JPH0867700A - Monoclonal antibody reactive with human lung cancer cells and method for detecting lung cancer - Google Patents

Abstract

(57) [Summary] [Object] To provide a monoclonal antibody useful for detection of human lung cancer and a hybridoma producing the antibody. [Structure] Monoclonal antibody that reacts with a mucin-type glycoprotein-derived glycopeptide obtained from human lung cancer cells, does not react with an asparagine-binding glycopeptide, and does not react with human lung cancer tissue and normal tissue, and the antibody A hybridoma capable of producing.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a monoclonal antibody that recognizes a sugar chain of a mucin-type glycoprotein of lung cancer cells and reacts with lung cancer tissue, a method for producing the same, and a method for detecting lung cancer using the monoclonal antibody. .

[0002]

2. Description of the Related Art It is known that the sugar chains of glycoproteins on the cell surface change in structure as cells become cancerous, and they are attracting attention as cancer-related antigens. Monoclonal antibodies against various cancer cells that have been obtained so far are selected using cancer cells as an immunogen and the binding properties between the produced antibody and the cancer cells as an index, or glycolipids extracted from the cancer cells are suitable. It was usual to prepare it as an immunogen by mixing it with various immuno-adjuvants. If a monoclonal antibody that recognizes the glycoprotein sugar chain on the surface of the cancerous cell is obtained, it will be possible to detect the cancerous cell more appropriately, and it will be useful for early detection of cancer. However, in the case of selecting an antibody using the binding property with such a glycoprotein as an index, most of the antibodies against the glycoprotein recognize the peptide portion, and therefore, a part of the glycoprotein that is present on the surface of the cancer cell and is partly secreted. There was nothing but chance to make an antibody that recognizes sugar chains.

The present inventors have previously invented a method of using a solid-phased glycopeptide for measuring the titer of an antibody against a glycoprotein sugar chain (Japanese Patent Laid-Open No. 62-212568). By using this method, it is possible to efficiently select, from a large number of monoclonal antibodies prepared by using cancer cells as an immunogen, those that identify the sugar chain of a glycoprotein.
According to this method, the present inventors recognize a sialyl-Tn antigen and have high reactivity with human colon cancer MLS102 (JP-A-63-33398),
Further, they have succeeded in producing a monoclonal antibody NNY128 (Japanese Patent Laid-Open No. 2-255097) which recognizes Tn antigen and also has high reactivity to human colorectal cancer.

Among sugar chain antigens, mucin-type glycoprotein sugar chains have diverse and unique structures not found in sugar chains of glycolipids, and are regarded as promising agents for cancerous changes, and as cancer-related antigens. It is useful. The above-mentioned Tn antigen and sialyl Tn antigen are exemplified as typical mucin-type glycoprotein sugar chains.

The present inventors believe that the mucin-type glycoprotein sugar chains on the cell surface of lung cancer tissue also show characteristic structural changes different from those of normal tissue due to canceration. When a monoclonal antibody reactive with the mucin-type glycopeptide prepared from SK-LU-1 was selected and prepared, an antibody reactive with lung cancer tissue and not reactive with normal tissue was obtained.

[0006]

[Problems to be Solved by the Invention] Lung cancer is a cancer that is difficult to detect at an early stage and currently ranks first in the mortality rate of cancer among boys in Japan. Therefore, if a monoclonal antibody that reacts specifically with lung cancer is obtained, it is useful for the diagnosis of lung cancer. To date, lung cancer monoclonal antibodies have been known, but the development of even better antibodies is desired.

[0007]

As a result of various studies, the present inventors have found that an antibody produced by a hybridoma obtained by cell fusion of spleen cells of mice immunized with human lung cancer cells and mouse myeloma cells is produced by human lung cancer. Succeeded in creating a monoclonal antibody with high specificity for human lung cancer, and a hybridoma producing this monoclonal antibody, by selecting as an index the reactivity with mucin-type glycoprotein-derived glycopeptides obtained from cells. Then, the present invention has been completed.

The method for preparing the hybridoma and the monoclonal antibody of the present invention will be described in detail below. In the following description, cancer cells used as an immunogen for producing antibody-producing hybridomas and various cells used for examining the reactivity of the monoclonal antibody of the present invention with each cell are all human cells. . In order to produce the monoclonal antibody of the present invention, a human lung cancer cell (immunogen) is immunized with an appropriate mammal, and spleen cells of the immunized animal, and a lymphoid cell of a mammal of the same or different species from the animal. Can be produced by fusing, and then cloning the fused cells. The specific manufacturing method is as follows. (Mammalian immunization with human lung cancer cells) Examples of lung cancer cells used as an immunogen in immunizing mammals include human lung cancer cells SK-LU-1 (ATCC No. HT).
B57), A42T (ATCC No. HTB53), CAL
U-3 (ATCC No. HTB55), SK-MES-
An established lung cancer cell line such as ATCC No. 1 (ATCC No. HTB58) can be used, and among them, a cell having a high mucin-type glycoprotein content is preferable. Examples of mammals for immunization with human lung cancer cells include, for example, mice, rats, guinea pigs, sheep, goats, etc. Of these, mice or rats are preferable. The route of administration of the immunogen at the time of immunization is not particularly limited, and it can be administered by any route such as subcutaneous, intraperitoneal, intravenous and intramuscular. In particular,
For example, BALB / c mice are inoculated with lung cancer cells or a homogenate thereof or an antigen (mucin-type glycopeptide or a sugar chain region-containing fragment thereof) collected therefrom several times every several days to several weeks. The inoculum is preferably 10 ⁵ to 10 ⁷ cells per animal at one time.

The spleen is excised from the mouse immunized with the above cancer cells and centrifuged to obtain antibody-producing cells (spleen cells). Since this cell is not proliferative, it is fused with a cell capable of self-proliferation. Lymphoid cells such as myeloma cells are particularly preferable as the cells having the self-proliferating ability. As the myeloma cells, it is preferable to use those of the same animal type as the animal from which the antibody-producing cells were obtained, and it is preferable to select those that do not secrete the antibody. Examples of such myeloma cells include mouse myeloma cells Sp2 / 0-
Ag14, P3 / NS1 / 1-Ag4-1, P3X63A
Examples include g8U.1. The fusion of antibody-producing cells with myeloma cells or the like can be carried out by a conventional method, for example, by treating these cells with a solution (or suspension) containing a cell fusion agent such as polyethylene glycol. The ratio of antibody-producing cells and myeloma cells is about 3: 1.
It is preferably set to 10: 1.

(Cloning of hybridoma) The obtained fused cells are separated by the limiting dilution method, the separated fused cells are grown, and the antibodies produced in each well are mucin-type glycopeptide solid phase prepared from cancer cells. It is possible to examine the reactivity with the lung cancer tissue by the immunohistochemical staining method by the ELISA method using the modified plate, and select the hybridoma producing the desired antibody from the results. That is, a mucin-type glycoprotein is isolated from human lung cancer cells by a conventional method, and a glycopeptide is prepared by thoroughly digesting it with a protease or the like to remove the peptide portion as much as possible, and then preparing the glycopeptide with alkylenedialdehyde and basic By immobilizing in the presence of a polypeptide or a basic synthetic polymer, a mucin-type glycopeptide sugar chain-specific antibody assay plate is prepared (JP-A-62-212568).
(See Japanese Patent Publication). In addition, the asparagine-binding glycopeptide derived from the asparagine-binding glycoprotein obtained from other fractions obtained during the isolation of the mucin-type glycoprotein from the human lung cancer cells was acylated with a higher fatty acid and immobilized. An asparagine-linked glycopeptide sugar chain-specific antibody assay plate is prepared (see the above-mentioned patent publication). Using these assay plates, the antibodies produced by the fused cells are tested by the ELISA method, and hybridomas producing antibodies that react with mucin-type glycopeptides but do not react with asparagine-binding glycopeptides are selected.

(Preparation of Monoclonal Antibody) The monoclonal antibody of the present invention is prepared by culturing the hybridoma selected as described above using an appropriate medium from a culture supernatant obtained by a conventional method (for example, ammonium sulfate fractionation, It can be easily obtained by ion exchange chromatography, affinity chromatography using protein A, etc.). Alternatively, the hybridoma may be inoculated into the peritoneal cavity of an appropriate mammal (eg, mouse) to allow the hybridoma cells to proliferate, and then the desired monoclonal antibody may be collected from the ascites, serum or the like of the mammal.

The monoclonal antibody thus obtained belongs to the IgG class and has a molecular weight of about 150,000. The present inventors have named this monoclonal antibody MA51.

As shown in Examples described later, the monoclonal antibody MA51 of the present invention reacts with human lung cancer cells and various lung cancer tissues, but hardly reacts with cancer tissues other than lung cancer and shows normal adult and fetal tissues. There was no reaction to the tissue. Therefore, the monoclonal antibody M of the present invention
The A51-producing hybridoma is a hybridoma producing a monoclonal antibody against an antigen present in a mucin-type glycoprotein of lung cancer.

The monoclonal antibody of the present invention can prove several types of lung cancer by one type, and thus can be said to be an extremely useful antibody in view of clinical application. That is, the MA51 of the present invention measures the antigen concentration in body fluids by a method such as radioimmunoassay or ELISA, and detects sputum and lung cancer cells in a biopsy sample by immunohistochemical staining or cytoplasmic fluorescent antibody method. , Can be used for diagnostic purposes. By using this monoclonal antibody MA51, it is possible to diagnose various lung cancers with extremely high accuracy. In addition, the monoclonal antibody of the present invention is a monoclonal antibody-anticancer agent complex in which the monoclonal antibody targeting the cancer tissue carrying the corresponding antigen is used alone or the monoclonal antibody is used as a means for specifically delivering the anticancer agent to the cancer tissue. Can also be used for missile therapy.

Details of the diagnostic method by the ELISA method, the immunohistological staining method and the cytoplasmic fluorescent antibody method using the monoclonal antibody of the present invention are as follows.

ELISA method A 96-well EIA plate is coated with 10 to 100 of the first antibody.
Dispense 50 to 200 μl / well of μg / ml, and
Leave at 1-2 ° C. for 1-2 nights or at room temperature for 2-4 hours. P
After washing with BS, a serum sample is diluted 1 to 100 times and 50 to 100 μl is added to each well. After standing overnight at 4 ° C or at room temperature for 2 hours, wash well with PBS. Next, biotinylated secondary antibody (10 to 100 μg / ml) was added to 50 to 1
Add 00 μl / well and let stand overnight at 4 ° C. or at room temperature for 2-4 hours. When a biotinylated antibody was used as the second antibody, the plate was thoroughly washed with PBS and then avidin-biotin-peroxidase (10
50-100 μl / well is added, and the mixture is left at room temperature for 30 minutes and washed with PBS. Substrate solution
(ABTS substrate) was added at 50-100 μl / well, left at room temperature for 30 minutes, and added with P containing 0.2% sodium azide.
Stop the reaction by adding 10 μl / well of BS solution. The OD ₄₀₅ value of each well is measured, and the amount of antigen in the serum sample is calculated from the degree of color development. The normal value is determined by comparing the amount of antigen in the lung cancer patient serum thus obtained with the amount of antigen in the standard (healthy person) serum, and those exceeding the normal value are regarded as positive for lung cancer.

Immunohistological Staining Method Tissue staining was performed using Vectstein ABC Kit (Ve
Ctor, Burlingame CA, USA). The staining procedure was carried out by blocking the plates with tissue sections with 30% H ₂ O ₂ in order to block endogenous peroxidase.
And a mixture of methyl alcohol at a ratio of 1: 100 for 20 minutes. Then, the cells are washed three times with PBS, and normal horse serum diluted 10-fold with PBS containing 1% BSA is added to block nonspecific reactions. After incubation for 20 minutes at room temperature, monoclonal antibody is added. The monoclonal antibody used is diluted with PBS containing 1% BSA from 2 μg / ml to 10 μg / ml. After leaving it at room temperature for 1 hour or at 4 ° C. overnight, it is washed 3 times with PBS. After washing 3 times with PBS,
PB containing biotinylated horse anti-mouse antibody containing 1% BSA
Dilute 1000 times with S and add. After leaving it for 30 minutes at room temperature, it is washed 3 times with PBS. Then, an avidin-biotin conjugate to which peroxidase is bound is added. The concentration is adjusted at least 30 minutes before adding 1000-fold diluted with PBS. After reacting for 30 minutes at room temperature, washing with PBS is performed three times. For color development, 20 mg of diaminobenzidine hydrochloride was dissolved in 25 ml of 0.2 M trisaminomethane and 19.2 ml of 0.2N hydrochloric acid to which distilled water was added to make 100 ml. Furthermore, H ₂ O ₂ is 0.03 m
Add at a rate of about l. The plate is allowed to react with the above solution for 7 minutes. Then, the color development is stopped. Nuclear staining is performed with hematoxylin.

Cytoplasmic fluorescent antibody method 1 to 5 × 10 ⁴ target cells were put on a slide glass.
The cells are fixed with% acetone for 10 minutes and stored at −70 ° C. and used as target cells. 25 μl of hybridoma culture supernatant was used as the primary antibody at 4 ° C. or 25
React at 30 ° C. for 30 minutes. After washing with PBS, a 20-40-fold diluted fluorescence-labeled anti-mouse rabbit antibody is reacted as a secondary antibody at 25 ° C for 30 minutes. The reaction is judged by the presence or absence of fluorescence under a fluorescence microscope.

[0019]

EXAMPLES The present invention will be described in more detail with reference to Preparation Examples and Examples, but the scope of the present invention is not limited thereto.

Preparation Example 1 Preparation of mucin-type glycopeptide G-50-I and serotype glycopeptide G-50-II: Mucin-type glycopeptide G-50- used for selecting the monoclonal antibody-producing hybridoma of the present invention I is a human lung cancer cell SK-LU-1
(ATCC No. HTB57) is a glycopeptide prepared from a mucin-type glycoprotein that constitutes the cell membrane (plasma membrane) of (ATCC No. HTB57) and is also secreted extracellularly, and was prepared by the following method. Human lung cancer cell SK obtained from ATCC, USA
-LU-1 (ATCC No. HTB57) was cultured in an Eagle medium containing 10% fetal bovine serum for 7 days, then collected,
After repeated washing with PBS, 1% Triton X-100
PBS containing was added, and the mixture was stirred under ice-cooling, and the supernatant obtained by centrifugation was dialyzed and freeze-dried. This was degreased with chloroform / methanol (2: 1, V / V) to give 0.01M
Suspended in an acetic acid buffer containing calcium acetate, and added with 1/50 parts by weight of pronase (Actinase E, Kaken Pharmaceutical Co., Ltd.) and a small amount of toluene to the above lyophilized product to prepare 3
After standing at 7 ° C. for 3 days, sufficient protein degradation was performed. Approximately 10 volumes of clear solubilized cell digestive fluid
% Trichloroacetic acid aqueous solution was added and stirred, and then the insoluble matter produced by standing was centrifuged, and an equal volume of ether was added to the resulting supernatant, mixed well, and then centrifuged to remove the supernatant. . Ether was added to the aqueous layer and trichloroacetic acid was extracted and removed three times, and after confirming that the aqueous layer had a pH of 5, it was preliminarily treated with 0.5M pyridine-acetic acid buffer (pH 5.0). Equilibrated Sephadex G-2
5 columns (2.6 x 90 cm) were injected, developed with the same buffer and the effluent was collected with a fraction collector. Fractions positive for the orcinol-sulfuric acid reaction were collected and lyophilized. The obtained dry powder was dissolved in 0.5M pyridine-acetic acid buffer (pH 5.0), and then Sephadex G-
Gel filtration was performed using 50 in the same manner as described above, and fractionation was performed with a fraction collector into 5 ml fractions. The fraction (G-50-I) passed through at this time contains glycoprotein derived from mucin type (O-glycoside type) glycoprotein as a main component. The solvent was removed from the fractions of fraction numbers 24 to 30 by freeze-drying to obtain mucin-type glycopeptide G-50-I. Incidentally, the fractions of fraction numbers 31 to 58 (G-50-II)
Is a glycopeptide fraction derived from an asparagine-linked (N-glycoside type) glycoprotein, which was designated asparagine-linked glycopeptide G-50-II.

The sugar composition of each of the above glycopeptides was analyzed using Dionex anion exchange chromatography. The analysis results are shown in Table 1 below.

[Table 1] The abbreviations in the table have the following meanings. Fuc: Fucose GalNAc: N-acetylgalactosamine Gal: galactose GlcNAc: N-acetylglucosamine Xyl: xylose Man: mannose As shown in Table 1, G-50-I is Gal, GlcNAc,
The proportion of GalNAc was high, indicating the presence of mucin-type glycopeptides. Further, since mannose (Man) was not detected, it was shown that the asparagine-linked sugar chain was not contained in G-50-I. G-50-II is M
The high proportion of an and GlcNAc indicated that Asn type sugar chains were mainly present. From the above, G-50-
It was confirmed that I is a mucin-type glycopeptide and G-50-II is mainly an asparagine-binding glycopeptide.

Preparation Example 2 Immobilization of mucin-type glycopeptide G-50-I on a plate: 1 mg / ml of mucin-type glycopeptide (G-50-I)
To 100 μl, 1.9 ml of PBS solution and 33 μl of 0.25% glutaraldehyde solution were added, and 20 μl of the solution was dispensed into a 96-well plate, and then reacted at room temperature for about 1 hour.
After the reaction, 0.25 mg / ml polylysine (manufactured by Sigma)
After mixing with 10 μl of a PBS solution containing the above, the mixture was left standing at 4 ° C. for 24 hours. Under these conditions, 5 of the glycopeptides used
0% is immobilized.

Preparation Example 3 Immobilization of asparagine-binding peptide G-50-II on a plate: 60 mg of 17 mg / ml asparagine-binding peptide (G-50-II) in pyridine containing 10 mg / ml palmitic anhydride. Add 0.5 ml of solution and mix
The acylation reaction was carried out at 7 ° C. for 6 hours. After the reaction completion liquid was dried and solidified, extraction was performed 6 times with diethyl ether, and the residue was air-dried. The dried product was added with an 8% pyridine solution at 100 ug /
After making up to 20 ml, 20 μl of the solution was dispensed into a 96-well plate and dried at room temperature. Under this condition, 10% of the glycopeptide used is immobilized.

Example 1 Preparation of hybridoma: Human lung cancer cell SK-LU-1 (AT) producing a large amount of mucin on the cell surface as an immunogen
CC No. HTB57) was used. 6-week-old BALB /
SK-LU-cultured in Eagle's medium containing 10% fetal bovine serum intraperitoneally in c mice (female, Charles River)
1 cells were injected 4 times 1 to 2 × 10 ⁶ cells per animal per dose was suspended in saline 0.5ml every three weeks, three months later, the spleens from mice immunized I removed it.

The method of cell fusion was carried out according to the method of Watanabe et al. (Immune Experimental Procedure VII, 2963-2967, 1978). That is, the spleen of the isolated immunized mouse was cut into small pieces, passed through a stainless mesh, and spun down at 1500 rpm to obtain a precipitate, and 10 ml of 0.7% NH ₄ Cl was added to remove red blood cells.
The cells were washed twice with RPMI-1640, and the resulting live cell suspension 3.9 × 10 ⁷ cells / 22 ml was washed with RPMI-1640 2 times.
Mouse myeloma cells Sp2 / 0-Ag1 obtained by washing twice
4 (hereinafter referred to as SP2) 7.8 × 10 ⁶ pieces / 2.5 ml (5:
1) was mixed and spun down at 2000 rpm for 10 minutes. After precipitating the cells well, RPMI-1640 at 37 ° C and pH 7.4 containing 42.5% (W / V) polyethylene glycol (PEG1000, manufactured by Sigma), 1
Cell fusion was performed by gradually adding ml while rotating over 1 minute. One minute after the reaction, RPMI-1640 was gradually added to complete the cell fusion. After centrifugation at 2000 rpm, 1
HT (hypoxanthine-thymidine) containing 0% fetal bovine serum
Add 50 ml of medium and add 1 to 96 well microplate
Dispense 0.1 ml per well at 37 ° C, 5% CO ₂
Cultured in an incubator. After 24 hours, half of the supernatant was replaced with HAT medium (hypoxanthine, aminopterin, thymidine, 10% fetal bovine serum) every 2 days. On the 10th day, the supernatant was taken out and the ELISA method using the plate on which the above-described mucin-type glycopeptide was immobilized (Preparation Example 2) and the plate on which the asparagine-binding glycopeptide was immobilized (Preparation Example 3) was used. The presence or absence of antibody production was confirmed, and the number of hybridomas in the wells showing positive antibody production was adjusted to 0.3 to 0.6 per well by the limiting dilution method. The medium is HT (hypoxanthine, thymidine, 10%
(Fetal bovine serum) medium, using a feeder layer
Balb / c mouse spleen cells 5 × 10 ⁵ / well were added. Cloning by such limiting dilution method
G-50-I reacts strongly with G-50-I
A plurality of clones producing antibodies that did not react with I at all were selected. Furthermore, using the culture supernatants of these clones, a clone that reacts with lung cancer tissue but not with normal tissue was finally selected by the immunohistological staining method and cytoplasmic fluorescent antibody method described above, and the antibody was designated as MA51. I named it.

Example 3 (1) Preparation and purification method of monoclonal antibody: The hybridoma obtained in Example 2 was cultivated in the same manner as usual animal cell culture. That is, streptomycin (50 to 10
0 μg / dl), penicillin (50-100 units), glutamine (3.5-4.0 g / l), fetal bovine serum (10-20)
%) RPMI 1640 medium containing 10 to 20 ml, in the presence of 5% CO ₂ in a flask, 35 to 37 ° C., 3
Cultured for ~ 7 days. Alternatively, 0.5 to 1.0 ml of pristane was intraperitoneally injected into a mouse, and one month later, 5 to 10 × 10 ⁶ hybridomas were intraperitoneally transplanted.

The supernatant liquid of the culture of the hybridoma or the ascites fluid stored in the abdominal cavity of the pristane-treated mouse
Centrifugation was performed at 00 rpm for 20 minutes at 4 ° C. The supernatant was collected, diluted 10 times with PBS, and ammonium sulfate was added with stirring with a stirrer for about 20 minutes until 50% saturation was reached. Then, stir for 30 minutes, and then Falcon 205
The mixture was transferred to 9 tubes and centrifuged at 4500C for 9 minutes at 9500 rpm. 0.025M phosphate buffer (pH 8.0) for the precipitate
Was added and dissolved in the same amount as the initial ascites fluid or the supernatant of the culture. This was dialyzed against 0.025M phosphate buffer (pH 8.0). DEAE Cellulose column
man) DE52, first wash the column at 0.025M phosphate buffer (pH 8.0), 4 ° C., then
Antibodies semi-purified with ammonium sulfate were run through. Then
1M sodium chloride-0.025M phosphate buffer (pH
8.0) Collect the elution fractions, put them in a dialysis membrane, and
Concentrated by 0000. After removing PEG, the product was dialyzed against PBS to obtain a purified monoclonal antibody MA51.

(2) Analysis of antigens recognized by monoclonal antibody MA51: In order to analyze the antigens recognized by the monoclonal antibody MA51, solid-phased SK-LU-1 cells were treated with the following various enzymes and reagents, and then MA51 Was examined for reactivity. Enzymes and Reagents (1) Sialidase (Neuraminidase manufactured by Seikagaku Corporation) 0.25M acetate buffer containing 50 mM calcium chloride (p
20mU / ml in H4.5) (2) α-L-fucosidase (manufactured by TAKARA) 0.2mU in 50mM potassium phosphate buffer (pH 6.0)
/ Ml (3) Trypsin (manufactured by GIBCO) 0.25% in PBS (4) Pronase (Actinase E manufactured by Kaken Pharmaceutical Co., Ltd.) 0.1M borate buffer containing 10 mM calcium acetate (p)
10 μg / ml in H8.0) (5) Periodic acid (manufactured by Wako Pure Chemical Industries, Ltd.) 50 mM SK-LU-1 cells in PBS were added to 1 of 96 well microplate.
2 × 10 ⁴ cells were dispensed per well, the cells were cultured for 2 days, and then immobilized using a 0.1% glutaraldehyde solution. After blocking with a PBS solution containing 0.2% gelatin, 0.1% BSA, 100 mM glycine, and 0.05% sodium azide, it was washed 3 times with PBS-T (PBS solution containing 0.1% Tween 20). 50 μl / well of the above enzyme solution or reagent solution (1) to (5) was dispensed and reacted at 37 ° C. for 1 hour. Then, the plate was washed 3 times with PBS-T and 50 μl / ml of monoclonal antibody MA51 (2.5 μg / ml) was added.
Wells were dispensed and reacted at 37 ° C for 1 hour. After washing 3 times with PBS-T, biotin-labeled anti-mouse IgG (4000
50 μl / well was dispensed and the reaction was carried out at 37 ° C. for 30 minutes. After washing three times with PBS-T, avidin-
Biotin-peroxidase (ABC kit, manufactured by Vector) was dispensed at 50 μl / well and reacted at 37 ° C. for 30 minutes. After washing 4 times with PBS-T, ABTS substrate was added to 1
00 μl was dispensed, and after reacting for 30 minutes, the absorbance at 405 nm was measured. As a result, the reactivity of MA51 to SK-LU-1 cells was completely abolished by the periodate treatment with trypsin and pronase, and thus the monoclonal antibody MA51 recognizes the sugar chain portion of the mucin-type glycoprotein. it is conceivable that.

Example 4 Reactivity of monoclonal antibody MA51 against various lung cancer cultures: Various cell lines shown in Table 2 were immobilized on a plate,
The binding of MA51 to the cell line was examined by the ELISA method. Table 3 shows the results. As is clear from Table 3, MA51 showed a binding ability to 7 of 8 lung cancer cell lines, whereas MA51 showed a binding ability to all cell lines other than lung cancer. There wasn't. From this, MA
It is clear that 51 is an antibody specific for lung cancer cells.

[0030]

[Table 2]

[0031]

[Table 3]

Example 5 Reactivity of monoclonal antibody MA51 against various lung cancer tissues: Immunohistochemical staining of lung cancer tissues was performed using the monoclonal antibody MA51. That is, the paraffin-embedded block sections of various lung cancer paraffin-embedded block sections extracted from 10 lung cancer patients and 16 tissues other than lung cancer (7 cancers other than lung cancer, 4 lung fibrosis cases, 5 normal lungs) shown in Table 4 were paraffin-embedded. The embedded block sections were used for testing by the immunohistological staining method described above. As is clear from the results shown in Table 4, in the lung cancer tissue, cancer cells were strongly stained in 8 out of 10 cases. On the other hand, in tissues other than lung cancer, only 1 case out of 16 cases stained weakly. From the above results, it is found that the immunohistochemical staining using MA51 enables the pathological diagnosis of lung cancer.

[0033]

[Table 4]

[0034]

INDUSTRIAL APPLICABILITY According to the present invention, there are provided a monoclonal antibody which reacts with human lung cancer tissue but hardly reacts with cancer tissues other than lung cancer and does not react with normal tissue at all, and a hybridoma cell producing the same. It Therefore, human lung cancer is specifically detected by using such a monoclonal antibody, and is useful for early detection of lung cancer.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 21/08 9358-4B G01N 33/574 D 33/577 B // A61K 39/395 T (C12P 21/08 C12R 1:91) (72) Inventor Takeji Shibata, 3-18-5, Uzumoridai, Higashinada-ku, Kobe-shi, Hyogo Prefecture (72) Miki Aoki, 3-chome, Shioya, Tarumi-ku, Kobe-shi, Hyogo 25-304

Claims (11)

[Claims] 1. A monoclonal antibody that reacts with human lung cancer cells. 2. The monoclonal antibody according to claim 1, which reacts with a mucin-type glycopeptide derived from human lung cancer cells. 3. The monoclonal antibody according to claim 1 or 2, which binds to a sugar chain region of a mucin-type glycopeptide. 4. The monoclonal antibody according to claim 3, wherein the immunoglobulin class is IgG. 5. Obtained by fusing a spleen cell of a mammal immunized with a human lung cancer cell with a cell having self-proliferation ability derived from an animal of the same species or different species as the mammal, and cloning the fused cell. A hybridoma producing the monoclonal antibody according to claim 1. 6. The hybridoma according to claim 5, wherein the mammal is a mouse. 7. Human lung cancer cells are established lung cancer cells SK-LU
6. The hybridoma according to claim 5, wherein the hybridoma is -1 (ATCC No. HTB57), and the cell having a self-proliferating ability is a mouse myeloma cell. 8. A method for producing a monoclonal antibody, which comprises culturing the hybridoma according to claim 5 and collecting the monoclonal antibody according to claim 1 from the culture supernatant. 9. The method according to claim 8, wherein the hybridoma is inoculated into a mammal and cultured, and then the monoclonal antibody accumulated in the mammal is collected. 10. A method for detecting human lung cancer cells, which comprises using the monoclonal antibody according to claim 1. 11. The detection method according to claim 10, wherein human lung cancer cells are detected by an ELISA method, a radioimmunoassay method, an immunohistological staining method or a fluorescent antibody method.