JP2000186099A - Monoclonal antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new monoclonal antibody which is obtained by immunizing and sensitizing a mammalian with a specific protein capable of inducing the production of interferon-γ(IFN-γ) in an immunity-charging cell, is useful for purifying or detecting the protein by immunoaffinity chromatography, and immunologically reacts with the protein. SOLUTION: This monoclonal antibody is obtained by immunizing and sensitizing a mammalian with a protein or its antigenic fragment. The protein has an amino acid sequence of the formula (Xaa is methionine or threonine) or an amino acid sequence of the formula (but one or more amino acids are lacked, replaced or added), and can induce the production of IFN-γ in immunity-charging cells. The antibody is obtained by fusing antibody-producing cells collected from a mammalian immunized and sensitized with the protein or its antigenic fragment with mammalian cells capable of unlimitedly growing, selecting a clone capable of producing the objective antibody from the obtained hybridomas, and then culturing the clone.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel monoclonal antibody, and more particularly, to interferon-γ (hereinafter referred to as “IFN-γ”) in immunocompetent cells.
Abbreviated. The present invention relates to a monoclonal antibody specific to a protein that induces the production of

[0002]

2. Description of the Related Art IFN-γ is known as a protein having an antiviral action, an antitumor action, and an immunoregulatory action, and is said to be produced by immunocompetent cells stimulated by an antigen or mitogen. Because of these biological effects, IFN-
Since its discovery, γ has been put to practical use as an antitumor agent, and is currently being vigorously studied in clinical trials as a therapeutic agent for general malignant tumors such as brain tumors. Currently available IFN-γ is natural IFN produced by immunocompetent cells.
-Γ, and recombinant IFN-γ produced by a transformant obtained by introducing DNA encoding IFN-γ collected from an immunocompetent cell into Escherichia coli. Are administered as “foreign IFN-γ”.

[0003] Among them, natural IFN-γ is usually produced by culturing immunocompetent cells established in a culture in a culture medium containing an IFN-γ inducer and purifying the culture. In this method, the type of IFN-γ inducer is IFN-γ
-It is said that it has a great effect on the production amount of γ, the ease of purification, and the safety of the product. Usually, concanavalin A, lentil lectin, American pokeweed lectin, endotoxin, lipopolysaccharide, etc. Mitogens are frequently used. However, all of these substances have a problem in that the molecules are diverse, the quality is easily varied depending on the source and the purification method, and it is difficult to obtain a desired amount of an IFN-γ inducer having a constant inducibility. In addition, many of the above substances show remarkable side effects when administered to a living body or even show toxicity depending on the substance, and it is extremely difficult to induce IFN-γ production by directly administering to a living body. there were.

The present inventors have been studying cytokines produced by mammalian cells, and have found that there is a completely unknown substance which induces IFN-γ production in mouse liver. Was. This substance was isolated by a combination of various purification methods, mainly column chromatography, and its properties and properties were examined.It was found that the substance was a protein and had the following physicochemical properties. found. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces IFN-γ production in immunocompetent cells.

[0005] Proteins having such physicochemical properties are not yet known and are considered to be novel substances. Then, the present inventors continued a thorough search for mouse hepatocytes, and succeeded in isolating a DNA encoding this protein. Upon decoding, it was found that this DNA was composed of 471 base pairs and encoded the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing. Then this D
When NA was introduced into Escherichia coli and expressed, the protein was produced in a high yield in the culture. The above findings are disclosed in Japanese Patent Application No. 6-184162 filed by the same applicant.

As described above, the protein has a property of inducing the production of IFN-γ in immunocompetent cells, and is a general-purpose IFN-γ inducer, an antiviral agent,
A wide variety of uses are expected as antitumor agents, antibacterial agents, immunomodulators, platelet increasing agents and the like. In general, if a bioactive protein is to be blended and used in a pharmaceutical product, it is essential to develop a method that can purify the protein highly and efficiently and a method for assaying many test samples at once. The best material that allows such purification and assay is a monoclonal antibody, but no monoclonal antibody specific to the protein has been established.

[0007]

In view of such circumstances, an object of the present invention is to provide a monoclonal antibody specific to a protein having physicochemical properties as described above.

[0008] Another object of the present invention is to provide a hybridoma capable of producing such a monoclonal antibody.

[0009] Still another object of the present invention is to provide a method for producing such a monoclonal antibody.

[0010] Still another object of the present invention is to provide a method for purifying the protein using the monoclonal antibody.

[0011] Still another object of the present invention is to provide a method for detecting the protein using the monoclonal antibody.

[0012]

The present invention solves the first problem by using a monoclonal antibody specific to a protein having the following physicochemical properties. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces the production of interferon-γ in immunocompetent cells.

The present invention solves the second problem by a hybridoma capable of producing such a monoclonal antibody.

The third object of the present invention comprises a step of culturing a hybridoma capable of producing such a monoclonal antibody in vitro or in vivo, and a step of collecting the monoclonal antibody from the culture or body fluid. The problem is solved by a method for producing a monoclonal antibody.

[0015] The present invention provides a fourth object of the present invention, which comprises contacting the monoclonal antibody with a mixture containing the protein and a contaminant to adsorb the protein to the monoclonal antibody, and desorbing the adsorbed protein from the monoclonal antibody. The problem is solved by a protein purification method comprising a step.

The present invention solves the fifth problem by a method for detecting a protein, which comprises bringing a monoclonal antibody into contact with a test sample and detecting the protein by an immune reaction.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention reacts specifically with a protein having specific physicochemical properties.

The hybridoma of the present invention produces such a monoclonal antibody when cultured in and out of a living body.

According to the manufacturing method of the present invention,
The desired amount of such a monoclonal antibody is easily obtained.

According to the purification method of the present invention,
From the mixture containing the protein and the contaminants, the protein is efficiently collected with high purity.

According to the detection method of the present invention,
Since only the protein in the test sample gives an immune response,
The protein in the test sample can be qualitatively or quantitatively detected by measuring the immune reaction by an appropriate technique.

The monoclonal antibody referred to in the present invention is:
Includes all monoclonal antibodies specific to proteins having the aforementioned physicochemical properties, their sources and origins,
Classes do not matter. As individual proteins of interest,
For example, a protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing or an amino acid sequence homologous thereto can be used. An amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing refers to one or more amino acids in the amino acid sequence of SEQ ID NO: 3 without substantially changing the aforementioned physicochemical properties. In which one or more amino acids are added to the N-terminal and / or C-terminal in the amino acid sequence of SEQ ID NO: 3, and one or more amino acids at the N-terminal and / or C-terminal thereof are missing. Include what you lost.

The monoclonal antibody of the present invention can be obtained by using such a protein or an antigenic fragment thereof as an antigen. Specifically, for example, a hybridoma of an antibody-producing cell collected from a mammal immunized with such an antigen and a cell derived from a mammal capable of infinite growth is prepared, and the monoclonal antibody of the present invention is thereby prepared. The clone can be obtained by selecting a hybridoma clone that can be produced and culturing the clone in and outside the living body.

The protein which can serve as an antigen can be obtained, for example, from mouse hepatocytes, as disclosed in Japanese Patent Application No. 6-184162, or the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing or an amino acid homologous thereto. It can be obtained by culturing a transformant into which the DNA encoding the sequence has been introduced, and it is usually used in a state of being completely purified or partially purified. In order to obtain an antigenic fragment, these purified or partially purified products may be chemically or enzymatically degraded, or a peptide may be synthesized based on the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.

The immunization may be carried out by a conventional method. For example, the above-mentioned antigen is injected alone or appropriately together with an adjuvant into a vein, intracutaneously, subcutaneously or intraperitoneally of a mammal, and bred for a certain period. The mammal is not particularly limited, and may be of any type, size, and sex, as long as the desired antibody-producing cells can be obtained. Normally, rodents such as rats, mice, and hamsters are used, and the optimal one is selected in consideration of compatibility with cells derived from mammals capable of infinitely growing, as described below. Depending on the type and size of the mammal, the total amount of antigen to be inoculated is usually about 5 to 500 μg / animal,
It is inoculated in 2 to 5 divided doses at intervals of about 1 to 2 weeks. Then, 3 to 5 days after the final inoculation, the spleen is removed and dispersed to obtain spleen cells as antibody-producing cells.

Next, the antibody-producing cells thus obtained are fused with cells derived from mammals capable of infinite growth to obtain a cell fusion product containing the desired hybridoma. Cells derived from mammals that can grow indefinitely usually include P3-NS1-A
g4-1 cells (ATCC TIB18), P3-X63
-Ag8 cells (ATCC TIB9) and SP2 / 0-
A cell line derived from a mouse bone marrow species such as Ag14 cell (ATCC CRL1581) or a mutant thereof is used. For cell fusion, for example, a conventional method using a fusion promoter such as polyethylene glycol or Sendai virus or an electric pulse is used.For example, infinite growth with antibody-producing cells in a fusion medium containing the fusion promoter is possible. Mammalian-derived cells are suspended at a ratio of about 1: 1 to 1:10, and incubated in this state at about 30 to 40 ° C. for about 1 to 5 minutes. The fusion medium includes, for example, MEM medium, R
Ordinary general media such as PMI1640 medium and Iskov-modified Dulbecco's medium can be used, but it is desirable to remove serums such as bovine serum.

To select a target hybridoma, first, the cell fusion product obtained as described above is transferred to a selection medium such as a HAT medium, and cultured at about 30 to 40 ° C. for about 3 days to 3 weeks. Kill cells other than hybridomas. Next, the hybridoma is cultured by a conventional method, and the antibody secreted into the culture is tested for reactivity with the protein. For the test, a conventional method for detecting an antibody such as an enzyme immunoassay, a radioimmunoassay, and a bioassay is used.
Tamoe Ando, Monoclonal Antibody Experiment Manual, 199
One year, published by Kodansha Scientific, 105th to 1st
On page 52, various methods are described in detail. A hybridoma producing an antibody specific to the protein is immediately cloned by a limiting dilution method or the like to obtain a single cloned hybridoma according to the present invention.

The monoclonal antibody of the present invention can be obtained by culturing such a hybridoma in and out of a living body. For the culture, a conventional method for culturing cells derived from mammals is used.For example, when culturing in an in vitro culture medium, the culture is transplanted to a non-human warm-blooded animal, and then cultured. When cultured in the body, a monoclonal antibody is collected from the ascites and / or blood. Hybridoma M-1 described below is characterized in that it has a high ability to produce a monoclonal antibody and that it can be easily cultured in and out of a living body. To collect monoclonal antibodies from cultures or ascites or blood, any conventional method in the art for purifying antibodies in general is used. Individual methods include, for example, salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, gel electrophoresis and isoelectric focusing. Electrophoresis, and these are applied in combination as needed. The purified monoclonal antibody is
After that, it is concentrated and dried to make it liquid or solid depending on the application.

The monoclonal antibody of the present invention is extremely useful for purifying the protein by immunoaffinity chromatography. Such a purification method comprises the steps of contacting the monoclonal antibody of the present invention with a mixture of the protein and a contaminant such as a contaminant protein other than the protein to adsorb only the protein to the monoclonal antibody; and The step of desorption from the monoclonal antibody, both steps usually being carried out in an aqueous medium. The monoclonal antibody of the present invention is usually used in the state of being bound to a gel-like water-insoluble carrier,
The water-insoluble carrier is packed in a column shape in a cylindrical tube or the like, and when, for example, an extract of mouse hepatocytes or a culture of a transformant or a partially purified product thereof is passed therethrough, substantially the protein alone is substantially removed. Adsorbs to the monoclonal antibody on the water-insoluble carrier. The adsorbed protein can be easily desorbed by changing the hydrogen ion concentration around the monoclonal antibody. For example, when a monoclonal antibody belonging to the class of IgG is used, the pH on the acidic side, usually pH 2 to 3, On the other hand, when a monoclonal antibody belonging to the class of IgM is used, elution is performed at a pH on the alkaline side, usually pH 10 to 11.

According to the purification method of the present invention, the protein can be highly purified with a minimum of labor and time. As described above, the protein is involved in IFN-mediated immunocompetent cells.
Since it has the property of inducing the production of -γ, the obtained purified protein is sensitive to IFN-γ as an inducer when producing IFN-γ by a cell culture method,
For example, it is useful as a therapeutic or prophylactic agent for viral diseases such as AIDS and condyloma acuminatum, malignant tumors such as kidney cancer, granulomas, mycosis fungoides, brain tumors, and immune diseases such as rheumatoid arthritis and allergic diseases. When the protein also has the property of enhancing cytotoxicity caused by killer cells, it may be used in combination with interleukin 2 or tumor necrosis factor as appropriate to include solid cancers such as lung cancer, kidney cancer and breast cancer by adoptive immunotherapy. Significant effects are obtained on the improvement of therapeutic effects and side effects in the treatment of malignant tumors.

The monoclonal antibody of the present invention has a wide variety of uses in various fields requiring detection of the protein. That is, when a labeled immunoassay such as a radioimmunoassay, an enzyme immunoassay, or a fluorescence immunoassay is applied to the monoclonal antibody of the present invention, the protein in the test sample can be quickly and accurately qualitatively or quantitatively analyzed. In such analysis, the monoclonal antibody of the present invention is used after being labeled with, for example, a radioactive substance, an enzyme, and / or a fluorescent substance. Since the monoclonal antibody of the present invention specifically reacts with the protein and gives an immune reaction, if the immune reaction is measured using these labeling substances as indices, a very small amount of the protein in the test sample can be accurately detected. be able to. Labeled immunoassays are characterized by being able to analyze a large number of test samples at a time, requiring less time and effort for analysis, and being more accurate than bioassays. Therefore, the detection method according to the present invention is extremely useful for process control and product quality control when producing the protein. Since the present invention does not relate to the labeling of the monoclonal antibody or the labeling assay itself, a detailed description is omitted. For example, P. Thyssen, Eiji Ishikawa, "Enzyme Immunoassay", 198
Ninth year, Tokyo Chemical Doujinshi, pp. 196-348, etc., various methods for that purpose are described in detail.

Hereinafter, the present invention will be described based on embodiments, but in the state of the art, such embodiments can be variously modified. In view of such a state of the art, it goes without saying that the present invention should not be limited to only these embodiments.

[0033]

Example 1 <Preparation of hybridoma M-1>

[0034]

Example 1-1 <Preparation of transformant KGFM5>
8 μl of 25 mM magnesium chloride in a 5 ml reaction tube,
10 μl of 10 × PCR buffer, 1 μl of 25 mM dNTP mix, 2.5 units / μl Amplitac DN
A set consisting of 1 μl of A polymerase, a DNA having the base sequence shown in SEQ ID NO: 4 in the sequence listing prepared from a phage DNA clone according to the method described in Japanese Patent Application No. 6-184162 and encoding the protein 1 ng of the recombinant DNA was chemically synthesized based on the amino acid sequence near the N-terminus and C-terminus in SEQ ID NO: 3 in the sequence listing.
'-GAGGAATTCTGGAGGAAGGTACC
ATGAACTTTGGCCCGACTTC-3 'and 5
'-GCGAAAGCTTCTACTACTTTGATGT
Appropriate amounts of the sense primer and antisense primer of the base sequence represented by AAG-3 'were added, and the mixture was made up to 100 µl with sterile distilled water. The mixture is heated to 94 ° C.
The cycle of incubation for 1 minute at 43 ° C., 1 minute at 43 ° C., and 1 minute at 72 ° C. was repeated three times. 37 cycles of sequential incubation
The PCR reaction was repeated once, and an EcoRI cleavage site and a HindIII cleavage site were provided at the 5 ′ end and the 3 ′ end of the nucleotide sequence shown in SEQ ID NO: 4, respectively.

PCR product, Stratagene plasmid vector "pCR-ScriptSK (+)" 10 n
g was ligated with DNA ligase by a conventional method, and
A was transformed into E. coli strain "XL-1 Blue MRF'Kan" manufactured by Stratagene by the competent cell method and transformed. 50 μg / ml of transformant
After inoculating an L-broth medium (pH 7.2) containing ampicillin, and culturing with shaking at 37 ° C. for 18 hours, the culture was centrifuged to collect a transformant.
The recombinant DNA was isolated by applying the method. This recombinant DN
When a part of A was analyzed by the dideoxy method,
The DNA having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing was ligated, and it was confirmed that the desired DNA was correctly amplified in the PCR reaction.

Therefore, the remaining recombinant D
After cutting NA with restriction enzymes Eco RI and Hind III, the same restriction enzyme as 0.1 μg of the obtained Eco RI-Hind III DNA fragment was obtained in advance using a DNA ligation kit “DNA Ligation Kit Version 2” manufactured by Takara Shuzo. Plasmid vector "pKK223-3" 10
ng was reacted at 16 ° C. for 30 minutes and ligated to obtain a replicable recombinant DNA “pKGFM5”. E. coli Y1090 strain (ATCC 37197) was transformed with this recombinant DNA pKGFM5 by the competent cell method.
The obtained transformant “KGFM5” was inoculated into an L-broth medium (pH 7.2) containing 50 μg / ml ampicillin, and cultured with shaking at 37 ° C. for 18 hours. The culture was centrifuged to collect a transformant, and a part of the transformant was subjected to the ordinary SDS-alkali method to extract the recombinant DNA pKGFM5. When analyzed by the dideoxy method, as shown in FIG. 1, in the recombinant DNA pKGFM5, KGFM5cD having the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing was obtained.
NA was linked downstream of the Tac promoter.

[0037]

Example 1-2 <Production of protein by transformant KGFM5> Ampicillin 50 µg /
sterilized L-broth medium (pH 7.2) containing
After cooling to 7 ° C., the transformant KG prepared in Example 1-1
FM5 was inoculated and seeded under shaking at the same temperature for 18 hours. 1 liter of the same fresh medium in a 20-liter jar fermenter
8 liters were similarly sterilized, cooled to 37 ° C., inoculated with the seed culture obtained above at 1% (v / v), and cultured with aeration and stirring at the same temperature for 8 hours. Centrifuge the culture to collect the cells,
A mixture containing 150 mM sodium chloride, 16 mM disodium hydrogen phosphate and 4 mM sodium dihydrogen phosphate (pH
7.3), and after sonication, crushed cells were removed by centrifugation, and the supernatant was collected.

The supernatant was added with 4 g of ammonium sulfate under ice-cooling.
0% (w / v), dissolve uniformly, leave for a while,
After centrifugation, the supernatant was collected. Ammonium sulfate was added to the newly formed supernatant up to 85% (w / v), the mixture was stirred at 4 ° C. for 25 hours, and after centrifugation, a precipitate containing the protein was collected. The precipitate was dissolved in a 150 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate, and the solution was previously equilibrated with a 10 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate (Pharmacia). Sepharose ”column, the column was washed with the same fresh buffer, and then passed through a 10 mM phosphate buffer (pH 6.6) under a concentration gradient of ammonium sulfate falling from 1.5 M to 0 M.

Next, the fractions eluted at an ammonium sulfate concentration of about 0.9 M were pooled, concentrated on a membrane, and dialyzed against 10 mM phosphate buffer (pH 6.5) at 4 ° C. for 18 hours.
The column was loaded on a column of "DEAE5PW" manufactured by Tosoh, which had been equilibrated with 10 mM phosphate buffer (pH 6.5) in advance, and the column was washed with the same fresh buffer, and then the column was washed from 0M to 0.
Under a concentration gradient of sodium chloride rising to 2M, 10 mM
After passing through a phosphate buffer (pH 6.5), a fraction eluted at a sodium chloride concentration of about 0.1 M was collected.

Thereafter, this fraction was concentrated on a membrane and loaded onto a Pharmacia "Super Dex 75" column which had been equilibrated with a phosphate buffer (hereinafter, referred to as "PBS") in advance, and was freshly loaded. When a fraction having a molecular weight of around 19,000 daltons eluted by passing through PBS was collected,
An aqueous solution containing about 4.7 mg of the purified protein was obtained. The yield over the entire purification step was about 26%.

When the protein was analyzed according to the method described in Japanese Patent Application No. 6-184162, the purified protein had the following physicochemical properties. That is, SDS-polyacrylamide gel electrophoresis under non-reducing conditions shows a main band capable of inducing IFN-γ at a position corresponding to a molecular weight of 19,000 ± 5,000 daltons. The isoelectric point is shown at 1.0. The two types of peptide fragments obtained by trypsin digestion had the amino acid sequences shown in SEQ ID NOS: 1 and 2 in the sequence listing.

[0042]

Example 1-3 <Preparation of Hybridoma M-1> 10
The purified protein obtained by the method of Example 1-2 was injected intraperitoneally into a week-old SD rat together with complete Freund's adjuvant for 20 days.
Inoculation was performed at a rate of μg / animal. Thereafter, the same amount was inoculated twice every two weeks, one week after the last inoculation, the same amount was further intravenously injected, and three days later, the spleen was removed and dispersed to obtain splenocytes.

The splenocytes were combined with mouse myeloma-derived SP2 /
0-Ag14 cells (ATCC CRL1581)
Cell density of 3 × 10 ⁴ cells / ml in serum-free RPMI1640 medium (pH 7.2) pre-warmed to
And 1 × 10 ⁴ cells / ml, and after centrifugation, the precipitate was collected. This precipitate has an average molecular weight of 1,5.
Serum-free RPMI 1640 medium containing 50 daltons (w / v) polyethylene glycol at pH 7.0 (pH 7.0).
2) Add 1 ml dropwise over 1 minute, incubate at 37 ° C. for 1 minute, then add serum-free RPMI1640 medium (pH 7.2) dropwise until the total volume becomes 50 ml, centrifuge, and remove the precipitate. Collected. HAT
Suspend in medium and place in 200 well microplate
The resulting mixture was dispensed in an amount of μl / well, and incubated at 37 ° C. for one week to select a hybridoma.

The antibody secreted into the culture supernatant in each well was examined for its reactivity with the purified protein obtained by the method of Example 1-2 by enzyme immunoassay to produce an antibody reactive with the purified protein. Hybridomas were selected. Subsequently, limiting dilution was repeatedly applied to the hybridoma according to a conventional method to obtain a hybridoma clone M- capable of producing the monoclonal antibody of the present invention.
1 was obtained.

[0045]

Example 2 <Preparation of monoclonal antibody M-1 mAb and Western blotting analysis>

[0046]

Example 2-1 <Preparation of monoclonal antibody M-1 mAb> Hybridoma M obtained by the method of Example 1-3
-1 is adjusted to 5% so that the cell density becomes about 1 × 10 ⁶ cells / ml.
(V / v) Suspended in RPMI 1640 medium (pH 7.2) supplemented with bovine serum,
The cells were cultured at 37 ° C. in a 5% CO ₂ incubator. When the desired cell density was reached, hybridoma M-1 was immunosuppressed in advance with rabbit-derived anti-hamster thymocyte antibody, and a 5-week-old hamster that had been injected intraperitoneally with pristane at 0.5 ml / animal. 1 × 10 ⁷ in the abdominal cavity of
Each animal was inoculated by injection and bred for 1 week in a usual manner.

[0047] Ascites was collected from the hamster, and 3 hours with PBS.
After double dilution, ammonium sulfate was added to 50% saturation, and the mixture was allowed to stand at 4 ° C. for 24 hours. After centrifugation, a precipitate was collected. This precipitate was dialyzed overnight at 4 ° C. against a 20 mM aqueous potassium dihydrogen phosphate solution (pH 6.7), and then loaded onto a hydroxyapatite column previously equilibrated with the same fresh aqueous solution to give a concentration of 20 mM to 300 mM.
Potassium dihydrogen phosphate aqueous solution (pH
After passing through 6.7), an aqueous solution containing the monoclonal antibody M-1 mAb of the present invention was obtained. The yield was about 5 mg per hamster. Analysis by a conventional method revealed that this monoclonal antibody M-1m
Abs belonged to the class of IgM.

[0048]

Example 2-2 <Western blotting analysis> Dithiothreitol 100 mg, 10% (w / v) SDS
1 μg of the purified protein obtained by the method of Example 1-2 was added to a mixed solution composed of 0.5 ml of an aqueous solution and 1 ml of glycerol, incubated at 37 ° C. for 1 hour, and subjected to SDS-polyacrylamide gel electrophoresis. The gel was transferred to a nitrocellulose membrane by a conventional method, and the nitrocellulose membrane was immersed in the culture supernatant of hybridoma M-1 for 1 hour.
50 mM Tris containing 05% (v / v) Tween 20
Excess antibody was removed by washing with a hydrochloric acid buffer (pH 7.5). PBS containing rabbit-derived anti-rat Ig antibody whose nitrocellulose membrane is labeled with horseradish peroxidase
In a 50 mM Tris-HCl buffer (pH 7.0) containing 0.05% (v / v) Tween 20.
After washing in 5), the plate was immersed in 50 mM Tris-HCl buffer (pH 7.5) containing 0.005% (v / v) hydrogen peroxide and 0.3 mg / ml diaminobenzidine to develop color.

At the same time, a system using a purified protein derived from mouse hepatocytes obtained by the method of Example 4 described below or recombinant human interleukin 12 was provided in place of the purified protein,
Controls were treated as described above. The molecular weight markers include bovine serum albumin (67,000 daltons),
Ovalbumin (45,000 daltons), carbonic anhydrase (30,000 daltons), trypsin inhibitor (20,100 daltons) and α-lactalbumin (14,400 daltons) were used. The results are shown in FIG.

As can be seen from the results in FIG. 2, the monoclonal antibody M-1 mAb was obtained from the purified protein obtained by the method of Example 1-2 (lane 1) and the purified protein obtained by the method of Example 4 (lane 2). ) Specifically, and did not react at all with human interleukin 12 (lane 3). This means that the monoclonal antibody of the present invention
Regardless of the production method, it supports that it specifically reacts with proteins having specific physicochemical properties.

[0051]

Example 3 <Purification of protein by immunoaffinity chromatography>

[0052]

Example 3-1 <Preparation of Gel for Immunoaffinity Chromatography> 80 mg of the monoclonal antibody M-1 mAb obtained by the method of Example 2-1 was added to 0.5 mg
0.1 M borate buffer (pH 8.
Dialysis was performed overnight at 4 ° C against 5). "CNBr-activated Sepharose 4B" manufactured by Pharmacia as a water-insoluble carrier
4 g was swollen in a 1 mM aqueous hydrochloric acid solution, washed in the order of a fresh same aqueous hydrochloric acid solution and a 0.1 M borate buffer (pH 8.5) containing 0.5 M sodium chloride, and then about 10 ml of the above monoclonal antibody aqueous solution was added. The mixture was stirred gently at room temperature for 2 hours and at 4 ° C. overnight. Thereafter, the gel was washed with a 1 M aqueous solution of ethanolamine (pH 8.0), and further, 0.1 M borate buffer (pH 8.5) containing 0.5 M sodium chloride and 0.1 M acetate buffer containing 0.5 M sodium chloride. The step of washing with the liquid (pH 4.0) in this order was repeated five times, and finally, the gel was washed with PBS to obtain a gel for immunoaffinity chromatography. Analysis by a conventional method revealed that about 6 mg of the monoclonal antibody M-1 mAb was bound per 1 ml of the gel.

[0053]

Example 3-2 <Purification of Protein by Immunoaffinity Chromatography> 10 ml of the gel for immunoaffinity chromatography obtained in Example 3-1 was packed in a cylindrical shape inside a plastic cylindrical tube, and washed with PBS. Phenyl Sepharose elution fraction 1 containing about 0.1 mg / ml of the protein obtained by the method of Example 1-2
0 ml was loaded. After washing with fresh PBS, a 35 mM ethylamine aqueous solution (pH 10.8) was passed through the column, and a fraction capable of inducing IFN-γ was collected. The collected fractions were pooled, concentrated, and then measured for IFN-γ inducing activity and protein content. As a result, it was found that a purified protein having a purity of 95% or more was obtained in a yield of almost 100% per raw material.

[0054]

Example 4 <Purification of Protein by Immunoaffinity Chromatography> Female CD-1 mouse 60 at the age of 8 weeks
No corynebacterium parvum (ATC)
C11827) was heated at 60 ° C. for 1 hour, and 1 mg / animal of dead cells was injected and administered. After breeding for 7 days in a usual manner, purified lipopolysaccharide derived from E. coli was injected intravenously at 1 μg / animal. . One to two hours later, the mouse was sacrificed, and after collecting the heart blood, the liver was excised and crushed and extracted in an eight-fold volume of 50 mM phosphate buffer (pH 7.3) using a homogenizer. The extract was centrifuged at about 8,000 rpm for 20 minutes, and about 9 liters of the obtained supernatant was added with 50 mM phosphate buffer (pH 7.3) containing saturated ammonium sulfate so that ammonium sulfate became 45% saturated. After standing for 18 hours, the mixture was centrifuged at about 8,000 rpm for 30 minutes to obtain about 19 l of a supernatant containing the protein.

The supernatant was pre-equilibrated with 50 mM phosphate buffer (pH 7.3) containing 1 M ammonium sulfate, and “Phasepharose” manufactured by Pharmacia was about 4.6.
After washing the column with fresh identical buffer, 50 mM phosphate buffer (pH 7.3) was added to the SV under a gradient of ammonium sulfate from 1 M to 0.2 M.
The solution was passed at 0.57. 0.8M ammonium sulfate concentration
Fraction containing the protein eluted at around 4.8 l
Was collected, membrane-concentrated, and 20 mM phosphate buffer (pH 6.
After dialyzing against 5) at 4 ° C. for 18 hours, the mixture was loaded on a column of about 250 ml of “DEAE-Sepharose” manufactured by Pharmacia which had been equilibrated with 20 mM phosphate buffer (pH 6.5) in advance. After washing the column with fresh identical buffer,
Under a concentration gradient of sodium chloride rising from
20 mM phosphate buffer (pH 6.5) was added to the column with SV1.
When the solution was passed through step 2, the protein was eluted at a sodium chloride concentration of about 0.13 M.

About 260 ml of the eluate containing the protein was collected, concentrated, and purified in the same manner as in Example 3. As a result, the purified protein having a purity of 95% or more was almost 100% per raw material.
In the yield.

[0057]

Example 5 <Detection of Protein by Enzyme Immunoassay> Rabbits were immunized with the purified protein obtained by the method of Example 1-2 according to a conventional method, and blood was collected to isolate an IgG antibody. . This IgG antibody was dissolved in PBS to a concentration of 20 μg / ml and dispensed into a 96-well microplate at 100 μl / well. After incubating the microplate at room temperature for 3 hours,
The gG solution was removed, PBS containing 1% (w / v) bovine serum albumin was added at 200 μl / well, and the mixture was allowed to stand at 4 ° C. overnight.

The PBS was removed from the microplate.
After washing with PBS containing 05% (v / v) Tween 20,
0.5% of purified protein obtained by the method of Example 1-2
(W / v) Dilute to an appropriate concentration with PBS containing bovine serum albumin, add 100 μl / well, and shake.
The reaction was performed at room temperature for 2 hours. After washing with PBS containing 0.05% (v / v) Tween 20, 100 μl / well of a biotin-labeled monoclonal antibody M-1 mAb was added thereto, and reacted at room temperature for 2 hours while shaking.
% (V / v) after washing with PBS containing Tween 20
100 μl / well of a complex of horseradish peroxidase and streptavidin was added, and the mixture was further reacted at room temperature with shaking for 2 hours. 0.05% (v / v)
After washing with PBS containing Tween 20, the activity of horseradish peroxidase bound to the purified protein was measured as an absorbance at a wavelength of 492 nm using o-phenylenediamine as a substrate. Table 1 shows the results.

[0059]

[Table 1]

As is evident from the results in Table 1, at least about 50 to 2,000 p.
g / ml of the protein can be accurately detected.

[0061]

Example 6 <Detection of protein by radioimmunoassay> After immunizing a rabbit with the purified protein obtained by the method of Example 1-2 according to a conventional method, blood was collected,
IgG antibodies were isolated. This IgG antibody is adsorbed to polystyrene beads for radioimmunoassay by a conventional method,
In PBS containing 2% (w / v) bovine serum albumin, 4
The mixture was allowed to stand at 0 ° C. overnight to obtain a solid phase antibody.

Each of the solid-phase antibodies was placed in a test tube one by one, and the purified protein obtained by the method of Example 1-2 was 0.5% (w
/ V) The mixture was appropriately diluted with PBS containing bovine serum albumin to an appropriate concentration, added in 0.2 ml portions, and allowed to stand at 4 ° C for 4 hours. After washing the solid phase antibody with PBS containing 0.05% (v / v) Tween 20 and 0.5% (w / v) bovine serum albumin, the monoclonal antibody M- obtained by the method of Example 2-1 was used. 1 mAb was labeled with ¹²⁵ I by a conventional method to give 0.1 mAb.
Each 2 ml (1 × 10 ⁵ cpm) was added and left at 4 ° C. overnight. Remove excess labeled antibody and remove 0.05% (v / v)
After washing with Tween 20 and PBS containing 0.5% (w / v) bovine serum albumin, the radioactivity of the beads was measured with a gamma counter. Table 2 shows the results.

[0063]

[Table 2]

As is apparent from the results in Table 2, at least about 100 to 1,200
pg / ml of the protein can be accurately detected.

[0065]

As described above, the monoclonal antibody of the present invention specifically reacts with a protein that induces the production of IFN-γ in immunocompetent cells. Therefore,
The monoclonal antibodies of the present invention will have a wide variety of uses for purifying and detecting such proteins. Such a useful monoclonal antibody of the present invention can be easily obtained in a desired amount by a production method using a hybridoma.

The present invention exerts such remarkable functions and effects, and it can be said that the present invention is a very significant invention to contribute to the art.

[0067]

[Sequence List] SEQ ID NO: 1 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Fragment type: Middle fragment Sequence Ile Ile Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln 1 5 10 15 Ser Asp Leu Ile Phe Phe Gln Lys 20 25

SEQ ID NO: 2 Sequence length: 18 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: middle fragment Sequence Gln Pro Val Phe Glu Asp Met Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro 1 5 10 15 Gln

SEQ ID NO: 3 Sequence length: 157 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn Asp 1 5 10 15 Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met Thr Asp 20 25 30 Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile Tyr Met Tyr 35 40 45 50 Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser Val Lys Asp Ser 55 60 65 Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile Ser Phe Glu Glu Met 70 75 80 85 Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser Asp Leu Ile Phe Phe Gln 90 95 100 Lys Arg Val Pro Gly His Asn Lys Met Glu Phe Glu Ser Ser Leu Tyr Glu 105 110 115 Gly His Phe Leu Ala Cys Gln Lys Glu Asp Asp Ala Phe Lys Leu Ile Leu 120 125 130 135 Lys Lys Lys Asp Glu Asn Gly Asp Lys Ser Val Met Phe Thr Leu Thr Asn 140 145 150 Leu His Gln Ser 155

SEQ ID NO: 4 Sequence length: 471 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism name: mouse Tissue name: liver Sequence characteristics Sequence Symbol indicating: mat peptide Location: 1..471 Method for determining characteristics: S sequence AAC TTT GGC CGA CTT CAC TGT ACA ACC GCA GTA ATA CGG AAT ATA AAT 48 Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn 1 5 10 15 GAC CAA GTT CTC TTC GTT GAC AAA AGA CAG CCT GTG TTC GAG GAT ATG 96 Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met 20 25 30 ACT GAT ATT GAT CAA AGT GCC AGT GAA CCC CAG ACC AGA CTG ATA ATA 144 Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile 35 40 45 TAC ATG TAC AAA GAC AGT GAA GTA AGA GGA CTG GCT GTG ACC CTC TCT 192 Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser 50 55 60 GTG AAG GAT AGT AAA AYG TCT ACC CTC TCC TGT AAG AAC AAG ATC ATT 240 Val Lys Asp Ser Lys Xaa Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile 65 70 75 80 TCC TTT GAG GAA ATG GAT CCA CCT GAA AAT ATT GAT GAT ATA CAA AGT 288 Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser 85 90 95 GAT CTC ATA TTC TTT CAG AAA CGT GTT CCA GGA CAC AAC AAG ATG GAG 336 Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu 100 105 110 TTT GAA TCT TCA CTG TAT GAA GGA CAC TTT CTT GCT TGC CAA AAG GAA 384 Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu 115 120 125 GAT GAT GCT TTC AAA CTC ATT CTG AAA AAA AAG GAT GAA AAT GGG GAT 432 Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp 130 135 140 AAA TCT GTA ATG TTC ACT CTC ACT AAC TTA CAT CAA AGT 471 Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser 145 150 155

[Brief description of the drawings]

FIG. 1 is a diagram showing the structure of recombinant DNA pKGFM5.

FIG. 2 shows the monoclonal antibody M-1 mA according to the present invention.
FIG. 4 is a western blotting diagram showing the reactivity of b with various proteins.

[Description of Signs] cDNA encoding KGFM5 cDNA protein Ptactac promoter rrnBT1T2 Transcription termination region of ribosomal RNA operon AmpR Ampicillin resistance gene pBR322ori Replication origin in E. coli

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/53 G01N 33/577 B 33/577 A61K 39/395 U // A61K 38/21 N 39/395 C12N 15/00 C A61K 37/66 C12N 5/10 C12N 5/00 B

Claims (12)

[Claims] [Claim 1] SEQ ID NO: 3 in the sequence listing (provided that
“Xaa” shall mean methionine or threonine. Or a protein or an antigenic fragment thereof which has an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence thereof, and induces the production of interferon-γ in immunocompetent cells. An antibody that immunoreacts with the protein obtained through a step of immunizing a mammal with the protein. 2. The antibody according to claim 1, which is obtained through a step of collecting a body fluid of a mammal. 3. The antibody according to claim 2, wherein the body fluid is blood or ascites. 4. SEQ ID NO: 3 in the sequence listing (provided that
“Xaa” shall mean methionine or threonine. ) Or a protein having an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence thereof, which induces the production of interferon-γ in immunocompetent cells, or an antigenic fragment thereof. A method for producing an antibody, comprising the step of immunizing a mammal using the method. 5. The method for producing an antibody according to claim 4, further comprising a step of collecting a body fluid of a mammal. 6. The antibody is subjected to salting out, dialysis, filtration, concentration, centrifugation, fractional precipitation, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography,
The method for producing an antibody according to claim 4, wherein the antibody is collected by affinity chromatography, gel electrophoresis, and / or isoelectric focusing. 7. A step of contacting the antibody according to any one of claims 1 to 3 with a mixture containing a protein having the following physicochemical properties and a contaminant to adsorb the protein to the antibody;
A method for purifying a protein, comprising a step of desorbing the adsorbed protein from the antibody. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces the production of interferon-γ in immunocompetent cells. 8. The antibody according to any one of claims 1 to 3, wherein the amino acid sequence shown in SEQ ID NO: 3 (where "Xaa" means methionine or threonine) or the amino acid sequence thereof is used. In the amino acid sequence, 1
Or having a plurality of amino acids deleted, substituted or added, having an amino acid sequence, and adsorbing the protein to the antibody by contacting with a mixture containing a protein and a contaminant that induces the production of interferon-γ in immunocompetent cells. ,
A method for purifying a protein, comprising a step of desorbing the adsorbed protein from the antibody. 9. The method for purifying a protein according to claim 7, wherein the antibody is bound to a water-insoluble carrier. 10. A method for detecting a protein, which comprises bringing the antibody according to claim 1 into contact with a test sample and detecting a protein having the following physicochemical properties by an immune reaction. (1) Molecular weight It shows a molecular weight of 19,000 ± 5,000 daltons when measured by SDS-polyacrylamide gel electrophoresis or gel filtration. (2) Isoelectric point When measured by the chromatofocusing method, 4.8 ± 1.
0 indicates the isoelectric point. (3) Partial amino acid sequence It has a partial amino acid sequence shown in SEQ ID NOS: 1 and 2 in the sequence listing. (4) Biological action Induces the production of interferon-γ in immunocompetent cells. 11. An antibody according to any one of claims 1 to 3, which is brought into contact with a test sample, and subjected to an immune reaction, whereby SEQ ID NO: 3 (where "Xaa" means methionine or threonine) in the sequence listing. ), Or an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in FIG.
A method for detecting a protein that detects a protein that induces the production of a protein. 12. The method for detecting a protein according to claim 10, wherein the antibody is labeled with a radioactive substance, an enzyme, and / or a fluorescent substance.