JPS6192566A - Preparation of antibody-forming cells - Google Patents

Abstract

PURPOSE:The mixed cultivation of B lymphocytes and an antigen is carried out in vivo, the resultant antibody-producing cells are fused with tumor cells to form a hybridoma whereby cells producing a specific monoclonal antibody are prepared. CONSTITUTION:The spleen is removed from a mouce, ground to form a suspen sion, the suspension is filtered, centrifuged to recover B lumphocytes (A). The component A is mixed with an antigen such as nucleic acid or protein and the mixture is cultured in a medium in test tubes (such as RPM1-I640) in the presence of about 5% carbon dioxide at about 37 deg.C for 2-7 days. The resultant culture mixture is subjected to enzyme immunoassay to select the desired antibody-producing cells and the cells are fused with tumor cells.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention relates to a method for preparing antibody-producing cells, and more specifically, to a method for preparing antibody-producing cells, and more specifically to a method for antigen stimulation by mixing phosphorus/brachial cells and an antigen in a test tube. The present invention also relates to a method for preparing cells that produce antibodies specific to a particular antigen.

(Prior Art) Recently, cell engineering has been attracting attention due to dramatic advances in immunology, and cell fusion methods are particularly popular as described by Keller and Milstein et al. (Natur@).

256.495 (1975)), it has become possible to produce a variety of monoclonal antibodies. Since these monoclonal antibodies have significantly higher specificity for specific antigens than conventional antisera, they are widely used in medicine. In the field, it is used as a clinical diagnostic agent, therapeutic agent, etc. (CanC
@r R@11.14 p 4749 (1981):
Anal, Blochem, 111 e 1 (
1981): 1 Mechanism of biological defense 1 Chemistry special issue, 121
(1981)), and in the field of biochemistry, it has been applied to affinity chromatography as a means of substance purification, and it has also been used for structural analysis of cell surface structures, and its application fields will continue to expand in the future. It is expected that demand will increase.

Monoclonal antibodies are generally produced by the cell fusion method described above, in which antibody-producing cells are fused with tumor cells capable of self-propagation for a long period of time to create a cell fusion cell line that has antibody-producing ability and is capable of self-propagation for a long period of time. This is carried out by culturing this cell fusion strain. The antibody-producing cells used in this cell fusion method are generally prepared by sensitizing an experimental animal such as a mouse with an antigen, killing the animal, and aseptically removing the spleen. Japanese Unexamined Patent Publication No. 1983
-502090, 58-502132, 58-20
6532, 58-179486, 5B-19283
1, 58-216125, 5j-20228, 5
9-39832, 59-4432, etc.).

However, the above antibody-producing cells have various problems due to the use of animals. For example,
During animal husbandry, animals can be naturally infected with microorganisms and become sensitized to these microorganisms - not only are antigens that are too toxic or carcinogenic not yet available; It is not possible to use an antigen solution containing impurities that are highly toxic to the extent that they impair the normal functions of the antigen. Furthermore, a large amount of antigen is required for immunization, and the immunization period generally lasts two weeks or more. Also, animals may die from infection during booster immunization. Furthermore, in order to produce human-like monoclonal antibodies, humans must be immunized, but humans cannot be subjected to artificial immune manipulation, so human-type monoclonal antibodies cannot be produced arbitrarily.

In order to solve these problems that animal sensitization methods have, in vitro immunization methods [e.g. Molscolar Immunol
ogy*17, 635-639 (1980))
has been proposed, but the antigen used in this method is a molecule [less than 9000 proteins ω'(Omt*oc
laat Actlvating Fartor) and Pseudomonas aeruginosa (Ps@udo
mona aeruginoam) (hereinafter referred to as pyosho), and human antibody 1gG, which is a human-derived protein.
There is no description of a method for using the Fab portion of as an antigen. Also, J, C11nlc, Invest,
, 71 r 1032-1040 (1983);
J, Im+nono1.131, 1229-1
233 (1983): J. rmmunol., 1
32, 928-935 (1984): and J. Im.
munol,, 132tl192-1201 (198
4) describes the preparation of antibody-producing cells by in vitro immunization, but does not describe the creation of cell fusion lines using these antibody-producing cells.
゛(Problems to be Solved by the Invention) The present invention provides a cell fusion strain capable of producing monoclonal antibodies by immunizing Cylindritus cells with various antigens including human-derived antigens in vitro. The purpose of the present invention is to provide a method for preparing antibody-producing cells that can be used as materials for the production of antibodies.

(Means for Solving the Problems) The above object of the present invention is to mix B phosphorus/4' cells and an antigen in a test tube, culture this, and extract antibody-producing cells from the culture solution. This is achieved by a method for preparing antibody-producing cells, which is characterized by recovering the antibody-producing cells.

Note that the term "in vitro" is used herein to refer to cases where treatment is performed outside a living body using a test tube, Wellgrate, or the like.

Preparation of B lymphoid cells B as a material for antibody producing cells used in the present invention
Possible phospho/4 cells include pancreatic cells, lymph node cells, and phospho/4' cells isolated from peripheral blood, but pancreatic cells are usually used. For example, the lungs are removed from experimental animals such as makus, and the lungs are adjusted.

Antigens Any antigen may be used as long as it induces an immune response in living organisms, such as nucleic acids, proteins (including glycoproteins), polysaccharides, glycolipids, and other compounds, or compounds containing these. However, these may be toxic or carcinogenic. It is generally preferable for the above-mentioned compound to have a molecular weight of 1000 or more. In the present invention, the Fab moiety of Pseudomonas aeruginosa and Human IgG will be specifically explained.

Immunization treatment and culture The B lymphoid cells and antigen are mixed in a test tube for immunization treatment, and then cultured.

In this case, the antigen is dissolved or suspended in a medium, and then the antigen is dissolved or suspended in a medium.
? system cells (mix with M suspension).As the above-mentioned medium, those commonly used for culturing animal cells in vitro, such as RPMI-1640 medium, can be used.

Culture is generally carried out at 37℃ in a carbon dioxide gas incubator for 50 minutes.
% CO2. The culture period is 2 to 7 days. In addition, in order to increase the immune effect in this culture, T
Cell-derived B cell differentiation inducing factors, B cell growth factors, etc. can be added. As such an additive, for example, TCCM (Thymus Cs1l Cond1ti
on@d Medium).

Next, target antibody-producing cells are selected from this culture solution. This selection is usually performed using an enzyme immunoassay method, such as ELJS.
Method A By fusion with CImmunochemlstry e 8 +, fused cells capable of producing monoclonal antibodies can be prepared.

(Effects of the Invention) According to the method of this invention, the following effects can be obtained: (a) The amount of antigen required to immunize B cells is extremely small compared to methods using animals; For example, compared to when using a mouse, the user's feet will fall at a rate of about 1/10 to 1,000 times.

(b) The period of immunity is significantly shortened. Generally, in methods using animals, it takes more than two weeks, but in the method of the present invention, it takes four to five days.

C. It is relatively easy to operate because there is no need to raise animals.

2) It is possible to prepare antibody-producing cells against toxic and carcinogenic substances.

(e) It is possible to prepare human antibody-producing cells.

(Example) Next, the method of the present invention will be explained in more detail with reference to Examples.

Example 1゜(1) Preparation of antibody-producing cells Aseptically plated from normal Bulb/e mice aged about 10 years.
1% was extracted, loosened, and made into IIfl cell suspension (RP).
A Shimawas lung cell suspension (2xlO
'1 hard/++A') was prepared. Pseudomonas aeruginosa (type F3 according to Fisher's classification) was cultured, the cells were fixed with formalin, and then transferred to TBS (-) RTMI.
After washing with -1640, RPMI-1640 medium (10% fetal bovine serum (Fe2), 5x10M2-
Mercaptoethanol, 100 U/subpenicillin, and U
The cell longitudinal waves were adjusted to 1 x 10 cells/d. Next, the mouse cells mentioned above are reduced once a month! 100μ of turbidity and 10μ of Pseudomonas aeruginosa suspension
0 μl was mixed on a 96-well plate and cultured for 3 days at 37° C. and 5% CO2 in a carbon dioxide gas incubator.

(2) Assay using radiolabeled thymidine It is generally known that antigen-stimulated B cells undergo active cell proliferation and produce antibodies.

Therefore, whether or not cell proliferation is actively occurring can be determined by
By checking at the synthesis level, it is possible to assay whether or not antibody-producing cells have been produced.

DNA synthesis can be investigated using C-thymidine uptake into cells as an indicator.

After culturing as described above, 0.2 μCI of 140-thymidine per I+7 well was added to each well, and 140-thymidine was added to each well.
Culture was performed for 5 hours. After culturing, the culture solution in the well was transferred to glass filter paper, washed with 5% trichloroacetic acid, physiological saline, and ethanol, transferred to a vial, and a scintillator was added, followed by measurement using a liquid scintillation counter. . The results are shown in Table 1 below.

Margin M1 table below Note (1): RTMI-1640 medium was added to a sample of the antigen (Pseudomonas aeruginosa suspension 't'11.) and the same treatment as above was performed (2): As an antigen Pseudomonas aeruginosa concentration 5 x 10' pieces/go.

In the above table, it is clear that mouse lung cells (B linnos and bulbs) were activated (immunized) by the antigen, since 14 cm thymidine O uptake increased with the addition of the antigen.

Example 2゜(1) Preparation of antibody-producing sainagi 100 μl of Pseudomonas aeruginosa suspension (IX10'i hard/go) prepared in the same manner as described in Example 1 and Matus tiles 11
Cells (2 x 10'l solid/d) 100 fil,
! : All were mixed in a 96-quel grade fer and cultured for 5 days at 37'C and 5% CO2.

In addition, in parallel with this, TCCM was added to the cells so that the final concentration became a bar, and the cells were cultured in the same manner as above. here,
TCCM (Thymu@Cs1l Condltlo
nedM15 di um) was prepared by injecting 10'i'L of Ba1b/c mawas (10-14 days old) thymocytes into 1 cell.
RPMI-1640 medium (10% FC8, 5xlO
- 5M 2-mercaptoethanol, 2mM glutamine, 100 U/ral penicillin, and 041 ~ ~ streptomycin)). As a control, a section in which RTMI-1640 medium was added to a Pseudomonas aeruginosa suspension was similarly fed with N.

Next, mouse pancreatic cells were washed with RPMI-11-1640 medium, and the cell El concentration was adjusted to RPMI-1640 (10% FC).
3.

The cells were cultured for an additional 7 days at 2×10′ cells/d in 2-mercaptoethanol, penicillin, and streptomycin.

(2) Assay for specific antibodies If B#1ljlx is immunized with an antigen, specific antibodies against that antigen should be produced. Therefore, by examining the presence or absence of specific antibody production, it is possible to determine whether B cells are immunized. You can know whether or not.

In order to confirm whether or not the above-mentioned cultured cells were immunized, a specific antibody was detected by the method (ELISA method) described by the present inventors in Japanese Patent Application No. 59-133942. In each well of the plate immobilized with Pseudomonas aeruginosa,
) After injecting 50 μl of each culture solution,
After being left at room temperature for 2 hours, it was washed three times with PBS-Tw, , n. Then, as a secondary antibody, rabbit anti-mouse immunoglobulin 50 conjugated with horseradish bran oxidase was used.
After adding μ to each well, the wells were washed three times with PBS-Tw@en. To this, 0.1% was added as a substrate for enzyme activity measurement.
ABTS [2,
2/-Azinobis(3-ethylbenzthiazoline)-6
A solution of Fusulfone 01, 32.5 mM and ■2025 mM was prepared immediately before use, and 100 μl of this was injected into each well. After reacting at room temperature for 5 to 15 minutes, 2 mM sodium azide was added to each well. 100μ in well
! Add Titertic Multiscan 7
(7o-company) was used. D 405□. . Absorbing niobium, 1
I decided.

Note that the PBS-Tween is a cleaning liquid having the following composition.

Sodium chloride 8.00:! Potassium chloride 1.15# Sodium hydrogen phosphate (anhydrous) 0.20.9 Potassium dihydrogen phosphate (anhydrous) 0.20&Tve@n -20
0.51M or more dissolved in distilled water to make 11.

The results are shown in Table 2 below.

The above table shows the results of immunizing lung cells with Pseudomonas aeruginosa.
This shows that n+ Kura cells produced specific antibodies against Pseudomonas aeruginosa.

Example 3゜(1) Preparation of antibody-producing cells By the same method as described in Example 1, normal 9al
Lung cell suspension from b/c mice (total number 1.46X1
08 pieces; Yoyo 3.5 x 10' pieces/rag), and 1i8J solution of Pseudomonas aeruginosa F3 (IXIO8 pieces/d) t-, J
Jt,, these were mixed and RPMI-1640 (1
The cells were cultured in a carbon dioxide gas incubator (37°C, 5% CO2) for 4 days in 0% FC812 (containing mercaptoethanol, penicillin and streptomycin). After culturing, shift using the same method as in the actual θa example above. 64 x 10' live cells were collected (recovery rate 5
2%).

(2) Cell fusion The immunized lung cells and Maxmieloma cells obtained in (1) above P3-X63-Ag8.U, 1 [hereinafter P
3Ul (abbreviated as Flow Inc.)]
1640 several times, mix the two at a ratio of 10:1, centrifuge the mixture, remove the supernatant, and place 4 cells at the bottom of the centrifuge tube. Expand, 3
The mixture was left at 7°C for 1 minute, and then 1 d of a 40% solution of PBS (→ medium polyethylene glycol (PEG4000)) at 37°C was slowly added dropwise. After this was left at 37°C for 4 minutes, RPMI- 1640 (contains 10% FC8)
The PEG was gradually diluted with

Centrifuge and remove the supernatant, and finally p3u, concentration 1, o
x to511. EtPMT so that z, /nLl
After diluting with -1640 (containing 10% FC3), it was dispensed into a 96-well grate at a rate of 200 μl/well.

In this medium, 1.0 x 10' of Ba1b/C mouse thymocytes within 3 weeks of age were added as feeder cells.
[above fusion operation]. Half i (100μ
1) was replaced with HAT medium. Thereafter, the medium was replaced every other day.

In addition, the said HAT medium is RPMI-1640 medium containing 100 μM hypoxanthine, 0.1 μM aminopterin, 16 μM thymidine, 10% FC8, 5×10 M2
-Mercaptoethanol, 2mM glutamine, 100U
/a/penicillin, and 0.1! n9//nl strectomycin was added.

After culturing for 14 days, colonies were formed in 285 out of 384 wells. These 285 samples were screened for anti-cancer antibodies by the ELISA method described in the patent application specification.

Two colonies had developed antibodies against Pseudomonas aeruginosa F3. The subclass of this antibody was rgM, and its longitudinal chain was single chain (rgM/ni).

Example 4゜(1) Preparation of antibody-producing cells Il'4! Visceral cells (1,
08 pieces: final concentration 3 x 10' pieces/me) and the Fab portion of human antibody IgG [Cabell, 10 fill/r
ut) and RPMI-16404 in the presence of TCCM.
Immunization was performed by culturing for 4 days in 0d. After completion of the culture, 6.8 x 107il ISII living cells were collected.

(2) Cell fusion with the above immunized Pai Sei cells and mouse myeloma cells P
, Ul and 4@ cell fusion was performed in the same manner as in Example 3. 14 days after the cell fusion, human light chain [IMi! EL described in the above
When υ antibodies were screened by the ISA method, antibodies against human light chain were produced in 8 out of 480 wells. The subclass of this antibody is I
gM/. Furthermore, this antibody reacted only with human light chains (kappa chain and lambda chain) and did not react with the heavy chain of human IgG or the Fc portion of human IgG.

Example 5 Preparation of EBV EBV was treated with B95-8 cells using RPMI-1640 (20
After culturing for 11 days in a carbon dioxide gas incubator at 37°C in the presence of 5% Co□ in a culture solution (containing %FC8), the B95-8 cells were removed by centrifugation and the supernatant was obtained. Ta. The activity of EVB thus prepared was TD5o10/-.

Preparation of antibody-producing cells Lymphocytes were separated from peripheral blood collected from a normal person by Ficoll-Conray density centrifugation.
Ten lymphocytes were obtained. Next, this cell 2O-(2
×106 pieces/7 pieces and autoclave treatment (120℃, 2
0 min) with Pseudomonas aeruginosa F7 (5 x 107 cells/go) at RPM
Culture was carried out in a carbon dioxide gas incubator for 4 days in I-1640 (containing 10% FC3, penicillin, and stregtomycin) at 37° C. in the presence of 5% CO□ (in vitro immunological manipulation). Next, after collecting phosphorus/4' sphere cells by centrifugation, RPMI-1640 (20% FC8, 2mM
(poor glutamine, penicillin, streptomycin) to make I x 106 cells/-.

One transformant of the above antibody-producing cells (I x 10'/-) was dispensed into a 24-well plate, and 1-1 of the above EBV supernatant was added to the plate.
After adding 00′ μl, at 37°C in the presence of 54 Co□,
Transformation was performed by culturing in a carbon dioxide incubator.

Track antibody production over time from 2 weeks after the start of selective culture of antibody-producing cells (II
Cells were grown using ELISA method. This EL
The ISA method was carried out according to the specification of Japanese Patent Application No. 59-133942 using a plate on which Pseudomonas aeruginosa cells were immobilized. That is, 50 μl of the culture supernatant of the transformed cells was injected into each well of the above-prepared grate, and the well was left for 2 hours at a southern temperature, and then washed 13 times with PBS-Tween. Next, it was washed with secondary antibody. Next, 0.1M citrate buffer (pi-
14,2) with ABTSC2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid 12.5mM
A solution of H20□5rrLM and H20 step by step)
Then, OD 405 was measured using Titertic Multiscan ■ (Flow Co., Ltd.).

After taking out cells from the wells that showed positive results in the above procedure, cloning was performed using the soft agar method.

Then, perform the above ELIS again while performing the cloning operation.
A clone producing a specific antibody was obtained by carrying out Method A.

The clones obtained at this time produced antibodies that specifically reacted with F7 (see the ELISA results in Table 3).

Table 3 in the margin below Antibody-producing cells obtained by cloning are RPMI-
After culturing in 1640 (M containing 20% FC8) medium at 37°C in the presence of 50% CO2 in a carbon dioxide incubator, the culture supernatant was subjected to ammonium sulfate fractionation to obtain a monoclonal antibody against υF7. When this antibody was characterized, its subclass was IgM/.

Example 6 Lymphocytes were adjusted to 2 x 10 cells/- from a human intraperitoneal linnon node removed during surgery in the same manner as described above. Next, Pseudomonas aeruginosa F1 to F7 sterilized by autoclaving was added to this at a final concentration of 2.5×1.
0 cells/1rLt, 37°C, 5% CO
Culture was performed for 2 days in a carbon dioxide gas incubator in the presence of 2.

After culturing, lymphocytes were collected by centrifugation and added to RPMI-1.
640 (204FC8, containing 2mM glutamine, inicillin, and streptomycin) to give 91 x 10' cells/-, and then dispensed in 100 μl portions into a 96-well plate. Next, 100 μl of the EBV supernatant was added to each well of the plate, and the cells were allowed to proliferate while monitoring antibody production over time using the ELISA method as in the previous example.

Wells showing positive results by ELISA were cloned by limiting dilution, and four clones producing specific antibodies against specific antigens were obtained. The types of clones at this time were respectively clones 3, 4.5 and 6 (EL
ISA results are shown in Table 4).

From the ependyma below, it was found that clone 3 produced antibodies specific to F6, clone 4 to F4, clone 5 to F, and clone 6 to F3.

In addition, the cells obtained here were cultured in the same manner as in the previous example, antibodies were collected, and the recovered antibodies were characterized. As a result, the subclasses of these antibodies were all Ig.
M/g.

Claims (1)

[Claims] 1. A method for preparing antibody-producing cells, which comprises mixing B lymphoid cells and an antigen in a test tube, culturing the same, and recovering the antibody-producing cells from the culture solution. . 2. The method according to claim 1, wherein the antibody-producing cells can form a cell fusion line capable of producing a monoclonal antibody specific to the antibody by fusion with a tumor cell.