Tumor cells and lymphocytes washed with RPMI1640 medium or isotonic buffer are mixed and then centrifuged to form a pellet. After loosening the pellet, cells are fused with HVJ (Sendai virus) or polyethylene glycol (PEG), but in general, PEG has an average molecular weight of 1000 to 8000, which is 40 to 60.
% solution is used. colchicine, dimethyl sulfoxide, poly-L to promote fusion.
-Arginine etc. may be added, but it is not essential. After performing the fusion reaction with the PEG solution for about 1 to 10 minutes, add DMEM medium, RPMI1640 medium, etc. for 10 to 50 minutes.
ml slowly to stop the fusion reaction. After stopping, centrifuge and remove the supernatant. Fetal calf serum (FCS) from 5 to
Add DMEM medium or RPMI1640 medium containing 20%
Lymphocytes were placed 1x per well in a 24-well culture plate.
Pour 1 ml each so that 10 ⁵ to 5 x 10 ⁶ cells are obtained. Or, place 1-2 lymphocytes per well in a 96-well culture plate.
Pour 0.1 ml per minute to make ⁶ x 10 pieces. It is preferable to add feeder cells to both. As the feeder cells, rat thymocytes, splenocytes, mouse thymocytes, splenocytes, etc. are used, and the feeder cells are added at a concentration of 0.5 to 2×10 ⁶ /ml. Next, Hyboxanthin 1×10 ⁴ M, Aminopterin 4×
10 ^-7 M, thymidine 1.6 x 10 ^-5 M, RPMI1640 medium (or DMEM medium), ie, HAT medium. The method for replacing HAT medium is generally to add the same volume to the culture plate the next day as that dispensed at the time of fusion.
Next day, half of the volume is replaced with HAT medium. Thereafter, replace half the volume with HAT medium every 2 to 3 days. HAT with aminopterin removed 10-14 days after fusion
Replace half of the medium with HT medium, and then add 1 to 3
After a few days, half of the medium is replaced with a normal HAT-free medium every 1 to 3 days. The cell culture supernatant of the well where hybridomas are actively proliferating is analyzed using various methods such as RIA, plaque method, agglutination reaction, ELISA, etc. to select hybridomas producing the antibody of interest.
Once a hybridoma is obtained, cloning is performed.
The cloning method is FACS (Fluorescent
Activated Cell Sorter) or Soft
There are methods to pick up colonies using Agar, and the commonly used limiting dilution method. Cloning is performed at cell concentrations such that colonies are formed from a single hybridoma. In the limiting dilution method
Perform so that the number of cells per well of a 96-well plate is 0.6 or less. Regardless of the method used, cloning is performed twice to produce a single clone. Once a clone has been established, antibodies can be produced by culturing in large quantities either in vitro or in vivo. Antibodies produced in vitro are free from contamination with other antibodies, but have low antibody titers. Antibodies produced in vivo are slightly contaminated with antibodies from the host, but the antibody titer is much higher than that produced in vitro. Which method to use to produce antibodies depends on the purpose. Anti-aflatoxin B ₁ monoclonal antibody can be applied to RIA, ELISA, etc. When used in ELISA, α-galactosidase, alkaline phosphatase, peroxidase, etc. can be used as standard enzymes. Examples will be given below to explain in more detail,
The following examples do not limit the scope of the invention. [Example] Example 1 Preparation of hybridoma and antibody producing anti-aflatoxin B ₁ monoclonal antibody: 1 Preparation of AFB ₁ -3-O-carboxymethyl oxime 10 mg of AFB ₁ was added to 5 to 7 ml of methanol and heated ( 60℃), add 100mg of carboxymethoxylamine hydrochloride, and PH with 5N-NaOH.
It was set to 7.0. The mixture was refluxed at 60°C for 2 hours to form an oxime. With this operation, AFB ₁ becomes AFB ₁ −3−
derived from O-carboxymethyl oxime (AFB ₁ -oxime). After methanol was distilled off under reduced pressure, the residue was dissolved in 5 ml of water, adjusted to pH 9.5 with 5N-NaOH, and unreacted AFB ₁ was extracted twice with 2 ml of ethyl acetate. 5N-HC1 was added to the aqueous layer to adjust the pH to 2.0 to precipitate only AFB ₁ -oxime, which was centrifuged and dried to obtain AFB ₁ -oxime. 2 Preparation of immune antigen and antigen for analysis Bovine serum albumin (BSA) (10mg) at 0.05M
It was dissolved in 4 ml of phosphate buffer (PH7.0), and 50 mg of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDPC) was added. AFB ₁ in this solution
- A solution of 1 mg of oxime in 0.1 ml of methanol was added dropwise under stirring at room temperature. Furthermore, 6 hours later,
After 12 hours, 50 mg of EDPC was added, and after stirring at room temperature for 24 hours, the reaction solution was dialyzed against physiological saline.
The non-dialyzed fraction was centrifuged at 2000 rpm for 10 minutes to precipitate some insoluble matter, and the supernatant was purified by AFB ₁ -oxime.
BSA in physiological saline solution. Also, ovalbumin (OVA) instead of BSA,
AFB ₁ -oxime-OVA was produced using the same procedure as above. 3 Immunity Mix 1 mg of physiological saline in which 1 mg of AFB ₁ -oxime-BSA has been dissolved and 1 ml of Freund's complete adjuvant to make an emulsion, and add 20μ of the mixture.
It was administered intradermally to the back of BALB/c mice (female, 4 weeks old). After 10 and 20 days, booster immunization was performed by intraperitoneal administration or tail vein injection of 20μ of AFB ₁ -oxime-BSA in physiological saline. 4 Cell fusion Three days after the final immunization, the spleen of the mouse was removed,
Splenic lymphocytes were disaggregated in a plastic jar containing 10 ml of MEM medium. Splenic lymphocytes were repeatedly centrifuged (1000 rpm, 10 minutes) and washed three times with MEM medium. 1.8 x 10 ⁵ splenic lymphocytes and 8-azaguanine-resistant myeloma SP2/0-Ag14 (SP2),
Or X63−Ag8.6532×10 ⁷ pieces mixed, 1000 rotations,
The mixture was centrifuged for 10 minutes and pelleted. The supernatant MEM medium was removed by suction and the pellet was loosened. 50%
1 ml of PEG4000 was added over 1 minute, and the mixture was reacted at 37°C for 1 minute while stirring with the pipette used. Subsequently, 1 ml of DMEM medium was added over 1 minute at 37°C. After performing the same operation again,
7 ml of DMEM medium warmed to 37°C was added over 2-3 minutes. Immediately centrifuge at 800 rpm for 6 minutes at room temperature and remove the supernatant. 30 ml of 20% fetal calf serum (FCS)-DMEM medium warmed to 37°C was added to suspend the pellet. Additionally 30ml of 20% FCS−
Add DMEM medium and suspend well, then add 0.1ml of this suspension per well to seven 96-well culture plates.
It was aliquoted and cultured in a CO ₂ incubator. Hereinafter, the day when cell fusion was performed will be described as day 0. 5 HAT selection On the first day, HAT medium (hypoxanthine 1×
10 ^-4 M, aminopterin 4x10 ^-7 M, thymidine
20% FCS-DMEM medium containing 1.6×10 ^-5 M)
Added 0.1 ml per hole. 2nd, 3rd, 5th, 8th and 11th
On day 1, aspirate half of the medium and 0.1ml of HAT medium.
added. Thereafter, the HT medium (HAT medium without aminopterin) was replaced every 3 or 4 days using the same procedure. Hybridomas have grown in almost all holes. 6 Selection of hybridomas From 2 weeks to 3 weeks after fusion,
Collect culture supernatant from each well every 3 or 4 days and perform ELISA
It was analyzed in First, AFB ₁ -oxime- was added to the ELISA plate.
75μ of OVA was dispensed and allowed to stand overnight at 4°C to immobilize the antigen on the plate. Contains 0.05% Tween20
After washing three times with PBS, use ovalbumin OVA to avoid nonspecific adsorption of proteins in the culture supernatant.
(500μg/100μ) was dispensed and allowed to stand at room temperature for 1 hour. Next, after washing three times with the same buffer solution, 50μ of each of the above cell culture supernatants was dispensed and allowed to stand at room temperature for 1 hour. 100μ of 20% FCS-DMEM medium as negative control
was dispensed. After further washing four times with the same buffer solution, 50μ of the anti-mouse immunoglobulin antibody-alkaline phosphatase complex solution was dispensed onto the plate and left at room temperature for 1 hour. After washing four times with the same buffer solution, P-nitrophenyl phosphate disodium 6H ₂ O (1
The alkaline phosphatase activity was determined by dispensing 100μ of the solution (mg/ml) and reacting for 1 hour at room temperature, then measuring OD405nm. In addition, in antigens prepared using carbodiimide, part of the carbodiimide undergoes intramolecular rearrangement, resulting in N
-OVA treated with carbodiimide only (EDPC-OVA) as it remains in the protein as substituted urea
EDPC-OVA was immobilized on a plate and ELISA was performed to find hybridomas growing in the culture supernatant that were positive for AFB ₁ -oxime OVA and negative for EDPC-OVA. It was selected as an anti-AFB ₁ Ab producing hybridoma. Anti-AFB ₁ antibody production was observed in 72 out of 2096 wells. Furthermore, in order to identify hybridomas that truly produce anti-aflatoxin antibodies, we conducted an inhibition test using free aflatoxin. That is, in the ELISA experimental system described in Examples 1 and 5, aflatoxin-HS in each well in the presence of 0.5 μg of aflatoxin.
-The activity against OA was measured, and those whose activity was inhibited by aflatoxin were determined to be true anti-aflatoxin antibody-producing hybridomas. Anti-aflatoxin antibody-producing hybridomas were finally found in 35 wells. 7. Expansion of the 1 ml culture scale Once it was determined in which well the anti-AFB ₁ antibody was being produced, the cells were transplanted to a 24-well culture plate and cultured on a 1 ml scale. At this time, thymus cells of BALB/c mice were used as supporting cells. Dispense 0.5 ml of HT medium into a 24-well culture plate.
Add 1-2 x 10 ⁷ thymus cells to each well. For this purpose, the thymus is removed from a 4- or 5-week-old mouse, washed at least three times, and then suspended in 1 ml of 20% FCS-DMEM medium per thymus. Add 50-100μ of this suspension to each well.
Then, transfer the cell suspension in the antibody-producing wells in the 96-well culture plate to a 24-well culture plate. Resuspend this and return 250μ of it to the original well of the 96-well culture plate. This can lead to replication and prevent loss of new cell lines. After 2 to 3 days, add 20% FCS to a 24-well culture plate.
Add 0.5 ml of DMEM medium. (Feeder cells are not needed here) After another 2 days, remove the supernatant and add fresh medium. Once the cells have spread over most of the area, test again for antibody activity. If antibodies continue to be produced, cloning will be performed immediately. If there are not that many wells producing antibodies, cloning can be performed directly from the stage of culturing in a 96-well culture plate. But 24
By cloning those that still produce antibodies even after being transplanted into well culture plates,
This eliminates the waste of cloning more unstable strains. 8. Monocloning The cloning medium was a 20% FCS-DMEM medium containing 10 ⁷ BALB/c mouse thymocytes/ml. If cloning is performed directly from a 96-well culture plate, use HT medium. Anti-AFB ₁ antibody-producing hybridomas were counted and diluted so that 1 ml of cloning medium contained 10 cells. Dispense 100μ of this suspension into 60 wells in a 96-well culture plate. on the fifth day
100μ of medium was added. On day 14, activity was measured by ELISA and active clones were grown in 24-well culture plates. Furthermore, recloning was performed using the same method to obtain 5 anti-aflatoxin B ₁ antibody-producing hybridoma clones (hereinafter referred to as AF
-1, AF-2, AF-3, AF-4 and AF-5
(referred to as stocks). 9 Production of monoclonal antibodies Monoclonal antibodies are present in culture supernatants at
Secreted at 50μg/ml. After growing the hybridomas, almost all the hybridomas were cultured until just before death, and the culture supernatant was collected. In addition, 2 × 10 ⁶ cultured cells of each of the 5 strains of AF-1 to AF-5 were suspended in 0.5 ml of DMEM medium, and BALB/c mice (female, 6 weeks old, 3 to 10 days old) were prepared with pristane. 0.5 ml was administered intraperitoneally) and the ascites was collected. 10 Determining the class of monoclonal antibodies The class of immunoglobulin produced by each hybridoma clone is determined by antiserum specific to each class (anti-IgG1, IgG2a, IgG2b, IgM, IgA).
It was determined by the Ouchterlony method and enzyme-linked immunosorbent assay. As a result, monoclonal antibodies (AF-1 to AF-5) produced by all five strains of AF-1 to AF-5 mentioned above were found.
All of them were found to belong to IgG ₁ . Furthermore, the short peptide chains of the monoclonal antibodies produced by each clone were λ type for AF-1 to AF-4, and κ type for AF-5. Example 2 Monoclonal antibody AF-1, 2, 3, 4, 5
In order to investigate the specificity of anti-aflatoxin B ₁ monoclonal antibodies AF-1 to AF-5, similar compounds of AFB ₁ such as AFB ₂ , AFB _2a , AFG ₁ , AFG ₂ ,
ELISA for cross-reactivity with AFM ₁ , AFP ₁ , AFQ ₁ and aflatoxycol I, (ColI,)
It was considered. The measurement operation was carried out in accordance with the method described in Example 1 6) Selection of hybridomas. However, instead of cell culture supernatant, multi-level dilutions of AFB ₁ or aflatoxin-like compounds can be used.
Each monoclonal antibody was used simultaneously with 50μ of a 10% methanol PBS-Tween solution. As an example, the results for AF-1 and AF-3 are shown in FIGS. 1 and 2, respectively. Table 1 shows the results summarized for all of AF-1 to AF-5.
In Table 1 below, the monoclonal antibodies labeled AF-3 and AF-4 belong to the present invention, and the others are monoclonal antibodies for reference. Furthermore, the hybridoma producing monoclonal antibody AF-3 has been deposited with the FERM under the FERM accession number FERM BP-1609.

【table】

[Table] *** Minimum measurement sensitivity

[Brief explanation of drawings]

FIG. 1 is a diagram showing the cross-reactivity of another monoclonal antibody (AF-1) of the present invention. FIG. 2 is a diagram showing the cross-reactivity of the monoclonal antibody (AF-3) of the present invention.

Claims (1)

[Scope of Claims] 1. An IgG type anti-aflatoxin monoclonal antibody produced from a hybridoma formed by cell fusion of mouse myeloma and lymphocytes of a mouse previously immunized with an antigen containing aflatoxin B ₁ as a hapten. Therefore, when the reactivity ratio with aflatoxin B ₁ is taken as 100%, the reactivity ratio with aflatoxin B ₂ is 1.4 to 2.3%, and the reactivity ratio with aflatoxin G ₁ is 1.2 to 3.1%,
The reactivity ratio with aflatoxin M ₁ is 1.7 to 4.5% and the reactivity ratio with aflatoxin B _2a is 0.8 to 4.5%.
A monoclonal antibody characterized by 1.3%. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody is an antibody produced by a hybridoma deposited with FERM under the FERM accession number FERM BP-1609. .