JPH0690785A - Monoclonal antibody against human mucin ovarian tumor and method for detecting human ovarian tumor using the same monoclonal antibody - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody specifically and strongly reacting with a mucin ovarian tumor, belonging to IgG1 isotype and to detect human ovarian tumor by using the monoclonal antibody. CONSTITUTION:A new anti-human ovarian tumor monoclonal antibody specifically reacts with human ovarian tumor. An anti-human ovarian tumor monoclonal antibody shows high specificity to mucin human ovarian tumor. In detecting human ovarian tumor cell by ELISA method, ABC enzyme antibody method, mouse mixed hemadsorption virus test, cell fluorescent antibody method, etc., an anti-human ovarian tumor monoclonal antibody OM-A is used to detect human ovarian tumor.

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]. Furthermore, treatment of cancer patients using 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. Have reported a CA125 monoclonal antibody that reacts with a carbohydrate antigen that is mainly present in serous ovarian tumors [J. Clin. Invest., 68:13.
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 a monoclonal antibody shown below 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 spleen cells immunized with human mucinous ovarian tumor cells and mouse myeloma cells, and having the following properties.

Immunoglobulin class: IgG ₁ Reacts with mucinous ovarian tumors. Serous tumor, mesothelioma, germ cell ovarian tumor, cervical cancer, endometrial cancer, uterine fibroid, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, lung cancer, breast cancer, sarcoma, melanoma, carcinoid,
Does not respond to nervous system tumors or lymphomas.

It does not react with normal cells of the ovary, fallopian tube, cervix, endometrium, placenta, esophagus, gallbladder, liver, kidney, mammary gland, thyroid gland, spleen, brain and skin. (2) The monoclonal antibody according to (1), which is produced from the hybridoma strain OM-A (FERM P-9875). (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-A. Such a monoclonal antibody belongs to the class (isotype) IgG ₁ of immunoglobulin (Ig). (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 (198
2)]

BALB / c mice
F1 mouse, such as a mouse, BALB / c mouse and other mouse
Can be Immunization: 1 mouse (4-8 weeks old, 20-30g)
Against mucinous ovarian tumor cells 10 ^7-82-5 using pieces
Do 2-3 times every week. Rearing mice and collecting splenocytes
Follows the usual law. Cell fusion Splenocytes and myeloma cells are mixed at a ratio of 1: 1 to 10: 1.
Combined and fused with NaCl (about 0.85%), dimethylsulfoxy
Poly [10-20% (V / V)] and molecular weight 1000-6000
Phosphate buffer containing ren glycol (hereinafter referred to as PEG)
Perform in saline (hereinafter referred to as PBS) (pH 7.2-7.4)
U

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)], S
194/5, 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 / dl)
, Thymidine (375-400 μg / dl), streptomycin
(50-100 μg / ml), penicillin (50-100 units),
Glutamine (3.5-4.0g / l), fetal bovine serum (10-20%)
Using basal medium containing, 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. Hybridoma culture Cloning of fused cells (hybridomas) is performed by the limiting dilution method at least three times.

When the hybridoma is cultured in the same manner as in the culture of normal animal cells, 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 , 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, 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 tetram
0.5 to 1 ml of ethyl pentadecane (manufactured by Aldrich, USA) 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. The 1M sodium chloride-0.025M phosphate buffer elution fractions are collected, placed on a dialysis membrane and concentrated with PEG 20000. 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-A (hereinafter referred to as OM-A antibody in principle) was resistant to sialidase treatment (0.5 U, 37 ° C, 2 hours). In the periodate treatment (10 mM, room temperature, 1 hour), the 0M-A antibody showed sensitivity. Regarding heat resistance (100 ° C, 15 minutes), the OM-A antibody was slightly sensitive.

Regarding the stability of the antibody in a section of a normal pathological tissue specimen treated with formalin, the OM-A antibody was stable. The immunohistological search for OM-A antibody is as described in Example 2. The OM-A antibody shows high specificity for mucinous ovarian tumors. OM-A antibody reacts with many mucinous ovarian tumors [10 (1) / 14 cases, 10% or less of tumor cells are positive in parentheses (the same applies below)], otherwise, It reacts only weakly [0 (2) / 3] with endometrioid tumors and not with serous tumors (0/16) and mesothelioma (0/9). OM-A antibody does not react with germ cell ovarian tumor (0/8) and is negative for cervical cancer (0/6), endometrial cancer (0/6) and uterine myoma (0/6) Is. Half of the ovarian metastases of gastrointestinal cancer are positive [1 (2) / 6]. In 71 cases of tumors outside the gynecological region, 10% or less of positive cells were found in 1 out of 5 cases of gastric cancer and 1 out of 8 cases of lung adenocarcinoma.

Regarding the reactivity to normal and fetal tissues, the OM-A antibody does not react with the ovary, fallopian tube, cervix, endometrium, placenta, and does not react with the fetal-derived ovary or fallopian tube. In normal tissues other than the gynecological region, it is positive in the epithelium of stomach, small intestine, acinar cells of pancreas, bronchial epithelium of lung, and small intestine and large intestine in fetal tissue. 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 specificity different from that of CEA antibody and CA125 antibody, and It is clearly different from the ABH-Lewis blood group antibody and other previously reported ovarian tumor associated antibodies.

The above-mentioned monoclonal antibody OM-A is a known ovarian tumor monoclonal antibody (1D ₃ , MO _V -1,4C7
The difference is as follows. i.OM-A antibody recognizes intracellular antigens and is a group of antibodies against well-analyzed tumor-associated antigens, many previously reported blood group-related antigens, CEA, CA125 antigens, etc. Is different from. ii. CA12, which is frequently used as an ovarian tumor-related antibody
The immunohistological reaction pattern differs from that of the 5 antibody and CEA antibody.
Compared with these antibodies, the prepared antibody OM-A has higher specificity for mucinous ovarian tumors and has a clear character and is useful. iii. Monoclonal antibodies with high specificity against mucinous ovarian tumors include 1D ₃ , MO _V -1, 4C7 and the like.

Both 1D ₃ and MO _V -1 antibodies are similar in reactivity to OM-A antibody, but the former responds to primary gastric cancer and colon cancer, whereas OM-A antibody Not responding to primary cancer,
Reacts to gastric cancer and colon cancer that have metastasized to the ovary. Also, OM-A antibody reacts with ovarian endometrium tumors but not the former two. Even in normal tissues, the former two react with normal colon epithelial cells. OM-A antibody reacts with fetal large intestine but not with normal colon epithelial cells, but only with gastric epithelium.

The 4C7 and OM-A antibodies have similar reactions to mucinous ovarian tumors, but the former shows a positive reaction with mesorenal tumors, while the latter shows a negative reaction. In the normal tissue, the former showed negative in both large intestine and gastric epithelium, which is different from the reactivity of the above-mentioned OM-A antibody to them. (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 diagnosis using the monoclonal antibody of the present invention, the ELISA method, the ABC enzyme antibody method, the mouse mixed blood cell adsorption test [M-MHA], the 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 is adjusted to 50 µg / ml with 0.05 M NaHCO ₃ -NaOH (pH 9.6) containing 0.1% NaN ₃ , and 50 µl of the solution is added to each well. After incubating at room temperature for 1 hour, wash 3 times with PBS. Blocking is 5
Use PBS containing% 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% Tween20,
50 μl peroxidase-conjugated anti-mouse IgG antibody (F (ab ') ₂ fragment) (Fc fragment specific) (Cappel) is added to each well. Dilute 1000 times with PBS containing 5% FBS. After incubating at room temperature for 1 hour,
Wash 3 times with PBS containing 05% Tween20. Next, in 54 mM acetate buffer (pH 5.5), O-phenylen-diamine 1.5 mg / m
l, H ₂ O ₂ 0.35 μl / ml to give a concentration of 1
Add 00 μl to each well to develop color. After reacting for 15 minutes, stop by adding 5 mM NaN ₃ . The absorbance of each well was measured with an MTP22 dual wavelength spectrophotometer (Corona, Japan). ABC enzyme antibody method (Avidin-biotin-peroxidase complex
Vectastain ABC kit (Vector, Burlingam)
e, 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.
robiol.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 the 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.

Hybridoma cell culture supernatant or hybridoma cell inoculated BALB / c mouse or BALB / c-derived nude mouse ascites (25 μl) was used as the primary antibody 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 with 10-100 mM metaperiodate for 1 hour at room temperature. Neuraminidase digestion of frozen samples is performed as follows. That is, neuraminidase is Behringw
erke AG, Marburg, West Germany test-neuraminidase is used, and its concentration is adjusted to two types, 0.1 U / ml and 1 U / ml. These neuraminidase and frozen sections are reacted on a plate for 2 hours at 37 ° C. 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.

Documents relating to the technology related to the present invention are shown below. 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 Investment (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 Et, 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.
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, 1
973. 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 Transactions (Biochem.Soc.Trans.), 12: 591-596,
1984.

[0028]

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

[0029]

[Example 1]

Production of Monoclonal Antibody OM-A (1) Preparation of Antibody-Producing Cells The surgical excision material of 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
Culture was performed at 7 ° C for 7 days.

The monoclonal antibody produced by the obtained cell line OM-A was designated as OM-A. The above-mentioned cell line OM-A was obtained from the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, Microtechnical Institute No. 9875.
Deposited as (FERM P-9875). When this cell line was cultured by the following method of intraperitoneally administering to mice, 3 to 15 mg / ml of OM-A antibody was produced. That is, 0.5 ml of pristane was injected into the abdominal cavity of the mouse, and then 5 x 10
^Six 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 administered at 12,000 rpm, 20 rpm.
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% saturated) 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 amount of ascites or serum 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. Fractions eluted with 1M sodium chloride-0.025M phosphate buffer are collected, placed on a dialysis membrane and concentrated with PEG20,000. After removing PEG, dialyz with PBS.

The selection of the monoclonal antibody is carried out 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] An immunohistological search was performed on 76 cases of gynecologic tumors using the monoclonal antibody OM-A and the control 2 antibody (CEA, CA125) by the ABC enzyme antibody method. The results are shown in Tables 1 to 4.

[0033]

[Table 1] Table 1 Immunohistochemical search of epithelial ovarian tumors using OM-A and control 2 antibody (CEA, CA125) ―─────────────────── ───────────── ABC enzyme-antibody method ^b) Case number Histological diagnosis ^a) ―─────────────────── OM- A CEA CA125 ―─────────────────────────────── 1 Mucinous tumor M 3 ++ ^c) 3 ++-2 M 3 ++ 3 ++ 3 ++ 3 M − 3 ++ 3 ++ 4 LPM 3 ++ 1 ++ − 5 LPM 2+ 2+ 2 ++ 6 LPM 2+ 2+ 2 ++ 7 LPM 2+ 3 ++ 3+ 8 LPM 2+ 3 ++ − 9 LPM 1+ 3 ++ − 10 LPM − 3 ++ 3 ++ 11 B 3 ++ − − 12 B 3+ 3 ++ − 13 B 2+ 3 ++ 2+ 14 B − 3+ − …………………………………………………………………………………… 15 Serous tumor M − − 3 ++ 16 M − 1+ 3 ++ 17 M − 2 ++ 3+ 18 M − 3 ++ − 19 M − 1 ++ 3 ++ 20 M − 1 ++ 3 ++ 21 M − 1 ++ 3 + 22 M − − 3 ++ 23 M − − 3 ++ 24 M − − 3 ++ 25 M − 1 ++ 3 ++ 26 M − 1+ 2+ 27 M − − 2 ++ 28 M − − 3 + 29 LPM − − 3 ++ 30 LPM − 2+ 3 ++ …………………………………………………………………………………… 31 Middle renal tumor M −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 − 2 ++ 3 ++ 41 M 1+ 2+ − 42 M 1+ 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.

[0035]

[Table 2] Table 2 Immunohistochemical search of tumors and normal tissues in gynecological region using OM-A antibody ―─────────────────────── ─── Number of positive staining tissues Number of tissues tested in tissue ―───────── OM-A ―────────────────────────── Tumor tissue Ovary, epithelial tumor ^a) 43 10 (3) ^b) Mucinous tumor 14 10 (1) Serous tumor 16 0 Mesorenal tumor 9 0 Intimal tumor 30 0 (2) Unclassifiable 10 Germ cell Tumor ^c) 80 Granulosa cell tumor 10 Metastatic tumor ^d) 61 ^e) 2 ^e) Uterine and cervical cancer 60 Adenocarcinoma 30 Squamous cell carcinoma 30 Intimal carcinoma 60 Myoma 60 Normal tissue Egg nest 4 0 Fallopian tube 4 0 Cervical gland duct 3 0 Endometrium 5 0 Placenta 20 Fetal tissue ^f) Egg nest 30 0 Fallopian tube 2 0 ―─────────────── ─────────── a) Summary of Table 1 b) The numbers in parentheses are the number of samples with 10% or less positive cells (see Note (c) in Table 1) c) embryonal carcinoma 2, dysgerminoma 1, immature Kikei carcinoma 1,
It consists of mature teratoma 3 and squamous cell carcinoma 1. d) Metastasis from gastric cancer 5 and metastasis from rectal cancer 1 e) Metastasis from gastric cancer f) Tissue obtained from 3 fetuses (30, 32 and 42 weeks old)

[0036]

[Table 3] Table 3 Immunohistochemical search of tumors other than gynecological region using OM-A antibody ―───────────────────────── ─ ― Number of positive staining tissues Number of tissues tested ────────── OM-A ―────────────────────────── -Epithelial tumor Esophageal cancer 20 Gastric cancer 50 (1) ^a) Colorectal cancer 50 Pancreatic cancer 20 Kidney cancer 20 Lung cancer, small cell cancer 80 Large cell cancer 70 Adenocarcinoma 80 (1) Squamous epithelium Cancer 70 Breast cancer 50 Sarcoma ^b) 40 Melanoma 30 Carcinoid 20 Nervous system tumor ^c) 70 Lymphoma T cell type 20 B cell type 2 0 ―───────────── ────────────── a) Number of samples with 10% positive cells in parentheses [See Note (c) in Table 1] b) Leiomyosarcoma 2, Osteosarcoma 1 and Liposarcoma 1 c) glioma 2, glioblastoma multiforme 1, medulloblastoma 1 terminal neuroma 1, meningioma 1 and neuroblastoma Cell tumor 1

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[Table 4] a) Tissue from 4 fetuses (11, 16, 20, 27 weeks old) b) Only a part of the epithelium is weakly positive c) Epithelium is positive d) 16 weeks old, −; 27 weeks old, + e) Secretory f ) Acinar cells g) Bronchial epithelium CEA, a control antibody, 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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─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12N 15/06 G01N 33/574 D 9015-2J (C12P 21/08 C12R 1:91)

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 tumors. Serous tumor, mesothelioma, germ cell ovarian tumor, cervical cancer, endometrial cancer, uterine fibroid, esophageal cancer, gastric cancer, colon cancer, pancreatic cancer, lung cancer, breast cancer, sarcoma, melanoma, carcinoid,
Does not respond to nervous system tumors or lymphomas. Does not react with normal cells of ovary, fallopian tube, cervix, endometrium, placenta, esophagus, gallbladder, liver, kidney, mammary gland, thyroid gland, thymus, spleen, brain and skin. 2. The monoclonal antibody according to claim 1, which is produced from the hybridoma strain OM-A (FERM P-9875). 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.