JPH0838192A - Monoclonal antibody-forming cell, production thereof, monoclonal antibody and production of the same - Google Patents

Abstract

PURPOSE:To obtain the cell capable of producing a hitherto hardly obtained antibody against a protein by fusing a myeloma cell with an antibody-forming cell of an animal immunized with either a native protein or a denaturated protein and subjected to a booster immunization with the other protein. CONSTITUTION:This new monoclonal antibody-forming cell capable of producing a hitherto hardly obtained monoclonal antibody against a protein is obtained by a method for producing an antibody-forming cell generating a monoclonal antibody having a specific reactivity with a low-antigenicity site, etc., of the principal proteins in an outer membrane of a Chlamydia-pneumoniae, etc. An animal such as mouse is immunized with either a native protein or a denaturated protein obtained by treating the native protein with a surface-active agent or a reducing agent and subjected to booster immunization with the other protein, antibody-forming cells are extracted from the immunized animal and the cells are fused with myeloma cells of mouse, etc., to obtain the aimed monoclonal antibody-forming cells.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody-producing cell having the ability to produce a monoclonal antibody specific to a protein which has been difficult to obtain conventionally, an efficient method for producing the same, a monoclonal antibody and a method for producing the same.

[0002]

2. Description of the Related Art The production of monoclonal antibodies and cells producing them has been reported a lot since Kohler and Milstein reported in 1975. However, although it is generally easy to produce a monoclonal antibody against a protein having a high antigenicity, it is difficult to produce a monoclonal antibody against a protein having a low antigenicity. Further, even a protein having a high antigenicity greatly differs in antigenicity depending on the antigenic site, and it is extremely difficult to produce a monoclonal antibody against the antigenic site having a low antigenicity.
For example, a major outer membrane protein (Major Outer Memblane Protein, hereinafter referred to as MOM), which is a protein having a molecular weight of 39.5 K daltons present in the outer membrane of Chlamydia pneumoniae.
(Abbreviated as P) has a species-specific antigen site and a genus-specific antigen site, and is contained in large quantity, and is therefore preferable as an antigen to be detected. Since a monoclonal antibody against a genus-specific antigen site has already been obtained, it is presumed that this antigen site has high antigenicity, but attempts have been made to produce a monoclonal antibody against Chlamydia pneumoniae. Nevertheless, since there is no report of species-specific monoclonal antibody, this antigenic site is
It is considered to have low antigenicity. It is difficult to obtain cells that produce a monoclonal antibody that specifically reacts with the antigenic site of MOMP having a low antigenicity by the conventional immunization method using Chlamydia pneumoniae basic body (EB), MOMP, etc. Up to now, a monoclonal antibody and its antibody-producing cells that are specifically reactive with the low antigenicity site of MOMP have not been obtained.

Various monoclonal antibodies against Chlamydia pneumoniae have been reported by Iijima et al. (J. Clinical Microbiology, 32 (3), 583-588.
(1994)), there is no report of a monoclonal antibody which specifically reacts with MOMP. Cho-chou Kuo et al. Reported a monoclonal antibody (RR402) having specific reactivity with Chlamydia pneumoniae (J. Clinical Micr
obiology, 24 (6), 1034-1037 (1986), Tokusho Sho-50-500083
No.), the antigen recognized by this monoclonal antibody (RR402) has not been clarified (Y. Iijima, J. Clinical.
Microbiology, 32 (3), 583-588 (1994)).

[0004]

The present inventors have examined MOMP derived from Chlamydia pneumoniae as a specific example. The three-dimensional structure (tertiary structure) of MOMP has high antigenicity, and a planar antigen structure or Assuming that the amino acid sequence itself has low antigenicity, as a result of intensive studies, it was possible to produce cells that produce a monoclonal antibody against the low antigenic site of MOMP, which was difficult to obtain in the past, and to complete the present invention. I arrived.

[0005]

[Means for Solving the Problems] That is, the present invention provides a method for producing an antibody-producing cell that produces a monoclonal antibody having a specific reactivity with a protein, by immunizing with either a native protein or a denatured protein. A method for producing a monoclonal antibody-producing cell characterized by fusing an antibody-producing cell obtained from an animal boosted with the other protein and then a myeloma cell, and a monoclonal antibody production obtained by the producing method The present invention relates to a cell, a method for producing a monoclonal antibody, which comprises culturing the monoclonal antibody-producing cell, and a monoclonal antibody obtained by the production method. The method for producing a monoclonal antibody-producing cell of the present invention is characterized by an immunization method for an animal to be immunized, but otherwise a general method, that is, immunization of animals, cell fusion, selection of fused cells,
It can be prepared through steps such as selection of specific antibody-producing cells and cloning. Examples of proteins to which the production method of the present invention can be applied include MOMPs of Chlamydia, particularly Chlamydia pneumoniae, various proteins derived from bacteria, viruses, humans and animals and plants.

[0006] The native protein is a protein in which the originally formed three-dimensional structure is not destroyed, and the organism itself such as chlamydia, bacteria, viruses, etc., for example, in the case of chlamydia, its basic body (EB) itself. Or a partially purified protein or a completely purified protein. In the case of Chlamydia pneumoniae, examples of the native protein antigen include Y
Examples include EB of K-41 strain and TWAR strain. The denatured protein refers to a protein in a state in which its three-dimensional structure originally formed in a physiological solution is destroyed by various treatments, and preferably, its disulfide bond (S-S bond) is also cleaved. It is a protein that can recognize itself as an antigenic site. By this immunity,
Even when it is difficult to obtain a monoclonal antibody-producing cell against the protein by immunization with the native protein alone, the antibody-producing cell against the low antigenic site of the protein is sufficiently amplified, so that the antibody-producing cell can be easily obtained. be able to. When only one of the proteins is used as an immunizing antigen, only the production of antibodies to the high antigenic site in the protein is induced, and it is difficult to efficiently induce the antibody to the target low antigenic site. Is. The method for denaturing the protein is not particularly limited, and there are a method of treating with a reducing agent or a surfactant, a method of heat treatment, a method of treating with an organic solvent and the like, but a method of treating with a reducing agent or a surfactant is preferable. The reducing agent is not particularly limited, but 2-mercaptol ethanol is preferable. The surfactant is not particularly limited, but S
DS (sodium dodecylbenzene sulfonate) is preferred. Mammals to be immunized include mice, rats,
Rabbits, sheep, chickens and the like can be mentioned, but mice are particularly preferable.

In the immunization method, one of the proteins is used to elicit immunity and the other protein is used as the final immunizing antigen, but the undenatured protein is used to elicit immunity and the denatured protein is used as the final immunizing antigen. Is preferred. For example, in the case of Chlamydia pneumoniae, EB
(Primitive body) or its native MOMP to induce immunity,
As a final immunizing antigen, it is recommended to use a denatured protein, for example, MOMP denatured by treating with a reducing agent and a surfactant in the case of Chlamydia pneumoniae. In addition, it is preferable to use an adjuvant at the time of immunization, and examples thereof include Freund's complete adjuvant (hereinafter abbreviated as FCA) and Freund's incomplete adjuvant (hereinafter abbreviated as FIA). The immunity can be usually elicited by performing the immunization operation once to about five times, but can also be elicited by the immunization operation of six times or more depending on the antigen to be immunized. The amount of antigen used for immunization is
Usually, 1 ng to 10 mg per animal can be used,
Higher and lower amounts of antigen can be used depending on the antigen. The final immunization can usually be performed by performing the immunization operation once to about five times, but the immunization can also be performed by immunizing six or more times depending on the antigen to be immunized. The amount of antigen used for immunization is
Usually, 1 ng to 10 mg per animal can be used,
Higher and lower amounts of antigen can be used depending on the antigen. In addition, 1/100 to 1/100 of the amount of antigen usually used for induction
It is used about 10 times as much. After sufficient immunization by the above method, spleen cells, lymphocyte B cells and the like are isolated from the immunized mammal as parent strains having antibody-producing ability.

Myeloma cells are generally used as the other parent strain for producing hybridomas.
For example, mouse-derived P3U1, NS-1, 653, S
Rat-derived AG1, such as P2, X63, and MPC-11,
Human-derived SK such as AG2, AG3, RCY3 and 210
O-007 and the like can be used, but mouse-derived cells are preferable, and P3U1, 653 and the like are particularly preferable. For cell fusion, a method using polyethylene glycol, a method using Sendai virus, an electrofusion method, or the like can be used, but a method using polyethylene glycol is preferable. Hybridomas can be selected by culturing in a selection medium in which only hybridomas can grow. For example H
Preferred examples include AT (hypoxanthine-aminopterin-thymidine) medium and HAz (hypoxanthine-azaserine) medium.

The selection of the specific antibody-producing cells is carried out by collecting the supernatant of the well in which the growth of the hybridoma is observed by the above culture and using the target protein or the like (for example, EB or MOMP in the case of Chlamydia pneumoniae). ,
It can be performed by examining the presence or absence of antibody production by an immunoblotting method or the like. Cloning refers to making the above-mentioned specific antibody-producing cells into a uniform cell population derived from one clone, and includes, for example, a limiting dilution method, a method for picking up a colony on soft agar, a single cell manipulation method, a FACS method. Etc.
The limiting dilution method is simple and preferable. Monoclonal antibody-producing cells can be obtained by the above method. Hybridoma CP-6, which is an example of the monoclonal antibody-producing cells obtained by the method of the present invention and which produces a monoclonal antibody having specific reactivity with Chlamydia pneumoniae-derived MOMP, was used as a biotechnology industrial research institute of the Institute of Industrial Science and Technology. And was deposited on July 20, 1994 under the deposit number: Life Science Research Institute No. 14436 (FERM P-14436).

The monoclonal antibody can be produced by culturing the monoclonal antibody-producing cells obtained by the above method in a suitable medium. Examples of the medium include denatured Eagle medium (MEM) supplemented with fetal bovine serum.
Medium), Dulbecco's modified Eagle medium (DMEM medium), RPMI1640 medium and the like are preferable. The monoclonal antibody is recovered by collecting the culture supernatant and using an affinity chromatography method using a protein A column, a protein G column, etc., an ion exchange chromatography method, a polyethylene glycol fractionation method,
It can be performed by an ethanol fractionation method, an ammonium sulfate fractionation method, etc., but an affinity chromatography method is preferable. It can be evaluated whether or not the obtained monoclonal antibody has a specific reactivity with the target protein. For example, an organism that is treated with an appropriate treatment solution, subjected to electrophoresis, and subjected to Western blotting using the same can select a substance that specifically reacts with the band of the protein.

According to the method for producing a monoclonal antibody of the present invention, any of the currently known types (globulin class) can be produced. Further, the monoclonal antibody obtained by the production method of the present invention, which can be used as a partially decomposed product of the monoclonal antibody or having a partial structure of the monoclonal antibody, has a low antigenic site of the protein which has been difficult to obtain conventionally. It is recognized as an antigen-determining site. Specifically, it recognizes the planar structure of the protein or the amino acid sequence itself as an antigen-determining site.

The monoclonal antibody obtained by the present invention can be used in a method for detecting a target protein in combination with another monoclonal antibody or a polyclonal antibody, if necessary. Specifically, it can be used in a detection method utilizing an immunoreaction such as a radioimmunoassay and an enzyme immunoassay. Further, it can be used as a component of a diagnostic agent or a research reagent therefor. Furthermore, it can be used in medicine directly or as a chimeric antibody or a drafted antibody. Further, the monoclonal antibody of the present invention can be used in a diagnostic kit in which polymerase chain reaction and the like are combined. The antibody-producing cells of the present invention can also be used as a source of antibody genes. Furthermore, this antibody gene can also be expressed in various cells.

[0013]

EXAMPLE The purpose of this example was to obtain a monoclonal antibody that specifically reacts with Chlamydia pneumoniae-derived MOMP that could not be obtained by the conventional method. 1. Preparation of Chlamydia pneumoniae EB YK-41 strain was used for the preparation of Chlamydia pneumoniae EB. Infection of human lung-derived cells (abbreviated as HL cells) with the YK-41 strain is performed by the method of Kishimoto et al.
(7), 959-964 (1990)). Infected cells cultured in a 24-well or 6-well dish were detached, the whole medium was collected, and then centrifuged at 6,000 rpm for 30 minutes. The supernatant was removed and the precipitate was collected with a sucrose-phosphate-glutamine solution (7.
5% (w / v) sucrose, 3.8 mM KH ₂ PO ₄ , 7 mM K ₂ HPO ₄ ,
The cells were suspended in 5 mM glutamic acid (pH 7.4), abbreviated as SPG. After crushing the suspension with a homogenizer, at 2,500 rpm
Centrifuged for 10 minutes. The supernatant was removed and the precipitate was suspended in SPG. After repeating this operation 5 times, the supernatant was collected. The recovered solution was mixed with 23% (v / v) urographine (Japan Schering Co., Ltd.) and 50% sucrose in a ratio of 2 volume: 2 volume: 1 volume and centrifuged (8,000 rpm, 1 hour). After removing the supernatant,
The lower layer (50% (w / v) sucrose layer) was collected, suspended in SPG, washed, centrifuged (10,000 rpm, 30 minutes), the supernatant was removed by suction, and the precipitate was suspended in SPG. , 26.6-38%
(v / v) Urographin Density Gradient was mixed at a ratio of 1: 4 by volume and centrifuged (8,000 rpm, 1 hour), and the intermediate layer was recovered and suspended in SPG. After centrifugation (10,000 rpm, 30 minutes), the supernatant was removed by suction, and the precipitate was suspended in an appropriate amount of SPG to give an EB solution. Storage was performed at 4 ° C or -70 ° C after dispensing.

2. Preparation of Chlamydia pneumoniae MOMP The EB solution obtained by the above method was separated and purified by disc preparative electrophoresis to prepare MOMP.
That is, the obtained EB solution (protein (EB) concentration 72
7 μg / ml) 0.5 ml and 0.5 ml double concentration of sample buffer (0.31 M Tris-hydrochloric acid (pH 6.8), 1.6% (w / v) sodium dodecyl sulfate (SDS), 1 mM dithiothreitol, 16 % (V / v) glycerin, 0.005% (w / v) bromophenol blue), and boil (100 ° C, 3 minutes)
After solubilizing the protein by, the total amount was added to a disc gel (concentrated gel 4%, separation gel 10%) and electrophoresed (device: disc preparative electrophoresis device NA-1800 type, Nippon Eido Co., Ltd., running condition 100V (constant Voltage)) and a fixed amount (0.26 ml / fraction) was collected. Perform SDS slab electrophoresis for each fraction (apparatus: electrophoresis apparatus for DNA-PAGE NB-5000 type, Nippon Eido Co., Ltd., electrophoresis condition 10
0V (constant voltage)) MOMP of molecular weight 39.5K is a fraction
It was confirmed that the protein was collected in Nos. 33 to 45 (protein concentration 0.21
mg / ml).

3. Production of hybridoma and monoclonal antibody 0.5 ml of physiological saline solution of EB obtained by the above method
Was mixed with 0.6 ml of FCA (Difco), and a water-in-oil type emulsion was prepared using an ultrasonic crusher (Branson) as an immunizing antigen, and BALB / c female mice of 8 weeks old 5
Each mouse was intraperitoneally injected with 200 μl (EB immunization amount of 10 μg / mouse each time). After 6 days and 12 days, immunization was performed using FIA (Difco). Six days later, a partial blood sample was taken and the antibody titer of each serum was determined by Dot Immuno Binding Assay.
Method (hereinafter abbreviated as DIBA method), the antibody with the highest antibody titer was purified by the above method as the final immunization for 3 days from the measurement day.
P immunity amount of 1 μg / mouse) was administered through the tail vein. On the day after the third immunization, the spleen of the mouse was dissected out after cervical dislocation and dispersed as single cells to obtain splenocytes for cell fusion. 10% The partner cells for cell fusion (v / v) were pre-cultured in fetal bovine serum pressurized Dulbecco MEM medium (abbreviated as 10F medium) (Napco manufactured CO ₂ incubator, 37 ° C., 5% carbon dioxide gas, saturated humidity ), And P3U1 mouse myeloma cells were used.

The above-mentioned splenocytes (1.1 × 10 ⁸ cells) and mouse myeloma P3U1 cells (2.1 × 10 ⁷ ) were mixed and centrifuged (1,00
After 10 minutes (0 rpm, 10 minutes), the supernatant was removed by suction, and the cells were gently shaken to loosen the cell pellet, and then a cell fusion agent (50% (v / v) polyethylene glycol; MW = 4000 Merck) was added. 0.
2 ml was added. After 1 minute and 45 seconds, 10 ml of the culture solution was added over 1 minute, 20 ml of the culture solution was further added, and after suspension, centrifugation was performed stepwise (300 rpm 3 minutes, 500 rpm 3 minutes, 700 rpm 3 minutes).
The supernatant was removed by suction, and 10F medium (abbreviated as 10F + Bri medium) containing 5% Blai clone (Dainippon Pharmaceutical Co., Ltd.) was added to the cell pellet to prepare a cell suspension. 96 cells suspension
It was dispensed at 4 × 10 ⁴ cells / well / 0.1 ml in terms of myeloma cells into a well flat plastic bottom multi-plate (Corning), and cultured at 37 ° C., 5% carbon dioxide gas, and saturated humidity. The next day, 10F medium containing HAT (1 × 10 ^-4 M hypoxanthine, 4 × 10 ^-7 M aminopterin, 1.6 × 10 ^-5 M
Dulbecco ME supplemented with thymidine and 10% fetal bovine serum
M medium, abbreviated as HAT medium) was added at 0.1 ml / well, and half of the culture supernatant was replaced with HAT medium at intervals of 3 or 4 days, so-called HAT selection was performed for about 2 weeks, and hybridoma growth was observed. Collect the supernatant from the wells and perform the Dot Immuno Binding Assay method (hereinafter referred to as DIBA
(Abbreviated as “method”) was screened for the presence or absence of antibody production against EB.

The DIBA method uses a 96-well U-bottom multi-plate (Corning Co.), and the dot size is set to 0.1 per dot in advance.
3mm square membrane filter (Advantech 47mm diameter filter with filter A0) with 02μg EB as antigen
45b047A) with 10% (v / v) FCS (Fetal Calf Serum) at room temperature for 15 minutes, and then the supernatant was removed, 100 μl of the hybridoma culture supernatant was added at room temperature for 1 hour, and then the supernatant was removed and then labeled with peroxidase. Add 100 μl of 500-fold diluted rabbit anti-mouse immunoglobulin antibody (Kappel) and repeat 1
After each reaction for 4 hours, 4-chloro 1 is used as the substrate.
Naphthol was added to develop the color. Those in which the color was visually recognized on the membrane filter on which the antigen was immobilized were determined to be positive for antibody production. As a result of this screening, hybridomas in wells positive for antibody production were further cloned by the limiting dilution method. That is, a suspension of the antibody-producing positive hybridoma in a HAT-containing medium was prepared, the cell concentration was measured, and the diluted solution was dispensed and cultured in a new microplate so that the hybridoma was 2 to 100 cells / 100 μl / well. . At this time, HT + 5% Bligh clone medium was used as a diluent.

When hybridoma growth is observed in wells of high dilution, antibody production is measured and observed under a microscope to confirm that it is a monoclone, and the same operation is repeated again to confirm that the antibody with high antibody continuous production ability is high. An EB monoclonal antibody producing clone was obtained. Each of the obtained clones was gradually raised in culture scale and finally cultured in 50 ml of 10F medium, and the properties of the antibody in the culture supernatant were examined. The above-mentioned experimental operation was repeated twice before and after, and finally 10 monoclonal antibody-producing clones shown in Table 1 were obtained. The globulin class of monoclonal antibody produced by 10 hybridoma clones is ISOTYPE Ab-STAT-I kit (Sang St
at Medical) (see Table 1). As shown in Table 1, monoclonal antibodies CP-1, CP-2, CP
-4, CP-5, CP-6, CP-7, CP-8 did not show reactivity to Chlamydia trachomatis, but showed reactivity to Chlamydia pneumoniae.

[0019]

[Table 1]

Furthermore, 10 F per hybridoma clone producing each of the monoclonal antibodies shown in Table 1.
Pristane (Wako Pure Chemical Industries) that grows cells cultured in a medium
The monoclonal antibody of the present invention was purified from the ascites obtained 2-3 weeks after intraperitoneal inoculation of BALB / c mice pre-treated with 0.5 ml / mouse by inoculation of 1 × 10 ⁷ cells each. Hybridoma clone No. Ascites of 6 is protein A as follows
Was purified by a method using an immobilized affinity column. That is, 1.5 M glycine +3 equivalent to mouse ascites
After adding MNaCl (pH 8.9) and passing through a 0.45 μm nylon filter (Corning Co.), it was applied to an affinity column of Protein A-Poros (NGK) and washed with 1.5 M glycine + 3 M NaCl (pH 8.9). Then 150mM
Elution was performed with a phosphate-citrate buffer solution (pH 6.0) containing NaCl. The eluate was neutralized with 1 M Tris-HCl buffer (pH 9.0), cooled, dialyzed overnight with PBS, and the absorbance of the recovered solution at 280 nm was measured (U-2000, Hitachi, Ltd.) to give 0.2 μm cellulose. Pass through an acetate filter (Corning) -80 ° C
And used as a stock solution of the purified antibody.

4. Specificity analysis of monoclonal antibody The specificity analysis of the monoclonal antibody was performed by Western blotting. Reactivity with Chlamydia pneumoniae is 1. The EB solution (EB concentration 727 μg / ml) obtained by the method described in 1. was used. Specifically, an EB solution containing 50 μg of EB and a sample treatment solution (0.063M Tris, 5% (v / v)
2-mercaptoethanol, 10% (v / v) glycerin, 2.3
% (W / v) SDS, 0.001% (w / v) BPB (Bromphenol Blue) (pH
6.8)) is mixed with 1 volume: 1 volume, boiled for 10 minutes, and then 100μ
l was added and electrophoresed (electrophoresis device: AE-6560 (Ato)), gel: Atopagel 1020DL, migration buffer: 0.025M Tris, 0.192M glycine, 0.1% (w / v) SDS
(PH 8.3), electrophoresis conditions: 20 mA constant current, 80 minutes electrophoresis). As the transfer device, NA-1510 (Japan Eido Co., Ltd.) was used. Transfer buffer (0.025M
Tris, 0.192M glycine, 20% methanol (pH8.3))
The gel after migration was dipped in 2 mm thick filter paper (Whatman Co., Ltd.) and a nitrocellulose membrane (Bio-Rad Co.) for 15 minutes. The filter paper, the gel, the nitrocellulose membrane, and the filter paper were placed in this order on the pad, and the pad was set on the scissors device. The device was placed in ice and a constant voltage of 50 V was applied for 2 hours to transfer the protein to the nitrocellulose membrane. The nitrocellulose membrane was washed with PBS (Nissui Pharmaceutical) (2 minutes, shake twice), and then blocked with 10% FCS / PBS (30 minutes, room temperature, shake). Wash the nitrocellulose membrane with PBS (Nissui Pharmaceutical) (2 minutes, 3 times,
After shaking, each of the culture supernatants of all the hybridomas shown in Table 1 was reacted (1 hour, room temperature, shaking). P
After washing with BS (2 minutes, shaking 3 times), react with 500-fold diluted peroxidase-labeled rabbit anti-mouse immunoglobulin antibody (Cappel) (1 hour, room temperature, shaking)
I let it. After washing with PBS (2 minutes, 3 times, shaking), coloring solution (3 mg / ml 4-chloro-1-naphthol 1 ml, P
After adding 5 ml of BS (2 ml of hydrogen peroxide solution) and reacting until a protein band appeared, the membrane was washed several times with distilled water to stop the reaction. Purified Chlamydia trachomatis E for analysis of reactivity with Chlamydia trachomatis
Western blotting was performed using B (L2 strain) in the same manner as the above conditions. As a result, the monoclonal antibody C of the present invention
P-6 and CP-7 are MO of Chlamydia trachomatis
It showed no reactivity with MP, only with MOMP of Chlamydia pneumoniae.

5. Analysis of antibodies by fluorescent antibody method (reactivity with Trachomatis, Sittasi, Pecolum) Chlamydia trachomatis, Sittasi, and Pecolum isolates were placed on a slide glass on which 1 μl of each hybridoma shown in Table 1 was cultured. 8 μl of the supernatant was placed, placed in a wet box, reacted at 37 ° C. for 3 hours, washed with PBS and distilled water, and air-dried. After air-drying, place 8 μl of 10-fold diluted FITC-labeled anti-mouse immunoglobulin / goat serum (KPL) so that all the spotted sites are covered, put in a wet box, react at 37 ° C for 30 minutes, wash, air-dry and mount liquid. (50% glycerin-PBS) 10
The cells were sealed with 0 μl and observed with a fluorescence microscope. As a result, it was found that the monoclonal antibodies CP-6 and CP-7 of the present invention brilliantly appeared only with EB of Chlamydia pneumoniae, and that the antibody reacted with EB. The hybridoma producing this monoclonal antibody CP-6 was designated as CP-
6 was deposited with the Institute of Biotechnology, Institute of Biotechnology (Deposit No .: Life Science Research Institute No. 14436 (FERM P-144).
36)).

[0023]

INDUSTRIAL APPLICABILITY According to the method for producing a monoclonal antibody and cells producing the same of the present invention, it is possible to efficiently produce a protein which has been difficult to produce by the conventional method. The obtained monoclonal antibody is useful for various diagnostic methods and various reagents for specifically detecting the target protein, and also useful as a medicine. The obtained antibody-producing cells are also useful as a source of antibody genes in addition to producing the above-mentioned antibody.

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[Procedure amendment]

[Document name] Consignment number change notification

[Submission date] July 20, 1995

[Former name of depositary institution] Institute of Biotechnology, Institute of Biotechnology, AIST

[Old consignment number] FERM P-14436

[Name of new depositary institution] Institute of Biotechnology, Industrial Technology Institute

[New contract number] FERM BP-5155

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location // C12N 15/02 (C12P 21/08 C12R 1:91) (72) Inventor Kiyotaka Kawagoe Ibaraki Prefecture 4-13-1 Higashimachi, Hitachi, Ltd. Inside the Pharmaceutical Research Laboratory, Hitachi Chemical Co., Ltd. (72) Inventor, Akifumi Iguchi 4-13-1, Higashimachi, Hitachi City, Ibaraki Hitachi Chemical Co., Ltd. (72) Inventor Toshihide Morikawa 4-13-1, Higashimachi, Hitachi, Ibaraki Hitachi Chemical Co., Ltd. Pharmaceutical Research Laboratory (72) Inventor Yoshiki Nakao 4-13-1, Higashimachi, Hitachi, Ibaraki Yamazaki Industry Co., Ltd.

Claims (8)

[Claims] 1. A method for producing an antibody-producing cell that produces a monoclonal antibody having specific reactivity to a protein, wherein immunization is induced by either a native protein or a denatured protein, and then the other protein is used. A method for producing a monoclonal antibody-producing cell, which comprises fusing an antibody-producing cell obtained from a boosted animal with a myeloma cell. 2. A method for producing an antibody-producing cell that produces a monoclonal antibody having a specific reactivity to a protein, which was obtained from an animal in which immunization was induced with a native protein and then boosted with the modified protein. A method for producing a monoclonal antibody-producing cell, which comprises fusing an antibody-producing cell and a myeloma cell. 3. The denatured protein is a non-denatured protein treated with a surfactant or a reducing agent.
Or the method for producing a monoclonal antibody-producing cell according to item 2. 4. The immunized animal to obtain antibody-producing cells is a mouse, and the myeloma cell is derived from a mouse.
2. The method for producing a monoclonal antibody-producing cell according to 2 or 3. 5. The method for producing a monoclonal antibody-producing cell according to claim 1, 2, 3 or 4, wherein the protein is a major protein of Chlamydia outer membrane. 6. A monoclonal antibody-producing cell produced by the method according to any one of claims 1 to 5. 7. A method for producing a monoclonal antibody, which comprises culturing the monoclonal antibody-producing cell according to claim 6. 8. A monoclonal antibody produced by the production method according to claim 7.