JPH0315395A - Monoclonal antibody and method for measuring monooxygenase-cytochrome p-450-related protein using the same - Google Patents

Abstract

PURPOSE:To enable advantageous practice of an immunological searching method, analysis of relationship between a human monooxygenase and a cancerous change, etc., by using an antimonooxygenase.cytochrome P-450 monoclonal antibody having reactivity with human in vivo ingredients. CONSTITUTION:A purified human cytochrome(Cyt.) P-450 obtained from human specimen viscera, etc., is administered to a mouse to provide a hybridoma by fusion of a splenic cell from the immunized animal to a myelomatous cell. A culture supernatant of the resultant hybridoma is then used to prepare a monoclonal antibody. The obtained monoclonal antibody capable of recognizing a monooxygenase and proteins related thereto is used in a method for measuring in vivo ingredients according to an immunological method using the antibody. Furthermore, the aforementioned antibody is used in an immunohistochemical method for searching a test specimen of the presence of the monooxygenase and related proteins in tissues thereof, a drug missile employing the above-mentioned antibody as a carrier for an anticancer agent or carcinostatic agent, judgment of diseased parts, such as progressive cancer, for imaging diagnosis, etc.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to monoclonal antibodies, and more particularly to cytochrome P-450, which is a human monooxygenase.
(Cyt.P-450) and related proteins. The present invention also provides hybridomas that produce such monoclonal antibodies, immunoassay methods, immunohistochemical search methods, and drug missiles using monoclonal antibodies, and furthermore, the present invention provides hybridomas that produce such monoclonal antibodies. Regarding antibody-compatible antigens.

[Prior art and its problems] Conventionally, various analyzes of monooxygenase in experimental animals have been carried out, and in particular, Cyt. A detailed analysis of the fluctuations in P-450 has been carried out. However, monooxygenes in the human body is mainly analyzed using models using materials derived from laboratory animals, and sufficient studies have not been conducted, and human-specific materials useful for analysis have not yet been obtained. There wasn't. On the other hand, in recent years, clinical diagnostic tests using monoclonal antibodies against proteins in human organs and body fluids (blood, tiiffl, urine, etc.) have been actively carried out, and these substances are known to increase in cancer. Similar efforts have been made using antibodies against carcinoembryonic antigen (CEA), α-7 ethobrotene, etc.
Its association with cancerous changes is insufficient, and detection of early-onset cancer remains problematic.

[Means for solving the problem] As a result of various studies in order to develop human-specific monooxygenase analysis materials, the present inventors found that purified human Cr
t. Anti-Cyt.P-450 was used as an immunogen. P-45
As a result of producing 0 monoclonal antibodies and conducting various studies,
We obtained a monoclonal antibody that has specific reactivity with human biological components. As a class of such monoclonal antibodies, IgG is preferred, and among these, IgG antibodies are practically advantageous because of their excellent specificity and high antibody titer. The immunological search method using this antibody is particularly useful for the analysis of human monooxygenase.

An example of the method for producing a monoclonal antibody capable of recognizing monooxygenase of the present invention is as follows <Cyt. P-450
A method for preparing a monoclonal antibody that recognizes .

That is, first, Cyt. Although the isolation of P-450,
Human specimen organ or Cyt. After homogenizing various cultured cell lines in which the presence of P-450 is observed, solubilization using a surfactant, octylamino Sepharose 4B,
Hydroxyapatite, LH-20, DEAE-Cellulose, DEAE Sepharose, CM-Cellulose, C
Cyt. P-450 can be purified. Further, purification is also possible using an immunoaffinity column that utilizes cross-reactivity with animal-derived antibodies. It can also be obtained by purifying the cDNA expression product, and the immunogen does not interfere with the presence of contaminants.

The obtained Cyt. Animals to be immunized with P-450 include:
Examples include rats and mice, and mice are particularly advantageously used in practice. Hybridomas are obtained by fusion of the spleen cells of the obtained immunized animal with myeloma cells (myeloma). For fusion, any of the known methods using polyethylene glycol, fusion using Sendai virus, or electrical pulses may be used. May be used. The culture supernatant of the obtained hybridoma contains purified cyt, which is an immunogen.
．． ELISA using P-450, and I m@un
The reactivity is examined by oblot, and monoclonal antibodies are produced from positive hybridomas by limiting dilution. In addition, when producing monoclonal antibodies via hybridoma in this way, splenocytes and myeloma cells are
Cells from the same species of animal are preferred, and particularly mouse splenocytes and mouse-derived myeloma cells are practically preferred. The obtained monoclonal antibody-producing hybridoma can be frozen and stored, and can be cultured in large quantities by an appropriate method. Antibodies can be obtained from culture supernatants, or such hybridomas can be transplanted into animals to form tumors, and the desired monoclonal antibodies can be obtained from the ascites or serum. Purification of the monoclonal antibody of the present invention is performed using salting out and an affinity column or DEAE column.

The present researchers have developed a monoclonal antibody that can recognize such monooxygenases by l. 2. It has no reactivity with biological components of experimental animals other than humans or is below the detection limit; 2. It has high reactivity with the cytoplasm of normal human liver cells and does not react with cancerous lesions; 3. We discovered that proteins that selectively react with this antibody exist in tumor-bearing human biological components.

That is, the present invention is characterized in that a monoclonal antibody capable of recognizing monooxygenase and its related proteins is used in a method for measuring in-vivo components by an immunological method using antibodies. This is aimed at clinical application utilizing the above-mentioned properties of clonal antibodies.

In the measurement method of the present invention, a direct method, a sandwich method, etc. are used, but it is preferable to immobilize the biological sample or monoclonal antibody on a carrier. As the solid phase, for example, balls, beads, gears, microplates, etc. using polystyrene, polyethylene, polyacrylate, Teflon, polyacetate, polyacetal, latex, etc. are preferably used.

Furthermore, the methods and means for labeling monoclonal antibodies and the methods and means for detecting the same are not limited in any way, and include known methods and means, for example, anti-immunogenic antibodies labeled with radioactive substances, enzymes, or fluorescent substances. It can be measured by secondary reaction with globulin antibodies or staphylococcal protein A. As labeling agents, enzymes such as horseradish coperoxidase, β-D-galactosidase, and alkaline phosphatase, radioactive substances such as 'r and 'H, and fluorescent substances such as fluorescein isocyanate are usually used. Other substances may be used as long as the activity of the labeling agent can be measured. When the labeling agent is an enzyme, a chromogenic substrate is used to measure the activity. Examples of the substrate include horseradish peroxidase. As a substrate for 2,
2゜Azinodi[3-ethylbenzthiazolinesulfonic acid] ammonium acid (ABTS)-H,O,, 5-aminosalicylic acid-H,O,, 0-phenylenediamine 10,
O,,4-amino/antipyrine-H,O, etc., β-
Examples of substrates for D-galactosidase include fluorescein (β-D-galactopyranoside), o-nitrophenol, and β-D-galactopyranoside. In addition to these reagents, known reagents such as a solubilizer, a detergent, and a reaction terminator are used for the measurement.

As an embodiment of the immunoassay method of the present invention, a method for measuring monooxygenase in human liver biopsy will be exemplified.

That is, monoclonal antibody (a) is added to a 96-well microplate or latex beads at 5 μ9/well and then left at 4° C. for 12 to 24 hours.

Next, 100 μQ of phosphate buffered saline (PBS) containing 1% BSA is added to each well to prevent nonspecific adsorption of antibodies and other proteins. Next, 50 μe of a sample (human biopsy liver homogenate) is added to each well, stirred well, and then left at 37° C. for 30 minutes.

After thoroughly washing the reaction mixture with PBS, 50 μg of a biotin-labeled monoclonal antibody (1 μ9/μe) having a recognition site different from that of (a) for mo/oxygenase is added to each well. The monoclonal antibody was labeled with piotin using a known method. After stirring well, heat at 37°C for 3
Leave for 0 minutes. After thoroughly washing the reaction mixture with PBS, 50 μQ of a 5000-fold dilution of horseradish peroxidase (HRP)-labeled avidin is added to each well. After stirring well, leave at 37°C for 30 minutes.

After thoroughly washing the reaction mixture with PBS and draining the water, the substrate solution (orthophenylene di7mi:y(O P D)
l xy/t (1, pH 4.5 acetate buffer) was added in 100 μQ portions and reacted at 37° C. for 5 minutes. The amount of THP present in the sample is quantified by measuring the absorbance at 492 nm of each well with an EIA reader.

The present invention involves the detection of monooxygenase and related proteins in biological cells using monoclonal antibodies.
It also includes a detection method using the noblot method.

Immunoblot is performed using a known method, and there are no limitations on the method or materials. Specifically, the search method of the present invention detects monooxygenase in biological components by SD
Separated by S-PAGE, transferred to a known membrane such as nitrocellulose using electricity or capillarity, and then transferred onto the membrane by a known immunochemical search method using monoclonal antibodies. detects monooxygenase, determines the possibility of cancerous changes, determines the degree of cancer progression,
It is also used to determine drug dosage.

Furthermore, the present invention includes a method for immunohistochemically searching for the presence of monooxygenase and related proteins in a tissue, in which a monoclonal antibody capable of recognizing monooxygenase is attached to a part of the tissue by an immunoreaction. It also includes a search method characterized by combining. The term "tissue" used in such an immunohistochemical search method refers to cells or tissue sections of an animal, particularly, practically preferably, a human.

More specifically, the search method of the present invention involves causing a monoclonal antibody capable of recognizing monooxygenase to act on a tissue to generate an immune reaction, and targeting the site where monooxygenase exists in the tissue. It is characterized by binding to clonal antibodies. For example, by treating the monoclonal antibody with a biotinylated antibody that recognizes such a monoclonal antibody, and further treating it with an enzyme-labeled apidine, the site where monooxygenase exists in the tissue is labeled by staining, etc., and the normality of the tissue is searched. This makes it possible to search for the location of advanced cancer. Such an immunohistochemical search method is performed by a known method, and there are no limitations on the method or materials.

Furthermore, the present invention also includes a drug missile characterized in that a monoclonal antibody capable of recognizing monooxygenase is used as an anticancer agent or an anticancer agent.

Such anticancer drugs and anticancer drugs include chlorambutyl, ACN
U, CCNU, cisplatin, MTX, 5FU, F
UDR, ARA-C, vinblastine, pincristine, MMC, adriamycin, daunomycin, etc. are used, but any of them may be used as long as the monoclonal antibody of the present invention can be used as a carrier, and these are limited to these. isn't it.

Further, the monoclonal antibody of the present invention may be used in any form as a carrier, including, for example, a therapeutic method using a radioactive isotope as a carrier. Such radioisotopes include 212Bi, 2
11At, 1311, 90Y, etc. are used.

According to the drug missile of the present invention, anticancer drugs and anticancer drugs can be effectively administered to the affected area, and high therapeutic effects can be obtained and side effects can be reduced.

The present invention also includes a monoclonal antibody for imaging diagnosis that is labeled with a radioactive substance, a paramagnetic substance, a fluorescent substance, or the like, and is capable of recognizing the monoclonal antibody.

For example, the radioactive substance mentioned here is 1! ! ■、Il
Examples of the paramagnetic material include gadolinium (Gd) and the like.

In both of these methods, a labeled monoclonal antibody of the present invention is injected into the body of an animal, particularly preferably a human, to reach the site where monooxygenase exists in the body, and that site is labeled. If it can be used for imaging diagnosis in radiology or ultrasound diagnosis,
It can be anything.

According to the monoclonal antibody for imaging diagnosis of the present invention, it is possible to extremely easily diagnose the localization of monooxygenes in the body and the location of diseases such as advanced cancer that involve its fluctuations. be.

[Example] Next, the present research will be explained with reference to an example, but the present invention is not limited to this example.

Example 1 (1) Preparation of immunogen After homogenizing the liver of a human deceased specimen, a microsomal fraction is prepared according to a conventional method and solubilized in a phosphate buffer containing 0.8% cholic acid. After homogenization, the solubilized fraction was examined using octylamino Sepharose 4B, hydroxyapatite, LH-20 and OD4 17.
cyt. The fraction with p-450 activity was concentrated and S
Obtained as a single band on DS-PAGE. Obtained c
yt. P-450 was dialyzed against PBS and used as an immunogen.

(2) Production of monoclonal antibodies 1. Cyt. obtained in (1). P-450 was mixed with Freund's complete adjuvant (Freund's CoB1e).
ta Adjuvant) to form an emulsion, and then subcutaneously injected into BALB/C mice at 3 ml per mouse.
0 μ? administered. From the second time onward, use 0.2 m of sterile physiological saline.
(Dissolve 30μ9 of Cyt, P-450 in 1,
It was administered intravenously three times at daily intervals. For the final immunization, 50μ9 of crude THP was administered intravenously in the same solution and volume.

2. Splenocytes excised from hyperimmunized mice 3 days after final immunization and BALB/c mouse-derived myeloma cell line SP2/
0-Agl4 to polyethylene glycol (PEG)40
00 was used for fusion. Cells were placed in a 96-well plate.
After 24 hours, half of the medium was replaced with HAT medium, and the medium was replaced every two days. Growth of hat-resistant hybridomas is observed after 7 to 10 days. At this stage, the medium was changed to HT, and about 1
After culturing for 0 days, the medium was changed to hybridoma growth medium.

3. Screening for antibody-producing cells was performed using Cy used for immunization.
t. ELISA using P-450 as an antigen
) method, hybridomas that strongly react with the antigen can be selected, and roughly Cyt. As a result of Western blotting using P-450 as a sample, Cyt
．． We were able to select the hybridoma that reacts most strongly to P-450. The cell lines selected here were cloned by limiting dilution to establish monoclonal antibody-producing hybridomas. To measure THP, hybridomas were transplanted into BALB/c mice pre-administered with pristane.
Ascitic fluid was collected on the day, and a monoclonal antibody purified by 50% saturated ammonium sulfate salting out method was used.

(3) Properties of monoclonal antibodies Cyt. The class (subclass) of monoclonal antibodies against P-450 is IgG. Met. Antibody titer is 50
00 times (two-step dilution of antibody purified from mouse ascites,
ELISA (EL) in which purified THP is reacted with each
When performing ISA), the dilution ratio at which the highest absorbance persisted was taken as the antibody titer).

Claims (1)

[Claims] 1. Monoclonal antibody against monooxygenase. 2. The monoclonal antibody according to claim 1, wherein the monooxygenase is cytochrome P-450. 3. Cytochrome P-
Monoclonal antibody according to claim 1, wherein 450 is particularly human cytochrome P-450. 4. The monoclonal antibody according to claim 1, wherein the human cytochrome P-450 is present in a biological sample. 5. The monoclonal antibody according to claim 1, which is produced by a hybridoma obtained by fusing splenocytes and myeloma cells obtained using monooxygenase as an immunogen. 6. The monoclonal antibody according to claim 5, wherein the splenocytes are mouse splenocytes, and the myeloma cells are mouse-derived myeloma cells. 7. The monoclonal antibody according to claim 1, wherein the antibody class is IgG. 8. The monoclonal antibody according to claim 1, wherein the antibody class is IgG_1. 9. The monoclonal antibody for imaging diagnosis according to claim 1, which is labeled with at least one selected from the group of radioactive substances, paramagnetic substances, and fluorescent substances. 10. A hybridoma obtained by fusing myeloma cells with splenocytes, which were obtained using monooxygenase as an immunogen and have the ability to produce antibodies capable of recognizing monooxygenase. 11. A method for measuring monooxygenase and related proteins in a subject by an immunological method, which is characterized by using a monoclonal antibody capable of recognizing monooxygenase. 12. A reagent kit for measuring monooxygenase in a subject by an immunological method, characterized in that it uses a labeled monoclonal antibody capable of recognizing monooxygenase. . 13. In a method for immunohistochemically searching for the presence of monooxygenase and related proteins in tissues, a monoclonal antibody capable of recognizing monooxygenase and related proteins is bound to a part of the tissue by immunoreaction. A search method characterized by forcing. 14. A method for in vivo administration of a monoclonal antibody capable of recognizing monooxygenase and related proteins as a modulator of in vivo metabolic ability and for direct action. 15. A method for detecting human biological components by an immunological method, which utilizes the selective reactivity with human biological components of a monoclonal antibody capable of recognizing monooxygenase and related proteins. 16. A method for detecting human biological components by an immunological method, which utilizes the selective reactivity of monoclonal antibodies capable of recognizing monooxygenase and related proteins with human normal tissues. 17. A related protein that is reactive with a monoclonal antibody capable of recognizing monooxygenase and related proteins and is found in human organisms associated with tumor-bearing and cancerous changes. 18. Diagnosis of tumor bearing, cancerous change, and examination of the difficulty of cancerous change using the reaction between a monoclonal antibody capable of recognizing monooxygenase and related proteins and the related protein according to claim 17 An immunological search method aimed at preventive measures.