JPS63219394A - Antiaflatoxin monoclonal antibody and hybridoma producing said antibody - Google Patents

Abstract

PURPOSE:To obtain a hybridoma by cell fusion of lymphocyte of a mouse immobilized with an antigen containing aflatoxin B1 as a hapten with a myeloma of a mouse and produce IgG type titled antibody having high specificity and being practically useful from the resultant hybridoma. CONSTITUTION:Aflatoxin B1 is combined with a protein to afford an immune antigen and a mouse is previously immobilized using the immune antigen. A lymphocyte of the mouse is fused with a myeloma of a mouse using polyethylene glycol to provide a hybridoma, which is then subjected to cloning to give a monoclone, which is cultivated in vivo or in vitro to afford the aimed antiaflatoxin monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-aflatoxin monoclonal antibody and a hybridoma producing the same.

More specifically, the present invention relates to type IIG anti-aflatoxin monoclonal antibodies and hybridomas that produce the same.

[Prior art] Aflatoxins are produced by fungi of the genus Spergillus, and among them, Spergillus
s flavus and ^spergillus pa
It is a toxic secondary metabolite mainly produced by P. rasiticus.

Aflatoxin B+ (hereinafter sometimes referred to as AFB I) is one of the most potent carcinogenic substances among aflatoxins, and is considered to be a substance that induces liver muscle damage when orally administered in a certain amount or more.

Food contamination by aflatoxins has been seen in 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 aflatoxins 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 an antibody against a low-molecular compound such as aflatoxin B1, the low-molecular compound itself is not immunogenic, so it is usually combined with a polymeric carrier such as a protein and used as a hapten antigen to immunize animals. This is the method. However, when there are many related compounds such as aflatoxins.

It is difficult to produce antibodies that are specific for only one type of compound, and the compound usually cross-reacts with many other compounds in the group to which it belongs.

Antibodies against aflatoxin B1 have traditionally been produced by immunizing rabbits with a hapten antigen in which aflatoxin B is bound to bovine serum albumin (BSA), but the specificity of the antibodies obtained is not sufficient, and aflatoxin They generally showed cross-reactivity; for example, J, J,
I'estka, P., Gaur, F.

S, Chu, ^ppl, Environ, Micro
biof, 40.1027 (1980)].

Also, J. D. Groopman et al. [Proc. N.
atl, ^cad, sci.

USA, Male, 7728 (1984) Reference monoclonal antibodies against core flatoxin B also have cross-reactivity with other aflatoxins, and the type of monoclonal antibodies is 1gN type, 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. The creation of specific antibodies has been eagerly awaited ever since.

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel IgG type aflatoxin monoclonal antibody.

Another object of the present invention is to provide an IgG type aflatoxin monoclonal antibody that is highly specific for aflatoxins.

Further objects and advantages of the present invention will become apparent from the description below.

[Means and effects for solving the problem] The present inventors immunize with a hapten antigen, select a clone that produces an antibody that specifically acts on the target antigen, and create a monoclone, thereby improving specificity. The inventors have discovered that it is possible to produce antibodies with high levels of antibodies, and have completed the present invention based on these findings.

In addition, in the conventional method of producing a new hapten antigen each time an animal is immunized for the purpose of producing antiserum to -m,
Depending on how the hapten antigen is made, individual differences between animals, and immunization methods, antibodies with different titers, specificities, and antibody subclasses can be obtained each time. Therefore, when these antibodies are applied to measurement reagents, there may be subtle effects on measurement results. However, if antibodies are produced using this antibody-producing hybrid, there is no need to produce antigens, and antibodies of stable quality can always be obtained regardless of individual differences between animals. This is another advantage of the invention.

According to the present invention, the above objects and advantages of the present invention are as follows.
This is achieved using 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+ as a hapten.

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. Hybridoma selection and monocloning Aflatoxin B1 cannot be used alone as an antigen.
1 is combined with a protein and used as an immunogen. 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. The binding of aflatoxin B and protein itself can be carried out using known methods, such as the above-mentioned J, J, Pe5tk.
Method a can be effectively adopted.

Examples of protein binding methods include aflatoxin B,
was converted into oxime to give aflatoxin B1-0-carboxymethyl oxime, which was then converted to 1'-ethyl-3,3
- Bovine serum albumin (BSA) and ovalbumin (OVA) are
).

Immunized animals are determined by the tumor cell line used for cell fusion. Generally, rats and mice are often used. Among mouse types, the BALB/C strain, which is an established tumor cell line that does not produce immunoglobulin, is often used.

Hapten antigens are used by dissolving them in isotonic 11i solution or physiological saline, but at a time from 10 to 3 times per mouse.
It is preferable to administer 00ug. Immunization is carried out in several doses, but the first immunization is often administered with an adjuvant. Examples of adjuvants include alum, killed tuberculosis bacteria,
Nucleic acids, etc. are used. Immunization is carried out at intervals of 2 to 4 weeks, and the final immunization is administered intraperitoneally or intravenously without using an adjuvant, dissolved in physiological saline or the like.

Two to four days after the final immunization, the lymph nodes or pancreas are removed, and the resulting lymphocytes are subjected to cell fusion. On the other hand, the initial tumor cell lines used for cell fusion were MPC-11,
P3-X63-Ag8 etc., but since these produce their own immunoglobulin, recently P3-X63-Ag8
-Ul, P3-NS-1, SP210-Ag14 (SP
210), P3-X63-Ag8・653 (653)
etc. are commonly used.

During cell fusion, 5 to 20 times more Veri lymphocytes are used than tumor cells. DMEM medium, McCoy medium, RPMI
, 1640 medium, or isotonic suspension solution, etc., are mixed and centrifuged to form a pellet. After loosening the pellet, HVJ (Sendai virus) or
Cells are fused using polyethylene glycol (PEG), but in general, PEG has an average molecular weight of 1.0, which is convenient to handle.
Use 0.5 to 2 0.00 to 8,000 40 to 60% solutions. Colchicine, dimethyl sulfoxide, poly-L-arginine, etc. may be added to promote fusion, but this is not essential.

After carrying out the fusion reaction with the PEG solution for about 1 to 10 minutes, add DMEM medium, RPMr1640 medium, etc. for 10 to 50 m
/ Gradually add to stop the fusion reaction.

After stopping, centrifuge and remove the supernatant. Fetal calf serum (FCS>
DMEM medium containing 5-20% or RPM111640
Add medium and dispense 1-1 times a day to a 24-sized culture plate so that the lymphocytes are 1 x 10 to 5 x 10" per well. Alternatively, add 1 to 1 aliquot of lymphocytes per 16 wells to a 96-well culture plate.
Pour 0,1 w+1 aliquots so that ~2×10− pieces are obtained. It is preferable to add feeder cells to both. Rat thymocytes, tile cells, mouse thymocytes, tile cells, etc. are used as feeder cells, and the concentration is 0.
5 to 2 X I O”/yal.
Next, Hyboxanthin 1X10'M, Aminopterin 4X
RPMI 1640 medium (or DMEM medium) containing H
The method for replacing HAT medium is generally to dispense an equal volume to the culture plate on the next day as that dispensed at the time of fusion, and then replace half of the volume with HAT medium the next day. Thereafter, replace half the volume with HAT medium every 2 to 3 days. 10 after fusion
On day 14, half of the medium is replaced with H A 'I' medium, i.e., HT medium, which does not contain aminopterin, and from 1 to 3 days later, half of the medium is replaced with a normal medium that does not contain HAT every 1 to 3 days. .

Cell culture supernatants from wells where hybridomas are actively proliferating are analyzed using various analysis methods such as RIA, plaque method, agglutination reaction, and EL.
Select the desired antibody-producing hybridoma using ISA, etc. Once a hybridoma is obtained, cloning is performed.The cloning method is FACS (Fluor
essent Ativated Ce1l 5or
Cloning can be carried out at a cell concentration such that a colony is formed from a single hybridoma. In the limiting dilution method, the number of cells per well of a 96-well plate is 0.6 or less. No matter which method is used, cloning is repeated twice to obtain a single clone. Once a clone is established, antibodies can be produced by culturing in large quantities either in vitro or in vivQ. Antibodies produced in vitro are not contaminated 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 iTI in vitro. Which method to produce antibodies depends on the purpose. anti-aflatoxin B
, monoclonal antibodies can be applied to RIA, ELISA, etc. When used in ELISA, β-galactosidase, alkaline phosphatase, peroxidase, etc. can be used as standard enzymes.

Examples will be described in more detail below, but the scope of the invention is not limited by the following examples.

[Example] Example 1 Preparation of hybridoma and antibody producing anti-aflatoxin B1 monoclonal antibody: 1) Preparation of AFB, -0-hydroxymethyl oxime Add 10 mg of AFB+ to 5 to 7 III l of methanol and heat (60°C). ), 100 mg of carboxymethoxylamine hydrochloride was added thereto, and the mixture was diluted with 5N-NaOH.
It was set as H7.0. The mixture was refluxed at 60°C for 2 hours to form an oxime. With this operation, AFB.

is AFBI-0-carboxymethyloxime (AFB,
-oxime). After removing methanol under reduced pressure,
The residue was dissolved in 5 liters of water and adjusted to pH 9 with 5 N NaOH.
,5,2-! of unreacted A F twice with ethyl acetate
B+ was extracted. Add 5N-HCI to the aqueous layer to adjust the pH.
is set to 2.0, only AFB+-oxime is precipitated, centrifuged, and dried to obtain AFBI-oxime.

2) Preparation of immunization antigen and antigen for analysis Bovine serum albumin (BSA) (long) at 0°05
M phosphoric acid &! It was dissolved in 4 companies' solution (pH 7,0), and 50+*g of 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDPC) was added. Add AFBI to this solution.
A solution of 1 mg of -oxime dissolved in 0.1 - methanol was added dropwise while stirring at room temperature. Furthermore, after 6 hours and 12 hours, 50g of EDPC was added, and after stirring at room temperature for 24 hours, the reaction solution was dialyzed against physiological saline, and the non-dialyzed fraction was washed at 200ml.

p, m: Centrifugation was performed for 10 minutes to precipitate some insoluble matter, and the supernatant was used as a physiological saline solution of AFBI-oxime-BSA.

In addition, using ovalbumin (OVA) instead of BSA, A F B +-oxime-
0VA was created.

3) Mix 111 g of physiological saline in which Immune AFBI-oxime-BSAlmg was dissolved and 1 ml of 70 India complete adjuvant to make an emulsion, and add 20 pi of the emulsion to BAL.
It was administered intradermally to the back of B/c mice (female, 4 weeks old). After 10 and 20 days, booster immunization was performed by intraperitoneal administration or tail vein injection of 20 ui' of AFB+-oxime-BSA in physiological saline.

4) Cell fusion Three days after the final immunization, the lungs of the mice were removed and 10ml
The tile lymphocytes were loosened in a plastic Petri dish containing MEM medium of No. 1. The tile lymphocytes were centrifuged (1000
Rotate for 10 minutes) and wash three times with MEM medium.

1.8 x 10” lymphocytes and 8-azaguanine resistant myeloma SP210-Ag14 (SF3) or X63
- Mix 3.6532 x 10' Ag, 1000 rotations,
It was centrifuged for 10 minutes to form a pellet. Aspirate the MEM medium from the top and loosen the pellet. 50%)) E G
400 o1 mei' was added over 11 minutes, and reacted for 1 minute at 37° C. while stirring with the pipette used. followed by 1
1 mf of mN DMEM medium was added over 1 minute at 37°C. After performing the same operation once more, DMEM medium 7va1 warmed to 37°C was added for 2 to 3 minutes. Immediately centrifuge at 800 rpm for 6 minutes at room temperature, remove the supernatant, and add 20% fetal bovine serum (Fe2) that had been warmed to 37°C.
) -DMEM medium 30- was added to suspend the pellets. Furthermore, after adding 20% FC3-DMEM medium of 3C) al and suspending well, dispense this suspension into 7 96-well culture plates at 0 and 1 wlI per well, and add CO2.
The cells were cultured in a T incubator. Hereinafter, the day when cell fusion was performed will be described as day 0.

5) On the first day of HAT selection, HAT medium (hypoxanthine I x 10-'M
, aminopterin 4X10-'M, thymidine 1.6X
20% FC3-DMEM medium containing 10-'M)
Added 0.11 per hole. On days 2.3.5.8 and 11, half of the medium was aspirated and 0.1μ of HA'r medium was removed.
added. Thereafter, the HT medium (HAT medium without aminopterin) was replaced every 3.4 days using the same procedure. Hybridomas have proliferated almost like gold mines.

6) Selection of Biplodomas After fusion, culture supernatants were collected from each well every 3.4 days from 2 to 3 weeks after fusion, and analyzed by ELISA.

First, AFB, -oxime-0V was added to the ELISA plate.
75 μl of A was dispensed and incubated at 4° C. overnight to immobilize the antigen on the plate. PBS containing 0.05% Tween20
After washing three times with Ovalbumin 0VA (500 μg/1
00μ! ) was dispensed and left to stand at room temperature for 1 hour. After washing three times with street solution, 5Ou of each of the above cell culture supernatants was added.
N was dispensed, and the mixture was left standing at room temperature for 1 hour. 20 as a negative control
%F CS-I) MEM medium 100.1 was dispensed.

Furthermore, ditto M! After washing 4 times with buffer solution, add 50ul of anti-mouse immunoglobulin antibody-alkaline phosphatase complex solution.
Dispense onto a plate and leave at room temperature for 1 hour. Same as above [After washing 4 times with 8 liquids, disodium P-nitrophenyl phosphate.
6 HyO (1 mg/ml) solution was dispensed in 100 ui' portions, and after reacting at room temperature for 1 hour, O, D, and 405 nm were measured to quantify alkaline phosphatase activity.

In addition, in antigens prepared using carbodiimide, part of the carbodiimide undergoes intramolecular rearrangement and remains in the protein as N-substituted urea. O
VA was immobilized on the plate - (ELISA was performed and AF
B, -positive for oxime OVA and EDPC-
Biploidomas growing in the culture supernatant that were negative for OVA were treated with anti-A F B 1 A b prod.
This hybridoma was selected as a nucleating hybridoma.

Anti-AFB 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, the ELIS described in Example 1.5
In the experimental system A, the activity against aflatoxin-H3-OA in each well was measured in the presence of 0.5 ug of 77 aflatoxin, and hybridomas 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 1 ml culture scale Once it was determined which well was producing anti-AFB and antibodies, it was transplanted to a 24-well culture plate and cultured on a 1 ml scale. At this time, 1 in each hole of BALB/c.
Add ˜2×10′ thymocytes. For this purpose, remove the thymus from a mouse of 4.5 years of age, wash it at least three times, and then feed it with one thymus. Then, transfer the cell suspension in the antibody-producing wells of the 96-well culture plate to a 24-well culture plate, resuspend it, and return 250 μl of it to the original wells of the 96-well culture plate. This can lead to replication and prevent loss of new cell lines.

2. After 3 days, add 20% FCS-D to 24-well culture plate I.
Add 0.5s1 of MEM medium. (Feeder cells are not needed here) After another 2 days, remove the supernatant and add fresh medium. Once the cells have spread over the entire surface, test again for antibody activity.

If antibodies continue to be produced, cloning will be performed immediately.

If there are not that many holes producing antibodies, you can clone directly from the stage of culturing in a 96-well culture plate, but if the antibody is still being produced even after transplanting to a 24-well culture plate. By cloning from existing strains, you can avoid the waste of cloning more unstable strains.

8) Monocloning cloning medium was 20% FC8-DMEM containing 7 ml of 107 BAL B/c mouse leg cells.
It is a medium. If cloning is performed directly from a 96-well culture plate, use HT medium.

Anti-A F B + antibody producing hybridomas were counted and diluted to contain 10 cells in cloning medium ld. Dispense 100 μl of this suspension into 606 holes in a 96-well culture plate. On day 5, 100 ul 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, and five anti-aflatoxin B1 antibody-producing hybridoma clones (hereinafter referred to as AF-1, AF-2, AF-3, AP-4
and AF-5 strain) were obtained.

9) Production of monoclonal antibodies Monoclonal antibodies are produced at 10-501J9/
Kasu 1 is secreted.

After propagating the hybridomas, all hybridomas were cultured until just before death, and the culture supernatant was collected.

In addition, 2XIO' cells of each cultured cell line of 5 strains of AF-1 to AF-5 were suspended in DMEM medium 0.5+*N, and BAL B/c mice (female, 6 weeks old, aged 3 0.5 vsl of pristane was administered intraperitoneally 10 days prior), and the ascites was collected.

10) Determination of the class of monoclonal antibodies The class of immunoglobulin produced by each hybridoma clone is determined using antisera specific for each class (anti-1gG1, IgG1,
2a, IgG2b, IgM, IgA) and enzyme immunoassay.

As a result, all the monoclonal antibodies (AF-1 to AF-5) produced by all five strains of AF-1 to AF-5 mentioned above were all Ig.
It was found that it belongs to G+. In addition, the short peptide chains of the monoclonal antibodies produced by each clone are AF-1~
4 was the λ type, and AF-5 was the 2 type.

**Anti-aflatoxin B, monoclonal antibody AF-1
~5, analogous compounds of AFB, AF B2, AFBza, AFG+, AFG, AF
M,, AFPI, AFQ, and aflatoxycol■
, II (Coil, ■) was examined using ELrSA.

The measurement operation was carried out in accordance with the method described in Example 1-6) for selection of hybridomas.

However, instead of cell culture supernatant, multi-stage diluted AF
Each monoclonal antibody was used simultaneously with 50 uf of a 10% methanol PBS-Tween solution of B or an aflatoxin-like compound.

As an example, the results for AF-1 and AF-3 are shown in FIGS. 1 and 2, respectively. Further, Table 1 shows the results summarized for all AP-1 to AP-5.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the cross-reactivity of the monoclonal antibody (AF-1) of the present invention. Figure 2 shows another monoclonal antibody (AF-3) of the present invention.
FIG. 2 is a diagram showing the cross-reactivity of Others 1 person Figure 1 Og 0.5pg 5pg 50pg 5
00pg 5ng Aflatoxin 11 main body (/Gold Prayer) Fig. 2 Aflatoxin roast failure (/1it-, 斗q) Procedural amendment June 3, 1985 Director General of the Patent Office Kunio Ogawa 1, Indication of the case Showa 1962 Patent Application No. 52182 2, Name of the invention 3, Relationship with the case of the person making the amendment Patent applicant name (020) Ube Industries Co., Ltd. 4, Agent 107 5, Date of amendment order (voluntary) 6. Subject of amendment “Acceptance pressure regarding original deposit” 7. Contents of amendment

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. 2. The monoclonal antibody according to claim 1, which has a relative reactivity with aflatoxin B_1 and its analogues as shown in Table 1. 3. 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, and producing an IgG-type anti-aflatoxin monoclonal antibody.