JPH08217798A - Monoclonal antibody - Google Patents

Abstract

PURPOSE: To obtain the new antibody for detection, etc., of production-inducing protein of IFN-γ specific to a protein inducing production of interferon-γ (IFN-γ) and having antivirus, antitumor and immunemodulating action in an immunocompetent cell. CONSTITUTION: This monoclonal antibody is a new monoclonal antibody having 19000±5000 dalton when measured by SDS-polyacrylamide gel electrophoresis or gel permeation method and 4.8±1.0 isoelectric point measured by chromatofocusing method and has partial amino acid sequences expressed by formulas I and II and is specific to a protein having physicochemical properties for inducing production of IFN-γ in an immunocompetent cell and useful for detection, etc., of the protein. The monoclonal antibody is obtained by inoculating the protein inducing IFN-γ production into a rat abdominal cavity together with adjuvant by injection, collecting the spleen cell after final inoculation, fusing the cell with a myeloma cell, culturing the fused cell in HAT culture medium to afford hybridoma, cloning the hybridoma and culturing the cloned product.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel monoclonal antibody, and more specifically to a protein that induces the production of interferon-γ (hereinafter abbreviated as "IFN-γ") in immunocompetent cells. It relates to specific monoclonal antibodies.

[0002]

2. Description of the Related Art IFN-γ is known as a protein having an antiviral action, an antitumor action and an immunomodulatory action, and is said to be produced by immunocompetent cells stimulated by an antigen or a mitogen. Because of these biological effects, IFN-
γ has been put into practical use as an antitumor agent from the beginning of its discovery, and now clinical trials are vigorously carried out as a therapeutic agent for general malignant tumors including brain tumors. IFN-γ currently available is a natural type IFN produced by immunocompetent cells.
-Γ and recombinant IFN-γ produced by a transformant obtained by introducing DNA encoding IFN-γ collected from immunocompetent cells into Escherichia coli. Is administered as "foreign IFN-γ".

Of these, natural type IFN-γ is usually produced by culturing immunocompetent cells that have been cultured into culture cells in a culture medium containing an IFN-γ inducer, and purifying the culture. In this method, the type of IFN-γ inducer is IFN
-It is said that it has a great influence on the production amount of γ, the ease of purification, and the safety of the product. Usually, concanavalin A, lentil lectin, pokeweed lectin, endotoxin, lipopolysaccharide, etc. Mitogen is frequently used. However, all of these substances have a variety of molecules, their quality is likely to vary 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 inducing ability. In addition, many of the above substances show significant side effects when administered to living organisms, and even show toxicity depending on the substance, and it is extremely difficult to directly administer IFN-γ production to living organisms. there were.

When the present inventors have studied cytokines produced by mammalian cells, they found that there is a completely unknown novel substance that induces IFN-γ production in the liver of mice. It was When this substance was isolated by combining various purification methods centered on column chromatography and its properties and properties were investigated, it was found that the substance is a protein and has 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 shows the isoelectric point. (3) Partial amino acid sequence It has the partial amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing. (4) Biological action Induces IFN-γ production in immunocompetent cells.

A protein having such physicochemical properties has not been known yet, and it is considered to be a novel substance. Then, the inventors of the present invention continued to diligently search mouse hepatocytes, and succeeded in isolating the DNA encoding this protein. Upon decoding, it was revealed that this DNA consisted 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 E. coli and expressed, the protein was produced in good yield in the culture. The above findings are disclosed in Japanese Patent Application No. 6-184162 by the same applicant.

[0006] As described above, the protein has the property of inducing the production of IFN-γ in immunocompetent cells, and is a general-purpose IFN-γ inducer, and further an antiviral agent,
A wide variety of uses are expected as antitumor agents, antibacterial agents, immunomodulators, platelet proliferative agents, and the like. Generally, when a bioactive protein is to be used in combination with a drug, it is essential to develop a method capable of highly and efficiently purifying the protein and a method of assaying many test samples at once. Although the best material that enables such purification and assay is a monoclonal antibody, a monoclonal antibody specific to the protein has not been established yet.

[0007]

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

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

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

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

Still another object of the present invention is to provide a method for detecting the protein of interest using such a monoclonal antibody.

[0012]

The present invention solves the above-mentioned first problem by 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 shows the isoelectric point. (3) Partial amino acid sequence It has the partial amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing. (4) Biological action Induces interferon-γ production in immunocompetent cells.

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

The present invention has the above-mentioned third object, which 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. This is solved by a method for producing a monoclonal antibody.

The present invention is directed to the fourth object, wherein the monoclonal antibody is brought into contact with a mixture containing the protein and contaminants to adsorb the protein to the monoclonal antibody, and the adsorbed protein is desorbed from the monoclonal antibody. The problem is solved by a method for purifying a protein comprising steps.

The present invention solves the above-mentioned fifth problem by a method for detecting a protein in which a test sample is contacted with the monoclonal antibody and the protein is detected by an immune reaction.

[0017]

The monoclonal antibody of the present invention specifically reacts with a protein having specific physicochemical properties.

The hybridoma of the present invention produces such a monoclonal antibody when cultured in vitro.

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

According to the purification method of the present invention,
The protein is highly purified and efficiently collected from a mixture containing the protein and contaminants.

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

The monoclonal antibody referred to in the present invention is
It includes all monoclonal antibodies specific to the protein having the above-mentioned physicochemical properties, and its source and origin,
Class does 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 mentioned. An amino acid sequence homologous to the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing means one or more amino acids in the amino acid sequence of SEQ ID NO: 3 without altering the physicochemical properties described above. Of the amino acid sequence of SEQ ID NO: 3, one or more amino acids added to the N-terminal and / or C-terminal, and one or more amino acids at the N-terminal and / or C-terminal are deleted. Includes what you have lost.

The monoclonal antibody of the present invention can be obtained by using such protein or an antigenic fragment thereof as an antigen. Specifically, for example, a hybridoma of antibody-producing cells collected from a mammal that has been immunized with such an antigen and cells of mammalian origin capable of infinite growth is prepared, and the monoclonal antibody of the present invention is prepared from the hybridoma. It can be obtained by selecting a hybridoma clone that can be produced and culturing it in vitro or in vivo.

The protein which can be an antigen is, for example, as disclosed in Japanese Patent Application No. 6-184162, collected from mouse hepatocytes, 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 transformants into which the DNA encoding the sequence has been introduced, and they are usually used in the state of being completely purified or partially purified. To obtain an antigenic fragment, these completely purified products or partially purified products may be chemically or enzymatically decomposed, or peptides 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 alone or together with an appropriate adjuvant is inoculated into a mammal by intravenous, intradermal, subcutaneous or intraperitoneal injection, and the animal is bred for a certain period of time. 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. Usually, rodents such as rats, mice, and hamsters are used, and the optimum one is selected in consideration of compatibility with cells of mammalian origin capable of infinite growth described later. Depending on the type and size of mammal, the inoculation dose of the antigen is usually about 5 to 500 μg / animal,
This 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 splenocytes as antibody-producing cells.

Next, the antibody-producing cells thus obtained are fused with cells of mammalian origin capable of infinite growth to obtain a cell fusion product containing the target hybridoma. In general, cells of mammalian origin capable of indefinite growth normally contain P3-NS1-A.
g4-1 cells (ATCC TIB18), P3-X63
-Ag8 cells (ATCC TIB9) and SP2 / 0-
A cell line derived from a mouse myeloma such as Ag14 cell (ATCC CRL1581) or a mutant strain thereof is used. For the cell fusion, for example, a fusion accelerator including polyethylene glycol or Sendai virus, or a conventional method using an electric pulse is used. For example, the antibody-producing cells can be infinitely grown in a fusion medium containing the fusion accelerator. Mammalian cells are suspended at a ratio of about 1: 1 to 1:10, and in this state, they are incubated at about 30-40 ° C. for about 1-5 minutes. Examples of the fusion medium include MEM medium and R
Although generally used media such as PMI1640 medium and Iscove's modified Dulbecco's medium can be used, it is desirable to exclude sera such as bovine serum.

In order to select the desired hybridoma, first, the cell fusion product obtained as described above is transferred to a selection medium such as 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. For example, S. Tomiyama
Tamei Ando "Monoclonal Antibody Experiment Manual", 199
Published by Kodansha Scientific, 1st year, 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 monocloned hybridoma of the present invention.

The monoclonal antibody of the present invention can be obtained by culturing such hybridoma in vitro. For culturing, a conventional method for culturing cells of mammalian origin is used.For example, when culturing in an in vitro culture medium, from the culture, on the other hand, transplanted to a warm-blooded animal other than human, When cultured in the body, the monoclonal antibody is collected from the ascites and / or blood. The hybridoma M-1 described below is characterized in that it has a high monoclonal antibody-producing ability and that it can be easily cultured in vivo and in vivo. For harvesting monoclonal antibodies from cultures, ascites or blood, conventional methods in the art for purifying antibodies in general are used. Examples of individual methods include 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 is mentioned, and these are applied in combination as needed. The purified monoclonal antibody is
After that, it is concentrated and dried to be 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 a step of contacting the monoclonal antibody of the present invention with a mixture of the protein and a contaminant substance including a contaminant protein other than the protein to adsorb only the protein to the monoclonal antibody, and the adsorbed protein Comprising the step of desorbing 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 a state of being bound to a gel-like water-insoluble carrier,
When the water-insoluble carrier is column-shaped packed in a cylindrical tube or the like, and, for example, a mouse hepatocyte extract or a transformant culture solution or a partially purified product thereof is passed, substantially only the protein of interest is obtained. Adsorb 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 IgG class is used, the pH on the acidic side, usually pH 2 to 3, On the other hand, when using a monoclonal antibody belonging to the IgM class, elution is performed at an alkaline pH, 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 associated with IFN in immunocompetent cells.
Since it has the property of inducing the production of -γ, the obtained purified protein has a sensitivity to IFN-γ as an inducer in the production of IFN-γ by the cell culture method,
For example, it is useful as a therapeutic / prophylactic agent against viral diseases such as AIDS and Condyloma acuminata, renal cancer, granulomas, mycosis fungoides, malignant tumors such as brain tumors, and immune diseases such as rheumatoid arthritis and allergic diseases. When the protein also has the property of enhancing the cytotoxicity of killer cells, it may be used in combination with interleukin 2 or tumor necrosis factor to include solid cancers such as lung cancer, kidney cancer and breast cancer by adoptive immunotherapy. Remarkably effective in improving the therapeutic effects and side effects in the treatment of malignant tumors.

The monoclonal antibody of the present invention has a wide range of applications in various fields that require detection of the protein. That is, when a labeled immunoassay such as a radioimmunoassay, an enzyme immunoassay, or a fluorescent immunoassay is applied to the monoclonal antibody of the present invention, the protein in a test sample can be qualitatively or quantitatively analyzed rapidly and accurately. In such an 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 exhibits an immune reaction, if the immune reaction is measured using these labeling substances as an index, a very small amount of the protein in the test sample can be accurately detected. be able to. The labeled immunoassay is characterized in that a large number of test samples can be analyzed at one time, the time and labor required for the analysis are small, and the analysis is highly accurate as compared with the bioassay. Therefore, the detection method according to the present invention is extremely useful for process control and quality control of products when producing the protein. Since the present invention does not relate to the labeling of monoclonal antibodies or the labeling assay itself, a detailed description thereof will be omitted. For example, P. Thyssen, translated by Eiji Ishikawa, "Enzyme Immunoassay", 198
In 1989, Tokyo Kagaku Dojin, pp. 196 to 348, various methods 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 modified in various ways. Needless to say, the present invention should not be limited to these examples in view of the state of the art.

[0033]

Example 1 Preparation of hybridoma M-1

[0034]

Example 1-1 Preparation of transformant KGFM5 0.5
8 μl of 25 mM magnesium chloride in a ml reaction tube, 1
10 μl of 0 × PCR buffer, 1 μl of 25 mM dNTP mix, 2.5 units / μl AmpliTac DNA
Recombinant containing 1 μl of polymerase, DNA having the nucleotide 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. 5 ng of 1 ng of DNA was chemically synthesized based on the amino acid sequences near the N-terminal and C-terminal in SEQ ID NO: 3 in the sequence listing.
-GAGGAATTCTGGAGGAAGGTTACCA
TGAACTTTTGGGCCGAACTTC-3 'and 5'
-GCGAAAGCTTCTAACTTTGATGTA
Appropriate amounts of sense primer and antisense primer having the base sequence represented by AG-3 'were added, and the volume was adjusted to 100 µl with sterile distilled water. The mixture was incubated at 94 ° C. for 1 minute, 43 ° C. for 1 minute, 72 ° C. for 1 minute in this order, and the cycle was repeated 3 times, followed by 94 ° C. for 1 minute and 60 ° C. 37 cycles of 1 minute incubation at 70 ° C in this order
The PCR reaction was repeated twice, and an Eco RI cleavage site and a Hind III cleavage site were provided at the 5'end and 3'end of the base sequence shown in SEQ ID NO: 4, respectively.

PCR product, Stratagene plasmid vector "pCR-Script SK (+)" 10n
g was ligated with DNA ligase by a conventional method to prepare recombinant DN.
It was designated as A and was introduced into Escherichia coli "XL-1 Blue MRF'Kan" strain manufactured by Stratagene by the competent cell method for transformation. 50 μg / ml of transformant
L-broth medium (pH 7.2) containing ampicillin was inoculated and cultured with shaking at 37 ° C. for 18 hours, and then the culture was centrifuged to collect transformants.
The method was applied to isolate recombinant DNA. This recombinant DN
When part of A was taken and analyzed by the dideoxy method,
It was confirmed that the DNA having the nucleotide sequence represented by SEQ ID NO: 4 in the sequence listing was linked, and that the desired DNA was correctly gene-amplified in the PCR reaction.

Then, the remaining recombinant D is prepared according to a conventional method.
After cutting NA with restriction enzymes Eco RI and Hind III, DNA ligation kit "DNA" manufactured by Takara Shuzo
Ligation Kit Version 2 ",
0.1 μg of the obtained Eco RI-Hind III DNA fragment and a plasmid vector "pKK223-3" 10 manufactured by Pharmacia, which had been cleaved with the same restriction enzyme in advance.
ng was reacted at 16 ° C. for 30 minutes and ligated to obtain a replicable recombinant DNA “pKGFM5”. Escherichia coli Y1090 strain (ATCC37197) was transformed with this recombinant DNA pKGMF5 by the competent cell method, and the obtained transformant "KGFM5" was inoculated into L-broth medium (pH 7.2) containing 50 µ / ml ampicillin. Then
The cells were cultured at 37 ° C for 18 hours with shaking. The culture was centrifuged to collect a transformant, and a standard SDS-alkaline method was applied to a part of the transformant to extract the recombinant DNA pKGFM5. As a result of analysis by the dideoxy method, as shown in FIG. 1, in the recombinant DNA pKGFM5, KGFM5 cD of 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 μ / ml was autoclaved.
Sterilize L-broth medium (pH 7.2) containing
After cooling to 1, the transformant KGFM prepared in Example 1-1.
5 was inoculated and seeded under shaking at the same temperature for 18 hours.
18 liters of fresh identical medium in a 20 liter jar fermentor
After sterilization in the same manner and cooling to 37 ° C., 1% (v / v) of the seed culture obtained above was inoculated, and aeration-agitation culture was carried out at the same temperature for 8 hours. The culture was centrifuged to collect the bacterial cells,
Mixture containing 0 mM sodium chloride, 16 mM disodium hydrogen phosphate and 4 mM sodium dihydrogen phosphate (pH 7.
After suspending in 3) and ultrasonically disrupting, the disrupted cells were removed by centrifugation and the supernatant was collected.

Ammonium sulfate was added to this supernatant under ice cooling.
Add up to 0% (w / v), dissolve evenly, leave for a while,
After centrifugation, the supernatant was collected. Ammonium sulfate was added to this newly formed supernatant to 85% (w / v), the mixture was stirred at 4 ° C. for 25 hours, centrifuged, and the precipitate containing the protein was collected. This precipitate was dissolved in 150 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate, and the solution was preliminarily equilibrated with 10 mM phosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate. -Sepharose "column was loaded, the column was washed with the same fresh buffer solution, and then 10 mM phosphate buffer solution (pH 6.6) was passed under a gradient of ammonium sulfate concentration decreasing from 1.5 M to 0 M.

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

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

When analyzed in accordance with the method described in Japanese Patent Application No. 6-184162, the purified protein had the following physicochemical properties. That is, when subjected to SDS-polyacrylamide gel electrophoresis under non-reducing conditions, it shows a major band capable of inducing IFN-γ at a position corresponding to a molecular weight of 19,000 ± 5,000 daltons, while 4.8 ± when chromatofocused. The isoelectric point was shown at 1.0. In addition, the two types of peptide fragments obtained by digestion with trypsin 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] The purified protein obtained by the method of Example 1-2 was intraperitoneally administered to 10-week-old SD rats in an amount of 20 µm with complete Freund's adjuvant.
It was inoculated by injection at a rate of g / animal. Then, 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 excised and dispersed to obtain splenocytes.

This spleen cell and mouse myeloma-derived SP2 /
37 0-Ag14 cells (ATCC CRL1581)
Serum-free RPMI 1640 medium (pH 7.2) pre-heated to ℃ was used for cell density 3 × 10 ⁴ cells / ml, respectively.
And 1 × 10 ⁴ cells / ml were suspended, and the precipitate was collected after centrifugation. Average molecular weight of 1,5
Serum-free RPMI 1640 medium containing 50% (w / v) polyethylene glycol of 00 daltons (pH 7.
2) Add 1 ml dropwise over 1 minute, incubate at 37 ° C for 1 minute, add serum-free RPMI1640 medium (pH 7.2) dropwise until the total volume reaches 50 ml, centrifuge and precipitate the precipitate. It was collected. HAT this precipitate
Suspend in medium and add 200 to 96-well microplate.
μl / well was dispensed and incubated at 37 ° C. for 1 week to select hybridomas.

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

[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 to 5% so that the cell density becomes about 1 × 10 ⁶ cells / ml.
(V / v) Suspended in RPMI1640 medium (pH 7.2) supplemented with bovine serum, while expanding the culture scale,
Incubated at 37 ° C in a 5% CO ₂ incubator. At the time when the desired cell density was reached, the hybridoma M-1 was immunosuppressed with a rabbit-derived anti-hamster thymocyte antibody in advance, and 0.5 ml / mouse of pristane was intraperitoneally injected to the hamster at the age of 5 weeks. 1 x 10 ⁷ in the abdominal cavity of
Individual / animal injection was inoculated, and the animals were bred for 1 week by a usual method.

Ascites fluid was collected from the hamster, and the ascites was collected using PBS.
After doubling the dilution, ammonium sulfate was added to 50% saturation, the mixture was allowed to stand at 4 ° C. for 24 hours, centrifuged, and the precipitate was collected. The precipitate was dialyzed against a 20 mM potassium dihydrogen phosphate aqueous solution (pH 6.7) at 4 ° C. overnight, and then loaded on a hydroxyapatite column equilibrated with a fresh aqueous solution of the same, and the concentration was changed from 20 mM to 300 mM.
Aqueous potassium dihydrogen phosphate solution (pH
When 6.7) was passed through, an aqueous solution containing the monoclonal antibody M-1 mAb of the present invention was obtained. The yield was about 5 mg per hamster. This monoclonal antibody M-1m was analyzed by a conventional method.
Abs belonged to the IgM class.

[0048]

Example 2-2 Western Blotting Analysis 1 μg of the purified protein obtained by the method of Example 1-2 was added to a mixed solution of 100 mg of dithiothreitol and 0.5 ml of a 10% (w / v) SDS aqueous solution and 1 ml of glycerol, After incubating at 37 ° C. for 1 hour, SDS-polyacrylamide gel electrophoresis was performed. The gel was transferred to a nitrocellulose membrane by a conventional method, the nitrocellulose membrane was immersed in the culture supernatant of hybridoma M-1 for 1 hour, and
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
The reaction is performed by immersing in 50% tris-hydrochloric acid buffer solution (pH 7.) containing 0.05% (v / v) Tween 20 for 1 hour.
After washing with 5), the sample was immersed in 50 mM Tris-hydrochloric acid buffer solution (pH 7.5) containing 0.005% (v / v) hydrogen peroxide and 0.3 mg / ml diaminobenzidine to develop color.

At the same time, instead of the purified protein, a system using the purified protein derived from mouse hepatocytes or the recombinant human interleukin 12 obtained by the method of Example 4 described below is provided,
The same treatment as above was used as a control. The molecular weight markers include bovine serum albumin (67,000 dalton),
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 Figure 2.

As shown in the results of FIG. 2, the monoclonal antibody M-1mAb was purified protein obtained by the method of Example 1-2 (lane 1) and purified protein obtained by the method of example 4 (lane 2). ) Specifically reacted with human interleukin 12 (lane 3). This means that the monoclonal antibody of the present invention
It is proved that regardless of the production method, it specifically reacts with a protein having specific physicochemical properties.

[0051]

Example 3 Purification of protein by immunoaffinity chromatography [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 taken to 0.5 M.
0.1 M borate buffer containing sodium chloride (pH 8.
It was dialyzed against 5) overnight at 4 ° C. "CNBr-activated Sepharose 4B" manufactured by Pharmacia as a water-insoluble carrier
4 g was swollen in a 1 mM hydrochloric acid aqueous solution, washed with a fresh same hydrochloric acid aqueous solution and a 0.1 M borate buffer solution (pH 8.5) containing 0.5 M sodium chloride in this order, and then about 10 ml of the above monoclonal antibody aqueous solution was added. The mixture was gently stirred at room temperature for 2 hours and at 4 ° C. overnight. Then, the gel was washed with a 1 M aqueous solution of ethanolamine (pH 8.0), and further, a 0.1 M borate buffer solution (pH 8.5) containing 0.5 M sodium chloride and a 0.1 M acetate buffer solution containing 0.5 M sodium chloride. The step of washing with the liquid (pH 4.0) in this order was repeated 5 times, and finally with PBS to obtain a gel for immunoaffinity chromatography. When analyzed by a conventional method, 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 column made of plastic in a column shape and washed with PBS. Phenylsepharose elution fraction 1 containing about 0.1 mg / ml of the protein of interest obtained by the method 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 to collect a fraction capable of inducing IFN-γ. The collected fractions were pooled, concentrated, and then the IFN-γ-inducing activity and the protein content were measured. As a result, it was found that a purified protein having a purity of 95% or higher was obtained in a yield of almost 100% per raw material.

[0054]

Example 4 Purification of protein by immunoaffinity chromatography Example 4 Purification of protein by immunoaffinity chromatography 8-week-old female CD-1 mouse 600
Corynebacterium parvum (ATCC
1 187) was heated at 60 ° C. for 1 hour and injected with 1 mg / mouse of the killed cells, and after 7 days of normal rearing, the purified lipopolysaccharide derived from Escherichia coli was intravenously injected at 1 μg / mouse. . After 1 to 2 hours, the mice were sacrificed, the blood was collected from the heart, the liver was removed, and 8 volumes of 50 mM phosphate buffer (p
H7.3) was crushed and extracted by a homogenizer. The extract was centrifuged at about 8,000 rpm for 20 minutes, and about 9 l of the obtained supernatant was added with 50 mM phosphate buffer (pH 7.3) containing saturated ammonium sulfate so that the ammonium sulfate became 45% saturated. After standing for 18 hours, centrifugation was performed at about 8,000 rpm for 30 minutes to obtain about 19 l of a supernatant containing the protein.

This supernatant was previously equilibrated with 50 mM phosphate buffer (pH 7.3) containing 1 M ammonium sulfate, "Phenyl Sepharose" manufactured by Pharmacia, about 4.6.
After loading the column with 1 l of the column and washing the column with the same fresh buffer, SV was added with 50 mM phosphate buffer (pH 7.3) under a concentration gradient of ammonium sulfate decreasing from 1M to 0.2M.
The solution was passed at 0.57. Ammonium sulfate concentration is 0.8M
Fraction containing the protein of about 4.8 l eluted near
Was collected, the membrane was concentrated, and 20 mM phosphate buffer (pH 6.
After dialysis against 5) at 4 ° C. for 18 hours, it 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 the same fresh buffer,
From 0.2M to 0.2M under a concentration gradient of sodium chloride,
The column was loaded with 20 mM phosphate buffer (pH 6.5) SV1.
When the solution was passed through in 2, the protein was eluted at a sodium chloride concentration near 0.13M.

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

[0057]

Example 5 Detection of Protein by Enzyme Immunoassay According to a conventional method, rabbits were immunized with the purified protein obtained by the method of Example 1-2, blood was collected, and IgG antibody was isolated. This IgG antibody was dissolved in PBS at 20 μg / ml and dispensed at 100 μl / well into a 96-well microplate. After incubating the microplate at room temperature for 3 hours, I
The gG solution was removed, and 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.

Remove PBS from the microplate and
After washing with PBS containing 05% (v / v) Tween 20,
0.5% of the 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 carried out at room temperature for 2 hours. After washing with PBS containing 0.05% (v / v) Tween 20, 100 μl / well of biotin-labeled monoclonal antibody M-1 mAb was added, and the mixture was reacted at room temperature for 2 hours with shaking,
After washing with PBS containing% (v / v) Tween 20,
A complex of horseradish peroxidase and streptavidin was added at 100 μl / well, 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 the absorbance at a wavelength of 492 nm using o-phenylenediamine as a substrate. The results are shown in Table 1.

[0059]

[Table 1]

As is clear from the results of Table 1, at least about 50 to 2,000 p is obtained by this detection method.
It is possible to detect g / ml of the protein with high accuracy.

[0061]

Example 6 Detection of Protein by Radioimmunoassay According to a conventional method, rabbits were immunized with the purified protein obtained by the method of Example 1-2, and then blood was collected,
IgG antibody was isolated. This IgG antibody is adsorbed on polystyrene beads for radioimmunoassay by a conventional method,
4 in PBS containing 2% (w / v) bovine serum albumin
The mixture was left to stand overnight at 0 ° C to obtain a solid phase antibody.

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

[0063]

[Table 2]

As is clear from the results of Table 2, at least about 100 to 1,200 by the present detection method.
The protein of pg / ml can be detected accurately.

[0065]

Industrial Applicability As described above, the monoclonal antibody of the present invention specifically reacts with a protein that induces IFN-γ production in immunocompetent cells. Therefore,
The monoclonal antibodies of this invention will have a wide variety of uses in the purification and detection of 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.

It can be said that the present invention exerts such a remarkable action and effect, and has a great significance to contribute to the field.

[0067]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 25 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Fragment Type: Intermediate 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: Intermediate 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 strand Topology: Linear Sequence type: cDNA to mRNA Origin Organ name: Mouse Tissue name: Liver Sequence features Sequence Symbol: mat peptide Location: 1..471 Method of determining features: 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 L ys 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 drawings]

FIG. 1 shows the structure of recombinant DNA pKGFM5.

FIG. 2 Monoclonal antibody M-1 mA according to the invention
It is a western blotting figure which shows the reactivity of b and various proteins.

[Explanation of symbols]

CD encoding the KGFM5 cDNA protein
NA Ptac tac promoter rrnBT1T2 transcription termination region of ribosomal RNA operon AmpR ampicillin resistance gene ori replication origin in E. coli

[Procedure amendment]

[Submission date] August 31, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0036

[Correction method] Change

[Correction content]

Then, the remaining recombinant D is prepared according to a conventional method.
After cleaving NA with restriction enzymes Eco RI and Hind III, a DNA ligation kit “DNA Ligation Kit Version 2” manufactured by Takara Shuzo was used to obtain 0.1 μg of the same Eco RI-Hind III DNA fragment and the same restriction enzyme in advance. Pharmacia plasmid vector "pKK223-3" 10 that had been cleaved with
ng was reacted at 16 ° C. for 30 minutes and ligated to obtain a replicable recombinant DNA “pKGFM5”. Escherichia coli Y1090 strain (ATCC37197) was transformed with this recombinant DNA pKGFM5 by the competent cell method,
The obtained transformant "KGFM5" was inoculated into an L-broth medium (PH7.2) containing 50 µg / ml ampicillin, and shake-cultured at 37 ° C for 18 hours. The culture was centrifuged to collect a transformant, and a standard SDS-alkaline method was applied to a part of the transformant to extract the recombinant DNA pKGFM5. As a result of analysis 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.

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0037

[Correction method] Change

[Correction content]

[0037]

[Example 1-2: Production of protein by transformant KGFM5] Ampicillin 50 μg / m 2 by autoclave
sterilize L-broth medium (pH 7.2) containing 1
After cooling to 0 ° C., the transformant KGF prepared in Example 1-1
M5 was inoculated and seeded under shaking at the same temperature for 18 hours. 1 volume of fresh identical medium to 201 volume jar fermenter
8 liters were taken, sterilized in the same manner, cooled to 37 ° C., inoculated with 1% (v / v) of the seed culture obtained above, and cultivated with aeration and stirring at the same temperature for 8 hours. The culture was centrifuged to collect the cells,
Mixed solution containing 150 mM sodium chloride, 16 mM disodium hydrogen phosphate and 4 mM sodium dihydrogen phosphate (pH
The cells were suspended in 7.3), disrupted by ultrasonic waves, and the disrupted cells were removed by centrifugation to collect the supernatant.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] Explanation of code

[Correction method] Change

[Correction content]

[Explanation of symbols] cDNA encoding KGFM5 cDNA protein Ptac tac promoter rrnBT1T2 transcription termination region of ribosomal RNA operon AmpR ampicillin resistance gene pBR322ori origin of replication in E. coli

[Procedure amendment 4]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

FIG.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication location G01N 33/53 A61K 39/395 UN // A61K 38/21 9281-4B C12N 5/00 B 39/395 9162 -4B 15/00 CA 61K 37/66

Claims (15)

[Claims] 1. 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 shows the isoelectric point. (3) Partial amino acid sequence It has the partial amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing. (4) Biological action Induces interferon-γ production in immunocompetent cells. 2. The protein according to claim 1, wherein the protein has an amino acid sequence shown in SEQ ID NO: 3 in the sequence listing (wherein “Xaa” means methionine or threonine) or an amino acid sequence homologous thereto. Monoclonal antibody. 3. The monoclonal antibody according to claim 1, wherein the protein is derived from mouse. 4. The monoclonal antibody according to claim 1, 2 or 3, which belongs to the IgG or IgM class. 5. The monoclonal antibody according to claim 1, 2, 3 or 4, which is the monoclonal antibody M-1 mAb. 6. A hybridoma capable of producing the monoclonal antibody according to claim 1. 7. The hybridoma is hybridoma M-1.
The hybridoma according to claim 6, which is 8. A monoclonal antibody comprising the steps of culturing a hybridoma capable of producing the monoclonal antibody according to claim 1 in vitro or in vivo and collecting the monoclonal antibody from the culture or body fluid. Production method. 9. A monoclonal antibody from a culture or body fluid is salted out, dialyzed, filtered, concentrated, centrifuged, fractionated and precipitated, gel filtration chromatography, ion exchange chromatography, high performance liquid chromatography, affinity chromatography, gel electrophoresis. And / or the method for producing a monoclonal antibody according to claim 8, which is collected by isoelectric focusing. 10. The hybridoma is hybridoma M-.
The method for producing a monoclonal antibody according to claim 8 or 9, which is 1. 11. A step of contacting the monoclonal antibody according to any one of claims 1 to 5 with a mixture containing a protein having the physicochemical properties and a contaminant to adsorb the protein to the monoclonal antibody, and the adsorbed protein to the monoclonal antibody. A method for purifying a protein, comprising the step of desorbing from the protein. (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 shows the isoelectric point. (3) Partial amino acid sequence It has the partial amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing. (4) Biological action Induces interferon-γ production in immunocompetent cells. 12. The method for purifying a protein according to claim 11, wherein the monoclonal antibody is bound to a water-insoluble carrier. 13. The protein is SEQ ID NO: 3 in the sequence listing.
(However, "Xaa" means methionine or threonine.) The method for purifying a protein according to claim 11 or 12, which has an amino acid sequence shown in or a homologous amino acid sequence. 14. A method for detecting a protein, which comprises contacting a test sample with the monoclonal antibody according to any one of claims 1 to 5 and detecting a protein having the following physicochemical properties by an immunoreaction. (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 shows the isoelectric point. (3) Partial amino acid sequence It has the partial amino acid sequences shown in SEQ ID NOs: 1 and 2 in the sequence listing. (4) Biological action Induces interferon-γ production in immunocompetent cells. 15. The monoclonal antibody is labeled with a radioactive substance, an enzyme and / or a fluorescent substance.
The method for detecting a protein according to 1.