JPH0723400B2 - Monoclonal antibody against human mucinous ovarian tumor and method for detecting human ovarian tumor using the antibody - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel anti-human ovarian tumor monoclonal antibody that specifically reacts with human ovarian tumor, and a method for detecting human ovarian tumor using the monoclonal antibody.

[0002]

Mouse monoclonal antibodies against various human tumors are known and have already been applied to immunohistological diagnosis and serodiagnosis [Semin. Oncol., 11:26.
4-274,1984, Cancer Res., 46: 3225-3238, 1986, Am.
J. Pathol., 126: 230-242, 1987, Adv. Cancer Res., 4
9: 189-221, 1987, Adv.Immunol., 40: 323-378, 198
7]. In addition, treatment of cancer patients with monoclonal antibodies is also being performed [Lancet, i: 762-765, 1982, Proc. Nat.
L. Acad. Sci., USA, 82: 1242-1246, 1985]. Regarding ovarian tumors, Bast et al. [J. Clin. Invast., 68:13 have reported a CA125 monoclonal antibody that reacts with a carbohydrate antigen mainly present in serous ovarian tumors.
31-1337, 1981]. The CA125 antigen detected by the CA125 monoclonal antibody has been used for serodiagnosis of ovarian cancer patients. The CA125 antigen is an antigen often detected in non-mucinous tumors, especially serous tumors [Smin. Oncol., 1
1; 264-274, 1984, N.Engl.J.MeD., 309: 883-887, 198
3, Cancer.Res., 44: 1048-1053, 1984]. Immunohistochemically-reactive mucinous ovarian tumors are known [Cancer Res., 42: 1650-1654,1982, Cancer Re
s., 45: 2358-2362, 1985], but a tumor marker that specifically reacts strongly with mucinous tumor has not been found.

[0003]

The object of the present invention is to provide a monoclonal antibody that specifically reacts strongly with mucinous ovarian tumor and belongs to the IgG ₁ isotype, and to human ovarian tumor using this monoclonal antibody. To provide a detection method.

[0004]

The present invention provides the following monoclonal antibody and a method for detecting human ovarian tumor characterized by using the monoclonal antibody. (1) A monoclonal antibody produced by a hybridoma obtained by fusing mouse splenocytes immunized with human mucinous ovarian tumor cells and mouse myeloma cells and having the following properties.

Immunoglobulin class: IgG ₁ Reacts with mucinous ovarian tumor, cervical adenocarcinoma, gastric cancer, and colon cancer. It does not respond to serous tumors, mesothelioma, uterine fibroids, esophageal cancer, lung cancer, breast cancer, sarcoma, melanoma, carcinoid, nervous system tumors, lymphoma. It does not react with normal cells of the ovary, fallopian tube, endometrium, placenta, esophagus, liver, kidney, mammary gland, thyroid, spleen, brain and skin. (2) The monoclonal antibody according to (1), which is produced from the hybridoma strain OM-C (FERM P-9877). (3) Use the monoclonal antibody described in (1) or (2) when detecting human ovarian tumors using the ELISA method, the ABC enzyme-linked immunosorbent assay, the mouse mixed blood cell adsorption test, or the cytoplasmic fluorescent antibody method. A method for detecting a human ovarian tumor characterized by:

The present invention will be described in detail below. The novel anti-human ovarian tumor monoclonal antibody of the present invention specifically reacts with human ovarian tumor and exhibits high specificity for mucinous ovarian tumor. Specific examples of the monoclonal antibody of the present invention include a monoclonal antibody named OM-C. Such a monoclonal antibody belongs to IgG ₁ . (1) Production of Monoclonal Antibody The monoclonal antibody of the present invention can be produced by immunizing a mouse with ovarian cancer cells, removing splenocytes, and culturing hybridoma cells obtained by fusing the splenocytes with mouse myeloma cells. it can. Preparation of antibody-producing cells This hybridoma is produced by the method of Kohler and Milstein [Nature, 256 , 495-497 (1975)], which is an improved method of Ueda et al. [Proc. Natl. Acad. Sci. USA, 79 , 4386-]. 4390 (1982)
]

As the immunizing mouse, BALB / c mouse, F1 mouse of BALB / c mouse and other mouse, and the like are used. Immunization per mouse (4-8 weeks old, 20 to 30 g) performed 2-3 times every 2-5 weeks with 10 ^{7 8} mucinous ovarian tumor cells to. Rearing mice and collecting splenocytes follow conventional methods. Cell fusion Spleen cells and myeloma cells were mixed at a ratio of 1 to 1 to 10: 1, and fusion was performed by using NaCl (about 0.85%), dimethyl sulfoxide [10 to 20% (V / V)] and molecular weight of 1000 to 6000. It is carried out in a phosphate buffered saline solution (pH 7.2 to 7.4) containing polyethylene glycol (hereinafter referred to as PEG).

Fusion is carried out by incubating both cells at 35-37 ° C for 1-3 minutes. As myeloma cells, MOPC-21NS / 1 [Nature, 256 , 495-497 (1975)
], SP2 / 0-Ag14 [Nature, 277 , 131-133 (1979)],
S194 / 5, XXO.BU.1 [J.Exp.Med. 148 , 313-328 (1978)
] Etc. are used. Selection of hybridoma Hypoxanthine is used for selection of fused cells (hybridomas).
(1.3-1.4mg / dl), aminopterin (18-20μg / d)
l), thymidine (375-400 μg / dl), streptomycin (50-100 μg / ml), penicillin (50-100 units), glutamine (3.5-4.0 g / l), fetal bovine serum (10-20)
%), And select as growing cells.

As the basal medium, RPMI1640 medium, Eagle's MEM medium and the like which are generally used for culturing animal cells are used. Culture of hybridoma Cloning of fused cells (hybridomas) is repeated at least three times by the limiting dilution method.

When the hybridoma is cultured in the same manner as in normal animal cell culture, the antibody of the present invention is produced in the medium. For example, 2-5 × 10 ⁶ hybridoma cells were treated with streptomycin (50-100 μg / ml) and penicillin (5
0 to 100 units / ml), glutamine (3.5 to 4.0 g / l) and fetal bovine serum (10 to 20%) in 10 to 20 ml of RPMI1640 medium, and in a flask in the presence of 5% CO ₂ at 35 to 37 By culturing at 3 ° C for 3 to 7 days, the antibody is secreted and accumulated in the culture solution.

In addition, the hybridoma cells are treated with pristane (P
The antibody of the present invention can be accumulated in ascites by transplanting into the abdominal cavity of nude mice or BALB / c mice treated with ristane) and allowing the mice to grow. That is, pristane (Pristane, 2,6,10,14 tetramethy
(1 pentadecane, Aldrich, USA) 0.5 to 1 ml is injected, and 5 to 10 × 10 ⁶ hybridoma cells are transplanted into the abdominal cavity 2 to 3 weeks thereafter. Ascites usually accumulates after 7 to 10 days and is collected. Collection and Purification of Monoclonal Antibody The monoclonal antibody accumulated in the culture of hybridoma cells or ascites fluid is collected and purified as follows.

The hybridoma was administered intraperitoneally to the supernatant of the hybridoma culture or to the pristane-treated mouse, and the ascites or serum obtained was treated at 12000 rpm for 20 minutes.
Treat at 4 ° C. Collect the supernatant and dilute 10-fold with PBS. Then, while stirring with a stirrer, saturated ammonium sulfate is slowly added until the amount becomes double (50% saturated) in about 20 minutes. Then, the mixture is stirred for 30 minutes, transferred to a Falcon 2059 tube, and centrifuged at 4500C and 9500 rpm (14700 g) for 10 minutes. Dissolve the precipitate by adding 0.025 M phosphate buffer (pH 8.0) in the same amount as the initial ascites or serum amount. It is dialyzed against 0.025M phosphate buffer (pH 8.0). A DEAE-cellulose column is made using Whatman DE52 and the column is first washed with 0.025M phosphate buffer (pH 8.0) at 4 ° C and then the semi-purified antibody with ammonium sulfate is run. Fractions eluted with 1M sodium chloride-0.025M phosphate buffer are collected, placed on a dialysis membrane and concentrated with PEG20,000. PBS after removing PEG
Dialyze at. Monoclonal antibody selection is performed by ELISA (Enzyme-linked immunosorbent assay), ABC
Enzyme antibody method, mouse mixed adsorption test (M-MHA), cytoplasmic fluorescent antibody method, etc.

Selection of IgG isotype monoclonal antibodies is performed by the ELISA method, and selection of monoclonal antibodies reactive with mucinous tumors is performed by immunohistological staining. (2) Specificity of monoclonal antibody Monoclonal antibody OM-C (hereinafter referred to as OM-C antibody in principle) was resistant to sialidase treatment (0.5 U, 37 ° C, 2 hours). Periodate treatment (10 mM, room temperature, 1 hour)
The 0M-C antibody showed resistance. Thermal resistance (100 ℃, 15 minutes)
For, the OM-C antibody was sensitive.

Regarding the stability of the antibody in the section of a normal pathological tissue specimen treated with formalin, the OM-C antibody was somewhat unstable. Immunohistochemical search for OM-C antibody
This is as shown in Example 2. The OM-C antibody shows considerable specificity for mucinous ovarian tumors. OM-C antibody was mostly mucinous ovarian tumor (11/14 cases), endometrioid tumor [1 (1)
/ 3, the parentheses indicate cases in which only 10% or less of the tumor cells are positive (hereinafter the same)]. Responding to more than half of the serous tumors [0 (9) / 16], but less than 10% of positive cells. Mesothelioma was positive in only one case (1/9),
In germ cell tumors, 1 case each of fetal cancer and mature teratoma is positive (2/8). Half of the ovarian metastatic tumors respond [2 (1) / 6]. Cervical adenocarcinoma among uterine cancers [2 (1)
/ 3], and endometrial cancer is 10% in only 1 of 6 cases
The following positive cells are observed [0 (1) / 6].

For tumors other than gynecological, gastric cancer (4/5),
Positive for colorectal cancer (5/5), lung adenocarcinoma, lung squamous cell carcinoma, renal cancer,
Positive in a few cases of breast cancer, negative in others. In normal tissue, it reacts with cervical mucosal epithelium and mucus, and a few positive cells are also found in fallopian tube epithelium and endometrium. In fetal tissue, it is positive for fallopian tube epithelium. In tissues other than the gynecological region, it is positive for germ cells of gastric epithelium and metaplasia of the intestine, large intestine, small intestine, gallbladder epithelium, ductal epithelium of pancreas, and also positive for bronchial epithelium and medullary part of thymic epithelium. In fetal tissue,
It is positive for stomach, colon epithelium, small intestinal epithelial germ cells, etc.

As shown in Table 1 of Example 2, the antibody of the present invention showing the above-mentioned reactivity to various tumors and normal tissues shows a specificity different from that of CEA antibody and CA125 antibody. And is also distinct from the ABH-Lewis blood group antibody and other previously reported ovarian tumor associated antibodies. When the above monoclonal antibody OM-C is compared with the already known ovarian tumor monoclonal antibodies (1D ₃ , MO _V -1,4C7 etc.), the differences are as follows. i.OM-C antibody recognizes intracellular antigens, and is a group of antibodies against well-analyzed tumor-related antigens, many previously reported blood group-related antigens, CEA, CA125 antigens, etc. Is different from. ii. Also frequently used as an ovarian tumor-related antibody
The immunohistological reaction pattern is different from that of CA125 antibody and CEA antibody. Compared with these antibodies, the prepared antibody OM-C has higher specificity for mucinous ovarian tumor. (3) Diagnosis by Monoclonal Antibody The monoclonal antibody of the present invention is used for immunohistological diagnosis of human mucinous ovarian tumor, analysis of differentiation degree of ovarian tumor, and subclassification, diagnosis of body fluids such as serum and ovarian cyst fluid.

For the above-mentioned diagnosis using the monoclonal antibody of the present invention, an ELISA method, an ABC enzyme antibody method, a mouse mixed blood cell adsorption test [M-MHA], a cytoplasmic fluorescent antibody method and the like are used. The details of these methods are as follows. ELISA method This method selects IgG.

96-well plates (Nunc, Roskilde, Denmark) were plated with anti-mouse IgG antibody (specific for heavy and light chains) (Cappel
Company, Malvern, PA USA). The concentration was adjusted to 50 μg / ml with 0.05 M NaHCO ₃ -NaOH (pH 9.6) containing 0.1% NaN ₃ ,
Add 50 μl to each well. After incubating at room temperature for 1 hour, wash 3 times with PBS. Blocking
Use PBS containing 5% fetal bovine serum (FBS) and gelatin at a rate of 20 mg / ml. Incubate for 1 hour at room temperature and wash 3 times with PBS. Next, 50 μl of hybridoma culture supernatant is added to each well and incubated at room temperature for 1 hour. After washing 3 times with PBS containing 0.05% Tween 20, peroxidase-conjugated anti-mouse IgG antibody (F (ab ') ₂ fragment) (Fc fragment-specific) (Cappel) was added to 50μ.
l Add to each well. The concentration is 10 with PBS containing 5% FBS.
Dilute 00 times. After incubating at room temperature for 1 hour, the plate is washed 3 times with PBS containing 0.05% Tween20. next
O-phenylen-diamine 1.5mg / ml in 54mM acetate buffer,
Add H ₂ O _{2 to} a concentration of 0.35 μl / ml, and add 100
Add μl to each well to develop color. After reacting for 15 minutes, stop by adding 5 mM NaN ₃ . The absorbance of each well is
It was measured with a MTP22 dual wavelength spectrophotometer (Corona, Japan). ABC enzyme antibody method [Avidin-biotin-peroxidase complex method, ABC method]
Tissue staining is performed using the Vectastain ABC Kit (Vector, Burlinga
me, CA USA). The staining procedure was as follows: The plate on which the tissue section was placed was washed 3 times with PBS, and PBS containing 5% FBS was used.
Non-specific reactions are blocked by adding normal horse serum diluted 200-fold with. After incubation for 20 minutes at room temperature, the monoclonal antibody is added. 5 monoclonal antibodies
Use PBS diluted with% FBS from 2 μg / ml to 10 μg / ml. After incubating at room temperature for 1 hour or 4 ° C overnight, wash 3 times with PBS. In order to block the endogenous peroxidase, it is a mixture of 30% H ₂ O ₂ and methyl alcohol in a ratio of 1: 1000 20
Process for minutes. After washing 3 times with PBS, biotinylated horse anti-mouse Ig is diluted 200-fold with PBS containing 5% FBS and added. After incubating at room temperature for 30 minutes, wash 3 times with PBS. Then, an avidin-biotin conjugate to which peroxidase is bound is added. Prepare a 200-fold dilution with PBS at least 30 minutes before adding. After reacting for 45 minutes at room temperature, wash 3 times with PBS. Color development is 0.2M trisaminomethane 25ml, 0.2N hydrochloric acid 1
Distilled water was added to 9.2 ml to make 100 ml, and NaN ₃ 40 mg
And dissolve 20 mg of diaminobenzidine hydrochloride. Furthermore,
H ₂ O ₂ is added at a rate of about 0.03 μl / ml. The plate is reacted with the above solution for 7 minutes, and then the color development is stopped. Nuclear staining is performed with methyl green. Mouse mixed blood cell adsorption test [M-MHA] M-MHA is the original method of Espmark and Fagreus (Acta.Pathol.Micr
obiol.Scond.Suppl., 154 , 258-262, 1962).

The method for preparing the indicator red blood cells is as follows.
Washed sheep red blood cells 2% suspension and an equal amount of 1000-fold diluted mouse anti-sheep red blood cell antibody (prepared by hyperimmunizing BALB / c mice with sheep red blood cells) were reacted at 24 ° C for 45 minutes, and washed again to 2%. An equal volume of 200-fold diluted rabbit anti-mouse Ig (Cappel,
(US) for 45 minutes at 24 ° C., then washed twice and made into a 2% suspension again, which is used as so-called M-MHA indicator red blood cells.

As a method of the M-MHA assay, the cells to be searched are reacted with hybridoma cells, culture supernatant, or ascites of hybridoma cell-inoculated BALB / c mice or BALB / c-derived nude mice for 45 minutes at 24 ° C. After washing and removing the antibody, react with 0.2% indicator sheep red blood cells for 45 minutes at 24 ° C, lightly wash once with phosphate buffered saline (PBS), and then under the optical microscope, the presence or absence of rosette formation of the indicated red blood cells. Determine with. Cytoplasmic fluorescent antibody method 1 to 5 × 10 ⁴ target cells were fixed on a slide glass with 95% acetone for 10 minutes and stored at −70 ° C. and used as target cells.

As a primary antibody, 25 μl of hybridoma cell culture supernatant or hybridoma cell-inoculated BALB / c mouse or BALB / c-derived nude mouse ascites was used at 4 ° C.
Alternatively, react at 25 ° C for 30 minutes. After washing with PBS, the secondary antibody was diluted 20 to 40 times with fluorescently labeled rabbit mouse IgG + A + M (GA
M-FITC, Coulter, USA) is reacted at 25 ° C for 30 minutes. The reaction is judged by the presence or absence of fluorescence under a fluorescence microscope.

Periodate oxidation of frozen samples of antigen-positive tumors is carried out for 1 hour at room temperature using 10-100 mM metaperiodic acid. Neuraminidase digestion of frozen samples is performed as follows. That is, neuraminidase is Behringwer
Using the test-neuraminidase from ke AG, Marburg, West Germany,
Two kinds of concentration, 0.1 U / ml and 1 U / ml, are prepared. 2) These neuraminidase and frozen sections were plated on a plate.
Incubate at 37 ℃ for 24 hours Next, the monoclonal antibody is reacted, and the following procedure uses the Vectastain ABC kit for staining with the ABC method.

The dilution of neuraminidase is 154 mM.
50 mM acetate buffer (pH 5.5) containing NaCl. The thermostability of the antigen is determined by incubating the sample at 100 ° C for 15 minutes. Further, the stability test of the antigen in the section of the normal pathological tissue specimen treated with formalin is performed as follows. That is, a 5 μm section was prepared from a paraffin-embedded tissue block for normal histopathological examination, immersed in 100% xylene for 3 minutes to remove paraffin, and then
Wash with PBS and dry. Frozen sections from the same tissue are used as controls, and antibodies are diluted 100-fold and 1000-fold. After staining with the ABC enzyme antibody method, the staining properties of both sections are compared.

The following are references regarding the technology related to the present invention. 1. Bast, RC et al., Seminar in Oncology (Sem
in. Oncol.), 11: 264-274, 1984. Schlom, J., Cancer Res.,
46: 3225-3238, 1986. Nouwen, EJ et al., American Journal of
Pathology (Am.J.Pathol.), 126: 230-242, 1987. 4. Herlyn, M et al., Advance in Cancer Res., 49: 189-221, 1987. 5. Reisfeld, RA et al., Advance in Immunol., 40: 323-378, 1987. 6. Sears, HF et al., Lancet, i: 762-76
5, 1982. 7. Houghton, AN et al, Proceeding of the
National Academy of Science (Proc.Nat
L.Acad.Sci.), USA, 82: 1242-1246, 1985. 8. Bast, RC et al., Journal of Clinical Investigation (J.Clin.Invest.), 68: 1331-1.
337, 1981. 9. Bast, RC et al., New England Journal
Of Medicine (N.Engl.J.Med.), 309: 883-887, 198
3. 10. Klug, TL et al., Cancer Research
s.), 44: 1048-1053, 1984. 11. Bhattacharya, M. et al., Cancer Research (Cance
r Res.), 42: 1650-1654, 1982. 12. Mattes, MJ et al., Proceeding of the.
National Academy of Science (Proc.Nat
L. Acad. Sci.), USA, 81: 568-572, 1984. 13. Tagliabue, E. et al., Cancer Research
Res.), 45: 379-385, 1985. 14. Tsuji, Y. et al., Cancer Research
s.), 45: 2358-2362, 1985. 15. Sekine, H. et al., Journal of Clinical Oncol., 3: 1355-1363, 1985. 16. De Kretser, TA The European Journal
Of Cancer Clinical On Collology (Eur.
J. Cancer Clin. Oncol.), 21: 1019-1035, 1985. 17. Poels, LG et al., Journal of National.
Cancer Institute (J.Natl.Cancer Ins
t.), 76: 781-791, 1986. 18. Miotti, S. et al., International Journal.
Of Cancer (Int.J.Cancer.), 39: 297-303, 198
7. 19. Ward, BG et al., International Journal
Of, Cancer (Int. J. Cancer.), 39: 30-33, 1987. 20. Sakamoto, J. et al., Cancer Research
s.), 46: 1553-1561, 1986. 21. Kohler, G. et al., Nature (London), 25.
6: 495-497, 1975. 22. Ueda, R., et al., Proc. Natl. Aca Proceeding of the National Academy of Science.
d.Sci.), USA, 79: 4386-4390, 1982. 23. Kitagawa, H. et al., Japanese Journal Cancer Research (Jpn.J. Cancer Res.) Gann,
77: 922-930, 1986. 24. Namikawa, R. et al., Immunology, 57:
61-70, 1986. 25. Serov, SF et al., No. 9, Geneva: World Health
Organization (World Health Organization), 197
3. 26. Carey, TE et al., Proceeding of the
National Academy of Science (Proc.Nat
L. Acad. Sci.), USA, 73: 3278-3282, 1976. 27. Sakamoto, J. et al., Molecular Immunology (Mo.
l.Immunol.), 21: 1093-1098, 1984. 28. Gangopadhyay, A. et al., Cancer Research (Cance
r Res.), 45: 1744-1752, 1985. 29. Bara, J. et al., British Journal of.
Cancer (Br.J.Cancer), 36: 49-56, 1977. 30. Bara, J. et al., Cancer Research (Cancer Res.),
46: 3983-3989, 1986.31. Pant, KD et al., Hybridoma, 5: 129-135, 1986. 32. Pant, KD et al., American Journal of Clinical Pathology (Am.J.Clin.Pathol) .), 86: 1-9,
1986. 33. Scully, REIn: REScully (ed), Tumors of the
Ovary and MaldevelopedGonads, pp, 53-150, Washingt
on DC: Armed Force Institute
Armed Forces Institute of Patholo
gy), 1978. 34. Dallenbach-Hellweg, G., Journal of Cancer Research of Clinical Oncology
(J. Cancer Res. Clin. Oncol.), 107: 71-80, 1984. 35. Russel, P., Pathology, 17: 555-557,
1984. 36. Langley, FA et al., H. In: H. Fox (ed.) Haines and Tayler Obstetrical a
nd Gynaecological Pathology), Vol.l, pp.542-555, E
dinburgh: Churchill-Livingston, 1987. 37. Feizi, T. et al., Biochemical Society Trans Actions, 12: 591-596,
1984.

[0025]

INDUSTRIAL APPLICABILITY According to the present invention, a monoclonal antibody that specifically reacts strongly with mucinous ovarian tumor and belongs to the IgG ₁ isotype is provided, and a method for detecting human ovarian tumor using this monoclonal antibody is provided. It

[0026]

[Example 1]

Production of monoclonal antibody OM-C (1) Preparation of antibody-producing cells Surgical removal material for mucinous ovarian tumor was cut into small pieces with surgical scissors, and BALB / c mice (8 weeks old, 24 g) [Charles Ri
ver. Inc., Atsugi, Japan). After that, immunization was performed by inoculating the abdominal cavity three times every four weeks, and on the fourth day after the final immunization, the mouse was killed, the spleen was removed, and floating cells were obtained by a conventional method. (2) Cell fusion 10 ⁸ spleen floating cells and mouse myeloma MOPC-2
1NS / 1 and cell 2 × 10 ⁷ cells, 42% (W / V) PEG ( molecular weight 4,0
00, Koch-Light, Bucks, UK) and 15% (V / V) dimethylsulfoxide (Merck, Darmstadt, West Germany) in 0.2 ml of PBS [Proc. Natl. Acad. Sc.
i.USA, 78 , 5122-5126 (1981)]. (3) Selection of hybridoma Fused cells were prepared in HAT medium [hypoxanthine 1.36 mg / dl, aminopterin 19.1 μg / dl, thymidine 387 μg / dl, streptomycin 100 μg / ml, penicillin 100 units /
RPMI16 containing ml, glutamine 2mM and fetal bovine serum 10%
40 medium (pH 7.2)] and selected according to a conventional method (method of Ueda et al.). (4) Production of Monoclonal Antibody The three fused cells obtained were cloned by repeating the limiting dilution method three times. The obtained clones (3 clones) were cultured by the following method to obtain a strain producing a significant amount of a monoclonal antibody that specifically reacts with an ovarian tumor-associated antigen.
That is, RPMI1640 medium containing 5 × 10 ⁶ hybridoma cells containing streptomycin 100 μg / ml, penicillin 100 unit / ml, glutamine 3.8 g / l, and fetal calf serum 10% 15
ml in a 150 cm ² flask in the presence of 5% CO ₂
It was cultured at 7 ° C for 7 days.

The monoclonal antibody produced by the obtained cell line OM-C was designated as OM-C. The above-mentioned cell line OM-C was obtained from the Institute of Microbial Science and Technology, the Institute of Industrial Science and Technology, by the Micro Engineering Research Institute No. 9877.
Deposited as (FERM P-9877). When this cell line was cultured by the following method of intraperitoneally administering to mice, 3 to 15 mg / ml of OM-C antibody was produced. That is, 0.5 ml of pristane was injected into the abdominal cavity of the mouse, and then 5 × 10 ⁶ was injected into the abdominal cavity of the 3rd week.
The hybridoma cells were transplanted, and ascites was collected 10 days later. (5) Purification of monoclonal antibody Monoclonal antibody accumulated in the culture of hybridoma cells or ascites fluid is collected and purified as follows.

The hybridoma was administered to the supernatant of the culture of the hybridoma or to the abdominal cavity of a pristane-treated mouse, and the ascites or serum obtained was fed at 12,000 rpm, 20
Process at 4 ° C for min. Collect the supernatant and dilute 10-fold with PBS. Then, while stirring with a stirrer, saturated ammonium sulfate is slowly added until the amount becomes double (50% saturation) in about 20 minutes. Then, the mixture is stirred for 30 minutes, transferred to a Falcon 2059 tube, and centrifuged at 4 ° C., 9,500 rpm (14,700 g) for 10 minutes. Add 0.025 M phosphate buffer (pH 8.0) to the precipitate in the same amount as the initial ascites or serum amount to dissolve. 0.025M for this
Dialyze against phosphate buffer (pH 8.0). A DEAE cellulose column is prepared using Whatman DE52, and the column is first washed with 0.025 M phosphate buffer (pH 8.0) at 4 ° C., and then semi-purified antibody with ammonium sulfate is run. 1M sodium chloride
Collect the 0.025M phosphate buffer elution fractions, put them in a dialysis membrane, and concentrate with PEG20,000. After removing PEG, dialyz with PBS.

The monoclonal antibody selection is performed by the ELISA method (E
LISA: Enzyme-linked immunosorbent assay), ABC enzyme antibody method, mouse mixed adsorption test (M-MHA), cytoplasmic fluorescent antibody method, etc. Selection of IgG isotype monoclonal antibodies is performed by the ELISA method, and selection of monoclonal antibodies reactive with mucinous tumors is performed by immunohistological staining. [Example 2] 76 tumors in the gynecological region were immunohistologically searched by the above-mentioned ABC enzyme antibody method using the monoclonal antibody OM-C and the control 2 antibody (CEA, CA125). The results are shown in Tables 1 to 4.

[0030]

[Table 1] Table 1 Immunohistochemical search of epithelial ovarian tumors using OM-C antibody and control 2 antibody (CEA, CA125) ―────────────────── ────────────── ABC Staining result by enzyme antibody method ^b) Case number Histological diagnosis ^a) ―───────────────── OM-C CEA CA125 ──────────────────────────────── 1 Mucinous tumor M 2 ++ ^c) 3 ++-2 M 3 ++ 3 ++ 3 ++ 3 M 2 ++ 3 ++ 3 ++ 4 LPM 3 ++ 1 ++ − 5 LPM 3 ++ 2+ 2 ++ 6 LPM 3 ++ 2+ 2 ++ 7 LPM 3 ++ 3 ++ 3+ 8 LPM 3 ++ 3 ++ − 9 LPM 3 ++ 3 ++ − 10 LPM − 3 ++ 3 ++ 11 B − − − 12 B 3 ++ 3 ++ − 13 B 3 ++ 3 ++ 2+ 14 B − 3+ − …………………………………………………………………………………… 15 Serum Sexual tumor M 1+ − 3 ++ 16 M 1+ 1+ 3 ++ 17 M − 2 ++ 3+ 18 M 1 ++ 3 ++ − 19 M 1+ 1 ++ 3 ++ 20 M 1+ 1 ++ 3 ++ 21 M 1+ 1 ++ 3+ 22 M 1+ − 3 ++ 23 M 1+ − 3 ++ 24 M 1+ − 3 ++ 25 M − 1 ++ 3 ++ 26 M − 1+ 2+ 27 M − − 2 ++ 28 M − − 3+ 29 LPM − − 3 ++ 30 LPM − 2+ 3 ++ ……………………………………………………………… …………………… 31 Type M renal kidney tumor M 2+ 3+ 2 ++ 32 M − 2 ++ 1 ++ 33 M − 2 ++ − 34 M − 2 ++ 1 ++ 35 M − 1 ++ − 36 M − − 1 ++ 37 M − − 3 ++ 38 M − − 3 ++ 39 M − − 2+ ………………………………………………………… …………………………………… 40 Endometrial tumor M 1 ++ 2 ++ 3 ++ 41 M 2+ 2+ − 42 M − 1+ 2+ ……………………… …………………………………………………………… 43 Unclassifiable M − − − ───────────────────── ───────────── a) Organizational diagnosis is based on WHO classification. M: malignant tumor; LPM:
Low-grade tumor B: benign tumor b) Frozen tissue sections were stained with an antibody concentration of 5 μg / ml. c) Positive cell rate 1, <10%; 2, 10- <50%;
3, ≧ 50%.

Staining intensity: -negative, + positive, ++ strongly positive.

[0032]

[Table 2] Table 2 Immunohistological search of tumors and normal tissues in the gynecological region using 0M-C antibody ―─────────────────────── ──── Number of positive staining tissues Number of tissues tested in tissue ―────────── 0M-C ―───────────────────────── ─── Tumor Tissue Ovary, epithelial tumor ^a) 43 13 (10) Mucinous tumor 14 11 Serous tumor 16 0 (9) Mesorenal tumor 9 1 Intimal tumor 3 1 (1) Not classified 1 0 Germ cell tumor ^b) 8 2 ^c) Granulosa cell tumor 10 Metastatic tumor ^d) 6 2 ^e) (1) ^f) Uterine and cervical cancer 6 2 (1) Adenocarcinoma 3 2 (1) Squamous cell carcinoma 3 0 Endometrial cancer 60 (1) Myoma 6 0 Orthogonal tissue Ovary 40 0 Fallopian tube 40 (4) ^g) Cervical duct 30 ^h) Endometrium 50 (1) ⁱ⁾ Womb Board 20 Fetal tissue ^j) Egg nest 30 Fallopian tube 2 2 ^k) ―─────────────────────────── a) Table 1 Summary b) Embryonal carcinoma 2, Anaplastic germ cell tumor 1, Undifferentiated Mature teratoma 1,
It consists of mature teratoma 3 and squamous cell carcinoma 1. c) non-specific mucin glands of fetal and mature teratomas d) metastasis 5 from gastric cancer and metastasis from rectal cancer 1 e) metastasis from gastric cancer f) metastasis from rectal cancer g) reserve cells h) Mucosal epithelium and mucus i) A part of intimal epithelial cells j) Tissue obtained from 3 fetuses (30, 32 and 42 weeks old) k) Epithelium facing the gland duct lumen

[0033]

[Table 3] Table 3 Immunohistological search of tumors other than gynecological region using 0M-C antibody ────────────────────────── ─ Number of positive staining tissues Tissue count Number of tissues tested ───────── 0M-C ────────────────────────── Upper skin Gastrointestinal cancer Esophageal cancer 20 Gastric cancer 5 4 Large intestine cancer 5 5 Pancreatic cancer 20 Renal cancer 20 (1) ^a) Lung cancer, Small cell cancer 80 Large cell cancer 70 Adenocarcinoma 8 1 (1) ) Squamous cell carcinoma 70 (1) Breast cancer 5 1 Sarcoma ^b) 40 Melanoma 30 Carcinoid 20 Neurological tumor ^c) 70 Lymphoma T cell type 20 B cell type 20 ──────── ─────────────────── a) Number of samples with 10% positive cells in parentheses [See Note (c) in Table 1] b) Leiomyosarcoma 2, bone Sarcoma 1 and liposarcoma 1 c) glioma 2, glioblastoma multiforme 1, medulloblastoma 1 terminal neuroma 1, meningioma 1 and neuroblastoma Cell tumor 1

[0034]

[Table 4] a) Tissue from 4 fetuses (11, 16, 20, 27 weeks old) b) Epithelial positive c) Germ cells of intestinal metaplasia d) 11 weeks old, −; 16 weeks old, + e) Germ cells f ) Secretion g) Conduit epithelium h) Bronchial epithelium i) Thymic epithelium of medullary part The control antibody CEA was distributed by Dr. Kozo Imai, Department of Internal Medicine, Sapporo Medical University, and CA125 was purchased from Centocore. In addition, as tumors and tissues used in the examples, tissues surgically excised at the Aichi Cancer Center Gynecology Department and the Nagoya University Gynecology Department were used after explaining the purpose of retrieval to the patient or family and consent.

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Claims (3)

[Claims] 1. A monoclonal antibody produced by a hybridoma obtained by fusing mouse splenocytes immunized with human mucinous ovarian tumor cells and mouse myeloma cells and having the following properties. Immunoglobulin class: IgG ₁ Reacts with mucinous ovarian tumor, cervical adenocarcinoma, gastric cancer, and colon cancer. It does not respond to serous tumors, mesothelioma, uterine fibroids, esophageal cancer, lung cancer, breast cancer, sarcoma, melanoma, carcinoid, nervous system tumors, lymphoma. It does not react with normal cells of the ovary, fallopian tube, endometrium, placenta, esophagus, liver, kidney, mammary gland, thyroid, spleen, brain and skin. 2. The monoclonal antibody according to claim 1, which is produced from the hybridoma strain OM-C (FERM P-9877). 3. The monoclonal antibody according to claim 1 or 2 when detecting a human ovarian tumor using an ELISA method, an ABC enzyme antibody method, a mouse mixed blood cell adsorption test, or a cytoplasmic fluorescent antibody method. A method for detecting a human ovarian tumor characterized by being used.