Molecular and Cellular Probes (1991) 5, 55-63
Monoclonal antibodies against CA125-bearing antigenic molecule fragments ; reactivity with mucinous ovarian tumours and lung cancers Shunsuke Imai,'* Hiroko Maeda,' Yasuhiko Kiyozuka, 2 Junji Morimoto,' Satomi Haga,' Tsuneo Noda,2 Shingo Hiroishi 3 and Jo Hilgers4 'Department of Pathology, 'Department of Obstetrics and Gynecology, Nara Medical University, Kashihara, Nara, 634, Japan, 3Shiraimatsu Shinyaku Co . Ltd., Minakuchi-cho, Kohka-gun, Shiga, 528, Japan, and 4Academisch Ziekenhuis, Vrije Universiteit, De Boelelaan, Amsterdam, The Netherlands (Received 18 May 1990, Accepted 23 July 1990)
We first established a cell strain, SHIN-3, from human ovarian serous cystadenocarcinoma, and performed antigen analysis for CA125 which appeared to be massively secreted by the SHIN-3 cells . Protein digestion analysis revealed that a low molecular weight peptide of 49 kDa showed antigen activity . In the present study, we describe a mouse monoclonal antibody against this low molecular peptide, presumed to be part of the CA125-bearing antigenic molecule . Purified antigen prepared from culture supernatant was adsorbed to nitrocellulose membranes and injected intrasplenically in mice . Of the obtained 398 clones, 10 clones were selected by screening in an ELISA test . Of the 10, two were further selected, i .e . SH-9. This hybridoma produces IgG1 monoclonal antibodies . Immunoblotting analysis revealed that SH-9 recognizes the low molecular peptide used as the immunogen . Immunohistological examination with the SH-9 MAb revealed that the antigen reacted with the bronchial epithelium, cervical glands of the uterus and other various normal tissues . Of tumorous tissues, the antibody mainly reacted with ovarian tumours, but positive reactions were also observed with pulmonary adenocarcinomas or squamous cell carcinomas . Surprisingly the positive rate was high in mucinous tumour of the ovary, while no positive reaction was observed in serous tumours . Dot-blot assay using SH-9 revealed that 17/19 (90%) sera of lung cancer patients were positive for the titre suggesting that SH-9 may be useful to set up a serum test for lung cancers . KEYWORDS :
monoclonal antibody (SH9), CA125 associated antigen, ovarian tumour, lung cancer .
INTRODUCTION The CA125 antigenic molecule is recognized by a mouse monoclonal antibody, OC125, established by Bast et al .' by using human ovarian cancer culture cells as the immunogen . The CA125 assay is a very useful tumour marker test for the management of ovarian cancer patients with the serous adenocarcinoma. ',2 The test is not very useful for the differential diagnosis between benign and malignant tumours,
because the serum level of CA125 is elevated in cases with endometriosis, in women with early stages of .',',' pregnancy and during the menstrual phase We succeeded in establishing a cell strain (SHIN-3) constantly producing CA125 from human ovarian serous cystadenocarcinoma and have studied biochemical and physical characteristics of the CA125bearing molecule, secreted in the culture supernatant
`Author to whom correspondence should be addressed .
0890-8508/91/010055 + 09 $03 .00/0
55
© 1991 Academic Press Limited
56
S . Imai et al.
of these cells . 6 •7 We have previously reported that a low molecular peptide having molecular weight of about 49 kDa reveals the antigen activity . 1,7 In the
Cell fusion and cloning
MATERIALS AND METHODS
Cell fusion was performed in accordance with the method of KOhler & Milstein ." Spleen cells obtained from immunized mice were fused with P3-X63 myeloma cells at a ratio of 7 :1 by using 50% PEG 1540 (polyethylene glycol, Wako Pure Chemical Industries, Ltd . Kyoto, Japan) . The cells were washed and diluted with RPMI 1640 and pipetted into a 96 well microplate at the final concentration of 3 x 10 5 spleen cells per well . The cells were maintained by HAT medium .
Immunogen
After 2 or 3 weeks, culture supernatants were assayed for antibody activity by ELISA . Cells showing
present study, we have succeeded in preparing a monoclonal antibody against this antigen (so called SH-9), and obtained antibodies which have biological and immunohistological characteristics different from those of OC125 prepared by Bast et a! . 1 .2
The SHIN-3 cell strain was established from a specimen extirpated during surgery for ovarian cancer .' The cells were cultured in serum-free medium (HB 104; Hana Media Inc . Alameda, CA) and culture supernatant was collected . The supernatant was centrifuged at 1000 rpm for 10 min, concentrated by salting-out with half-saturated ammonium sulphate, dialyzed and lyophilized . The two materials described below were used as immunogens . (a) Group I . The lyophilized SHIN-3 culture supernatant was subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli et al .' and transferred to a nitrocellulose membrane . The desired protein fraction was detected by immunoblotting via Avidin-biotin peroxidase complex method (ABC method) using anti-CA125 antibody (HIST 125 KIT; Green Cross Corp.)' The fraction was cut into pieces in physiological saline and pulverized in a polytron (Kinematica, Switzerland) . (b) Group II . The lyophilized SHIN-3 culture supernatant was equilibrated in 10 mm Tris-HCI buffer (pH 7.4) containing 0-5m NaCl, 0-1% SDS and 6m urea and applied to a Sephacryl S-300 column . The eluted fractions were examined for CA125 activity by radioimmunoassay . CA125 active fractions were collected, concentrated, and directly adsorbed by a nitrocellulose membrane. The membrane was pulverized in the same manner as group I .
high activity were subjected to cloning twice by limiting dilution (0 . 5, 1 .0 cells well - ') using 3 x 105 cells well - ' of thymus cells from BALB/c mice as feeder cells .
ELISA Fraction II obtained by gel filtration with Sephacryl S-300 was resuspended in carbonate buffer (pH 9 .5) to 40 µg ml . - ' The suspension was pipetted into a 96 well microplate (100 µl well - ') and allowed to stand overnight at 4 ° C for coating . After washing with PBS, containing 0 . 01 % Tween-20, 50 tl of culture supernatant was placed and incubated at 37°C for 60 min . Then, peroxidase labelled rabbit anti-mouse IgG (Cappel) diluted 1500-fold was added and incubated for 60 min . Finally, 0 . 5 mg ml -1 of o-phenylene diamine was added and the reaction was stopped by 10% H 2S0 4.
Immunohistological examination (Avidin-biotin peroxidase complex; ABC method) Immunohistological examination was performed on formalin-fixed paraffin-embedded human tissue specimens with a Vectastain ABC Kit. 12
Competitive inhibition study Immunization method Eight-week-old male BALB/c mice were used for immunization . A volume of 0 . 2 ml of the antigen solution (about 5 .tg of protein) was injected into the spleen of each animal . 10 After 1 month, an equal amount of the antigen solution was intraperitoneally injected as a booster. A cell fusion experiment was performed 3 days after the booster injection .
A CA125 competition study against various antibodies (SH-9, OC125 or CEA) was performed in order to compare the epitopes of SH-9 with those of OC125 or CEA . Purified antigen, 50µl, and 50µl of 10-fold diluted unlabelled SH-9 and CEA antibody was incubated at room temperature for 2 h . Then, 50 gi of 12S 1OC125 was added . After incubation at 4° C overnight, 16. 7 gl of rabbit antimouse IgG and 37 . 5 tl of 10% suspension of Sct (Staphylococcus aureus) was added . After centrifugation, the pellets were counted .
57
Mucinous ovarian tumours and lung cancer Dot-blot immunoassay using SH-9 antibody Dot-blot assay was performed as follows ." ," Briefly, a dot-blot kit was set according to the manual . Samples were applied to each well and adsorbed by the nitrocellulose membrane. A volume of 3% BSA50 mm Tris-HCI buffer, pH 7.6, containing 0 . 5 M NaCl was added to each well as a blocking solution . Each well was washed with Tris-HCI buffer, pH 7 . 6, containing 0 . 5 M NaCl, three times for 10 min each . A SH-9 monoclonal antibody was reacted for 1 h at room temperature. Each well was washed three times for 10 min with the same buffer followed by the ABC method as described previously ."
was dissolved in a sample buffer containing SDS and 2ME and electrophoresed by 10% polyacrylamide gel . The sample on the gel was transferred to a nitrocellulose membrane and subjected to immunoblotting (Fig.1 ) . Immunological activity was observed at the high molecular fraction over 200,000 Da and the low molecular fraction around 49,000 Da . The protein fraction of lower molecular weight was cut from the nitrocellulose membrane and used as immunogen (I) . The lyophilized sample was fractioned by Sephacryl S-300 column chromatography . Radioimmunoassay with anti-CA125 antibody revealed activity in fraction II . The fractions were collected, concentrated and directly absorbed by a nitrocellulose membrane and used as immunogen (II) .
RESULTS Purification of CA125 antigen derived from SHIN-3 and preparation of immunogen
Preparation of monoclonal antibody
Culture supernatant of SHIN-3 was treated with 50% ammonium sulphate for salting-out and then dia-
Spleen cells of BALB/c mice sensitized by the intrasplenic immunization method were fused with P3X63 . Ag8 .653 myeloma cells . Produced clones were
lyzed followed by lyophilization . The obtained sample
subjected to primary screening by ELISA using frac-
TOP .+
. - 94 -0-67 -o-43
.0-30
f- 20
-14
Fig. 1 . SDS: 10% PAGE of CA125 antigen isolated from SHIN-3 supernatant . Immunoblotting analysis identified area of greater than 200,000 Da molecular mass ( I ) and a band near 50,000 Da ( a-- ) .
58
S . Imai et al.
tion II . Ten highly active clones (seven from Group I and three from Group II) were examined by the enzyme-labelled antibody technique (ABC method) using paraffin-embedded sliced specimens of ovarian serous or mucinous cystadenocarcinoma . All of the 10 clones showed reaction with the tumour cells . Of the 10 clones, five which were the most markedly
and the two IgG1 antibodies of Group II recognized only the low molecular bands . Without 2ME, antibodies of Group II reacted strongly with the upper gel .
positive were processed by limiting dilution, and finally, two monoclonal antibodies were obtained from Group I and three from Group II . Double immunodiffusion (ID) analysis using anti-mouse IgG1, IgG2a, IgG2b, IgG3 and IgM rabbit sera (Miles Laboratory Inc ., IL) revealed that two antibodies from Group II were IgG1 and The remaining three were IgM .
Fifty-fold diluted antibodies were immunohistologically assessed by the ABC method using formalinfixed paraffin-embedded blocks of various normal tissues and ovarian tumours. Since non-specific reaction is marked with IgM antibodies, only IgG1 antibodies were examined . Of normal tissues, only the cervical glands of the uterus and bronchial epithelium were strongly stained . The
Immunohistological examination
epithelial cells of the crypt were stained in one stomach cancer tissue (Table 1) . Of the tumour tissues, mucinous ovarian tumour tissue and lung cancer (adenocarcinoma and squamous cell carcin-
Characteristics of antibodies Immunoblotting
oma) were markedly stained (Fig . 3 and Table 2) . Table 3 compares the immunohistological reactivity between SH-9, our antibody and OC125 . While OC125 was relatively highly specific for serous tumours, SH-9 was characterized by marked reactivity
To compare the reactivities of antibodies obtained in this study and that of OC125 antibody, fraction II was electrophoresed with and without 2ME and subjected to immunoblotting (Fig . 2) . With 2ME, while OC125 reacted with both high molecular bands around 200,000 and low molecular bands around 49,000, the two antibodies of Group I
A
with mucinous tumours . Although OC125 stained normal endometrial glands and tissues with endometriosis, SH-9 did not react with these tissues at all .
C
B
a
b
a
b
a
b
Fig. 2. Immunoblotting analysis of fraction II with A ; IgM antibody (1), B; SH-9 and C; OC125. (a) 2ME(+),(b) 2ME(-) .
Mucinous ovarian tumours and lung cancer Table 1 .
MAb SH-9 reactivity with various organs
Tissues
Positive/total
Table 2. %
Female genital organ Ovary Fallopian tube Endometrium (secretory and proliferative) Endometrium (gestational) Endocervix Uterus cervical gland
0/10 0/2 2/2 6/7
0 0 100 86
Other tissues Brain Breast Bone marrow Artery Heart Lung-bronchus -pleura Thyroid Skin Oesophagus Stomach (foveolar epithelium) Duodenum Rectum Gallbladder Liver Adrenal Kidney Lymphnode Spleen Pancreas
0/3 0/5 0/3 0/3 0/3 5/5 0/5 0/3 0/3 0/2 1/1 0/2 0/3 0/1 0/3 0/2 0/2 0/3 0/3 0/2
0 0 0 0 0 100 0 0 0 0 100 0 0 0 0 0 0 0 0 0
0/20 0/5
0 0
Competitive inhibition study of SH-9 compared with those of OC125 and CEA Figure 4 shows the competitive inhibition study of SH-9 by use of labelled SH-9 and OC125 or unlabelled CEA . The curves obtained did not compete, showing that the epitopes of SH-9 are different from those of OC125 or CEA .
59
MAb SH-9 reactivity with various tumours
Tumours Female genital organ Ovarian tumour serous cystadenoma serous cystadenoma of borderline malignancy papillary serous cystadenocarcinoma mucinous cystadenoma mucinous cystadenoma of borderline malignancy mucinous cystadenocarcinoma endometrioid adenocarcinoma Brenner tumour granulosa cell tumour dermoid cyst (bronchial glands) Ovarian non-tumour region chocolate cyst paraovarian cyst simple cyst Cervix uteri condyloma dysplasia carcinoma in situ squamous cell carcinoma sarcoma Uterus endometrial hyperplasia well diff . adenocarcinoma adenomyosis leiomyoma Metastatic tumour Krukenberg tumour Other tissues Mammary carcinoma mastopathy gynecomastia Lymphoma Lung: squamous cell carcinoma adenocarcinoma Hepato-cellular carcinoma Gallbladder adenocarcinoma Rectal adenocarcinoma sarcoma
Positive/total
0/3
0
0/3
0
0/8 5/5
0 100
3/3 3/3 0/2 1/1 0/1 1/9
100 100 0 100 0 11
0/3 0/2 0/1
0 0 0
0/3 0/4 0/2 0/3 1/2
0 0 0 0 50
0/25 0/5 0/16 0/5
0 0 0 0
012
0
0/2 0/2 0/1 0/3 1/3 5/5 0/1 0/1 0/3 0/1
0 0 0 0 33 100 0 0 0 0
Dot-blot immunoassay using SH-9 in the sera from the patient with lung cancer The positive rates for adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma in the lung were 5/5 (100%), 9/10 (90%), 2/2 (100%), and 1/2 (50%) respectively (Table 4) .
DISCUSSION A cancer related-antigen, CA125, is reported to be a glycoprotein with molecular weight over 200 kDa which is recognized by a mouse monoclonal antibody, OC125, obtained by immunization using
human ovarian serous cystadenocarcinoma cells, OVCA 433 .' 5 The immunoradiometric assay (IRMA) with OC125 antibody is widely used for serological diagnosis of gynaecological malignant tumours . This antibody, however, is not necessarily specific to cancer, and positive reactions are also observed in the early stage of pregnancy, in some cases with normal endometrium and cases with benign diseases including cystic endometrial hyperplasia . Recently, Matsuoka et al ." reported that a monoclonal antibody, anti-CA130, derived from pulmonary adenocarcinoma cross-reacts with the CA125 antigen . They noted that their antibody specifically
60
S. Imai et al.
Fig. 3. (a) Immunoperoxidase staining shows strong positivity in the mucous as well as mucinous cystadenoma cells (-a) (ABC assay, original magnification x 120) . (b) Immunoperoxidase staining shows strong positivity in the mucous as well as mucinous cystadenocarcinoma cells (+) (ABC assay, original magnification x 200) . (c) Immunoperoxidase staining shows strong positivity in the luminar pulmonary adenocarcinoma cells ( .4) (ABC assay, original magnification x 200) .
Mucinous ovarian tumours and lung cancer Table 3 .
Comparison of MAb reactivity of SH-9 vs .
OC125
Tumours Serous tumours Benign Borderline Malignant Mucinous tumour Benign Borderline Malignant Endometrioid adenocarcinomas Endometrial hyperplasia Adenomyosis Krukenberg tumour
SH-9
OC125
Positive/ total %
Positive/ total %
0/3 0/3 0/9
(0) (0) (0)
3/3 3/3 6/9
(100) (100) (67)
5/5 3/3 3/3
(100) (100) (100)
0/5 0/3 0/3
(0) (0) (0)
0/2 0/25 0/16 0/2
(0) (0) (0) (0)
2/2 (100) 10/19 (53) 3/16 (19) 2/2 (100)
reacted with not only ovarian cancer (serous cystadenocarcinoma, in particular) as previously reported but also pulmonary cancer . It is thought that the feature of anti-CA130 antibody is essentially similar to that of the OC125 antibody . We have established a cell strain (SHIN-3) constantly producing CA125 from human ovarian serous cystadenocarcinoma and have studied the characteristics of CA125 . We previously reported that a low molecular component of molecular weight of about 49,000 was important for its antigenic activity .' We have focused our study on this peptide and attempted to prepare a new antibody which was more highly specific than currently reported monoclonal antibodies . In the present study, a purified low molecular protein fraction (Group I) and a crude high molecular protein fraction (Group II) were adsorbed by nitrocellulose membranes, and directly injected into the spleens of mice for immunization ." Of antibodies produced by obtained hybridoma, only IgG antibodies were immunohistologically studied because
61
Since antigens recognized by OC125 and antiCA130 are unstable at warmer temperatures, frozen sliced specimens have been used in immunohistological studies ."', " We used paraffin-embedded sliced specimens in the present study and obtained very favourable staining results by the ABC method . Application of paraffin-embedded specimens is advantageous from the viewpoints of permanent preservation and ease of handling . The antigen recognized by SH-9 antibody was stable under heating . This finding also suggests that the antibody is distinct from the currently used antibodies . When immunohistological characteristics were compared between SH-9 and OC125 by staining various tissues with the antibodies, marked differences were observed . Of normal tissues, SH-9 strongly reacted with the bronchial epithelium of the lung and the cervical glands of the uterus, as did OC125 . However, while OC125 stained the endometrium in the pregnant period or proliferation phase and tissues from cases with endometritis, endometrial cancer or ovarian serous cystadenocarcin."'" SH-9 did not react with these tissues . On the oma other hand, 100% of the reaction rate was obtained when tissues from cases with benign, intermediate and malignant mucinous ovarian tumour were treated with SH-9 . These findings indicate that the tissue staining specificity is definitely different between OC125 and SH-9 . Anti-CA130 antibody was reported to react strongly with pulmonary adenocarcinoma . 16 Positive reaction for SH-9 was also observed in many cases with lung cancer in the present study,' ,' and they were adenocarcinoma and squamous cell carcinoma . Moreover, in sera from patient with lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma), about 90%
non-specific reactions were marked in IgM antibodies . Analysis by immunoblotting revealed that anti-
was positive by dot-blot assay by use of SH-9 antibody . Therefore, SH-9 antibody is very useful to detect lung cancer patients by just applying it in the sera . Further studies are needed to differentiate between anti-CA130 and SH-9 antibodies in this respect. The antibodies prepared in the present study were established by using antigens derived from an ovarian
bodies of Group I recognized several bands around 50,000 although intensity of reaction varied between antibodies . Similar results were obtained with IgG antibodies of Group II . On the other hand, OC125 recognized both high and low molecular fractions (over 200,000 and about 50,000) . Without a reducing agent, our IgG antibodies reacted with very high molecular proteins . It is judged from these results that the antigen molecule recognized by our antibodies is apparently different from that for OC125 .
cystadenocarcinoma cell strain, but they were highly specific to ovarian mucinous tumour and lung cancers . In immunoblotting analysis, recognition of the low molecular component by the present antibodies was similar to that by OC125 . It is presumed from these results that high molecular components presenting antigens which histologically characterize ovarian tumours are essentially very similar to one another. Detailed analysis into this problem is projected for further studies .
S. Imai et al.
62
100
75
50
25
0 0 M0 M
100 75 50 25 0 0. 01
0.1
10 I SH-9(µg ml l )
100
1000
Fig. 4. (a) Competitive inhibition studies using (NH ,),SO, Ppt from SHIN-3 supernatant . Radioiodinated SH-9 antibody was incubated with increasing amounts of unlabelled SH-9 (0) and CEA ( •) antibodies . (b) Competitive inhibition studies using (NH,),SO, Ppt from SHIN-3 supernatant . Radioiodinated OC125 ( •) and SH-9 (0) antibodies were incubated with increasing amounts of unlabelled SH-9 antibody . The results were expressed as the percentage of radioactivity bound (B) in the absence of unlabelled SH-9.
ACKNOWLEDGEMENTS
Table 4 . Positive ratio of SH-9 using dot-blot assay in the sera with lung cancer patients Histological type Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Small cell carcinoma Normal human sera
This work was supported in part by a Grant-in-Aid from the Terumo Life Science.
Positive/total 5/5 9/10 1/2 2/2 0/15
100 90 50 100 0
REFERENCES 1 . Bast, R . C ., Feeney, M., Lazarus, H ., Nadler, L. M., Colvin, R. B . & Knapp, R . C. (1981). Reactivity of a monoclonal antibody with human ovarian carcinoma . Journal of Clinical Investigation 68, 1331-7 .
Mucinous ovarian tumours and lung cancer 2 . Bast, R . C . Jr ., Klug, T . L ., St . John, E. et al. (1983) . A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer . New England Journal of Medicine 309, 883-7 . 3 . Schilthus, M . S ., Aalders, J . G ., Bouma, J . et al. (1987) . Serum CA125 levels in epithelial ovarian cancer : Relation with findings at second-look operations and their role in the detection of tumour recurrence. British Journal of Obstetrics and Gynecology 94, 202-7 . 4. Kuzuya, K., Nozaki, M . & Chihara, T. (1986). Evaluation of CA125 as a circulating tumor marker for ovarian cancer . Acta Obstetrica et Gynaecologica japonica 38, 949-57. 5 . Takahashi, K., Kijima, S ., Yoshino, K., Shibukawa, T ., Maruo, T . & Kitao . M . (1986). Differential diagnosis between uterine myoma and endometriosis using CA125 as a new tumor marker of ovarian carcinoma . Asia-Oceania Journal of Obstetrics and Gynaecology 11,99-103 . 6 . Imai, S ., Maeda, H ., Kiyozuka, Y ., Noda, T ., Moriyama, I . & Ichijo, M . (1989) . Characterization of CA125 antigen secreted from a newly established human ovarian cancer cell line (SHIN-3). Acta Pathologica japonica 39, 43-9 . 7 . Imai, S ., Kiyozuka, Y ., Maeda, H ., Noda, T., Ichijo, M . & Hosick, H . L . (1990) . Establishment and characterization of a human ovarian serous cystadenocarcinoma cell line that produces the tumor marker, CA125 and TPA . Oncology 47, 177-84 . 8 . Laemmli U .K . (1970) . Cleavage of structure proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-5 . 9 . Burnette W . N . (1981) . Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gells to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated proteins . Analytical Biochemistry 112, 195-203 .
63
10 . Spitz, M ., Spitz, L ., Thorpe, R . & Eugui, E . (1984) . Intrasplenic primary immunization for the production of monoclonal antibodies . journal of Immunological Methods 70, 39-43. 11 . Kohler, G . & Milstein, C . (1975). Continuous cultures of fused cells secreting antibody of predifined specificity. Nature 256, 495-7. 12 . Hsu, J . M ., Raine, L., & Fanger, H . (1981). Use of avidinbiotin- peroxidase complex (ABC) in immunoperoxidase techniques : a comparison between ABC and unlabeled antibody (PAP) procedures. journal of Histochemistry and Cytochemistry 29, 577 . 13. Hawkes, R ., Niday, E . & Gordon, J . (1982) . A dotimmunobinding assay for monoclonal and other antibodies . Analytical Biochemistry 119, 142-7. 14. Bio-dot microfiltration apparatus instruction manual . Bio-Rad Laboratories, 32nd & Griffin Ave., Richmond, CA 94804 . 15 . Davis, H . M ., Zuzauski, V . R ., Bast, R . C ., Jr ., & Klug, T. L . (1986) . Characterization of the CA125 antigen association with human epithelial ovarian carcinomas . Cancer Research 46, 6143-8 . 16 . Matsuoka, Y ., Nakashima, T ., Endo, K . et al . (1987) . Recognition of ovarian cancer antigen CA125 by murine monoclonal antibody produced by immunization of lung cancer cells . Cancer Research 47, 6335-40 . 17 . Kabawat, S . E., Bast, R . C ., Jr ., Bhan, A . K ., Werch, W . R ., Knapp, R . C ., & Colvin, R . B. (1983). Tissue distribution of a coelomic epithelium related antigen recognized by the monoclonal antibody OC125. Gynecological Pathology 2, 275-85 . 18 . Kabawat, S . E ., Bast, R . C ., Welch, W . R ., Knapp, R. C ., & Colvin, R . B. (1983) . Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid, and clear cell types . American Society of Clinical Pathology 79, 98-104 .
Monoclonal antibodies against CA125-bearing antigenic molecule fragments ; reactivity with mucinous ovarian tumours and lung cancers Shunsuke Imai,'* Hiroko Maeda,' Yasuhiko Kiyozuka, 2 Junji Morimoto,' Satomi Haga,' Tsuneo Noda,2 Shingo Hiroishi 3 and Jo Hilgers4 'Department of Pathology, 'Department of Obstetrics and Gynecology, Nara Medical University, Kashihara, Nara, 634, Japan, 3Shiraimatsu Shinyaku Co . Ltd., Minakuchi-cho, Kohka-gun, Shiga, 528, Japan, and 4Academisch Ziekenhuis, Vrije Universiteit, De Boelelaan, Amsterdam, The Netherlands (Received 18 May 1990, Accepted 23 July 1990)
We first established a cell strain, SHIN-3, from human ovarian serous cystadenocarcinoma, and performed antigen analysis for CA125 which appeared to be massively secreted by the SHIN-3 cells . Protein digestion analysis revealed that a low molecular weight peptide of 49 kDa showed antigen activity . In the present study, we describe a mouse monoclonal antibody against this low molecular peptide, presumed to be part of the CA125-bearing antigenic molecule . Purified antigen prepared from culture supernatant was adsorbed to nitrocellulose membranes and injected intrasplenically in mice . Of the obtained 398 clones, 10 clones were selected by screening in an ELISA test . Of the 10, two were further selected, i .e . SH-9. This hybridoma produces IgG1 monoclonal antibodies . Immunoblotting analysis revealed that SH-9 recognizes the low molecular peptide used as the immunogen . Immunohistological examination with the SH-9 MAb revealed that the antigen reacted with the bronchial epithelium, cervical glands of the uterus and other various normal tissues . Of tumorous tissues, the antibody mainly reacted with ovarian tumours, but positive reactions were also observed with pulmonary adenocarcinomas or squamous cell carcinomas . Surprisingly the positive rate was high in mucinous tumour of the ovary, while no positive reaction was observed in serous tumours . Dot-blot assay using SH-9 revealed that 17/19 (90%) sera of lung cancer patients were positive for the titre suggesting that SH-9 may be useful to set up a serum test for lung cancers . KEYWORDS :
monoclonal antibody (SH9), CA125 associated antigen, ovarian tumour, lung cancer .
INTRODUCTION The CA125 antigenic molecule is recognized by a mouse monoclonal antibody, OC125, established by Bast et al .' by using human ovarian cancer culture cells as the immunogen . The CA125 assay is a very useful tumour marker test for the management of ovarian cancer patients with the serous adenocarcinoma. ',2 The test is not very useful for the differential diagnosis between benign and malignant tumours,
because the serum level of CA125 is elevated in cases with endometriosis, in women with early stages of .',',' pregnancy and during the menstrual phase We succeeded in establishing a cell strain (SHIN-3) constantly producing CA125 from human ovarian serous cystadenocarcinoma and have studied biochemical and physical characteristics of the CA125bearing molecule, secreted in the culture supernatant
`Author to whom correspondence should be addressed .
0890-8508/91/010055 + 09 $03 .00/0
55
© 1991 Academic Press Limited
56
S . Imai et al.
of these cells . 6 •7 We have previously reported that a low molecular peptide having molecular weight of about 49 kDa reveals the antigen activity . 1,7 In the
Cell fusion and cloning
MATERIALS AND METHODS
Cell fusion was performed in accordance with the method of KOhler & Milstein ." Spleen cells obtained from immunized mice were fused with P3-X63 myeloma cells at a ratio of 7 :1 by using 50% PEG 1540 (polyethylene glycol, Wako Pure Chemical Industries, Ltd . Kyoto, Japan) . The cells were washed and diluted with RPMI 1640 and pipetted into a 96 well microplate at the final concentration of 3 x 10 5 spleen cells per well . The cells were maintained by HAT medium .
Immunogen
After 2 or 3 weeks, culture supernatants were assayed for antibody activity by ELISA . Cells showing
present study, we have succeeded in preparing a monoclonal antibody against this antigen (so called SH-9), and obtained antibodies which have biological and immunohistological characteristics different from those of OC125 prepared by Bast et a! . 1 .2
The SHIN-3 cell strain was established from a specimen extirpated during surgery for ovarian cancer .' The cells were cultured in serum-free medium (HB 104; Hana Media Inc . Alameda, CA) and culture supernatant was collected . The supernatant was centrifuged at 1000 rpm for 10 min, concentrated by salting-out with half-saturated ammonium sulphate, dialyzed and lyophilized . The two materials described below were used as immunogens . (a) Group I . The lyophilized SHIN-3 culture supernatant was subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli et al .' and transferred to a nitrocellulose membrane . The desired protein fraction was detected by immunoblotting via Avidin-biotin peroxidase complex method (ABC method) using anti-CA125 antibody (HIST 125 KIT; Green Cross Corp.)' The fraction was cut into pieces in physiological saline and pulverized in a polytron (Kinematica, Switzerland) . (b) Group II . The lyophilized SHIN-3 culture supernatant was equilibrated in 10 mm Tris-HCI buffer (pH 7.4) containing 0-5m NaCl, 0-1% SDS and 6m urea and applied to a Sephacryl S-300 column . The eluted fractions were examined for CA125 activity by radioimmunoassay . CA125 active fractions were collected, concentrated, and directly adsorbed by a nitrocellulose membrane. The membrane was pulverized in the same manner as group I .
high activity were subjected to cloning twice by limiting dilution (0 . 5, 1 .0 cells well - ') using 3 x 105 cells well - ' of thymus cells from BALB/c mice as feeder cells .
ELISA Fraction II obtained by gel filtration with Sephacryl S-300 was resuspended in carbonate buffer (pH 9 .5) to 40 µg ml . - ' The suspension was pipetted into a 96 well microplate (100 µl well - ') and allowed to stand overnight at 4 ° C for coating . After washing with PBS, containing 0 . 01 % Tween-20, 50 tl of culture supernatant was placed and incubated at 37°C for 60 min . Then, peroxidase labelled rabbit anti-mouse IgG (Cappel) diluted 1500-fold was added and incubated for 60 min . Finally, 0 . 5 mg ml -1 of o-phenylene diamine was added and the reaction was stopped by 10% H 2S0 4.
Immunohistological examination (Avidin-biotin peroxidase complex; ABC method) Immunohistological examination was performed on formalin-fixed paraffin-embedded human tissue specimens with a Vectastain ABC Kit. 12
Competitive inhibition study Immunization method Eight-week-old male BALB/c mice were used for immunization . A volume of 0 . 2 ml of the antigen solution (about 5 .tg of protein) was injected into the spleen of each animal . 10 After 1 month, an equal amount of the antigen solution was intraperitoneally injected as a booster. A cell fusion experiment was performed 3 days after the booster injection .
A CA125 competition study against various antibodies (SH-9, OC125 or CEA) was performed in order to compare the epitopes of SH-9 with those of OC125 or CEA . Purified antigen, 50µl, and 50µl of 10-fold diluted unlabelled SH-9 and CEA antibody was incubated at room temperature for 2 h . Then, 50 gi of 12S 1OC125 was added . After incubation at 4° C overnight, 16. 7 gl of rabbit antimouse IgG and 37 . 5 tl of 10% suspension of Sct (Staphylococcus aureus) was added . After centrifugation, the pellets were counted .
57
Mucinous ovarian tumours and lung cancer Dot-blot immunoassay using SH-9 antibody Dot-blot assay was performed as follows ." ," Briefly, a dot-blot kit was set according to the manual . Samples were applied to each well and adsorbed by the nitrocellulose membrane. A volume of 3% BSA50 mm Tris-HCI buffer, pH 7.6, containing 0 . 5 M NaCl was added to each well as a blocking solution . Each well was washed with Tris-HCI buffer, pH 7 . 6, containing 0 . 5 M NaCl, three times for 10 min each . A SH-9 monoclonal antibody was reacted for 1 h at room temperature. Each well was washed three times for 10 min with the same buffer followed by the ABC method as described previously ."
was dissolved in a sample buffer containing SDS and 2ME and electrophoresed by 10% polyacrylamide gel . The sample on the gel was transferred to a nitrocellulose membrane and subjected to immunoblotting (Fig.1 ) . Immunological activity was observed at the high molecular fraction over 200,000 Da and the low molecular fraction around 49,000 Da . The protein fraction of lower molecular weight was cut from the nitrocellulose membrane and used as immunogen (I) . The lyophilized sample was fractioned by Sephacryl S-300 column chromatography . Radioimmunoassay with anti-CA125 antibody revealed activity in fraction II . The fractions were collected, concentrated and directly absorbed by a nitrocellulose membrane and used as immunogen (II) .
RESULTS Purification of CA125 antigen derived from SHIN-3 and preparation of immunogen
Preparation of monoclonal antibody
Culture supernatant of SHIN-3 was treated with 50% ammonium sulphate for salting-out and then dia-
Spleen cells of BALB/c mice sensitized by the intrasplenic immunization method were fused with P3X63 . Ag8 .653 myeloma cells . Produced clones were
lyzed followed by lyophilization . The obtained sample
subjected to primary screening by ELISA using frac-
TOP .+
. - 94 -0-67 -o-43
.0-30
f- 20
-14
Fig. 1 . SDS: 10% PAGE of CA125 antigen isolated from SHIN-3 supernatant . Immunoblotting analysis identified area of greater than 200,000 Da molecular mass ( I ) and a band near 50,000 Da ( a-- ) .
58
S . Imai et al.
tion II . Ten highly active clones (seven from Group I and three from Group II) were examined by the enzyme-labelled antibody technique (ABC method) using paraffin-embedded sliced specimens of ovarian serous or mucinous cystadenocarcinoma . All of the 10 clones showed reaction with the tumour cells . Of the 10 clones, five which were the most markedly
and the two IgG1 antibodies of Group II recognized only the low molecular bands . Without 2ME, antibodies of Group II reacted strongly with the upper gel .
positive were processed by limiting dilution, and finally, two monoclonal antibodies were obtained from Group I and three from Group II . Double immunodiffusion (ID) analysis using anti-mouse IgG1, IgG2a, IgG2b, IgG3 and IgM rabbit sera (Miles Laboratory Inc ., IL) revealed that two antibodies from Group II were IgG1 and The remaining three were IgM .
Fifty-fold diluted antibodies were immunohistologically assessed by the ABC method using formalinfixed paraffin-embedded blocks of various normal tissues and ovarian tumours. Since non-specific reaction is marked with IgM antibodies, only IgG1 antibodies were examined . Of normal tissues, only the cervical glands of the uterus and bronchial epithelium were strongly stained . The
Immunohistological examination
epithelial cells of the crypt were stained in one stomach cancer tissue (Table 1) . Of the tumour tissues, mucinous ovarian tumour tissue and lung cancer (adenocarcinoma and squamous cell carcin-
Characteristics of antibodies Immunoblotting
oma) were markedly stained (Fig . 3 and Table 2) . Table 3 compares the immunohistological reactivity between SH-9, our antibody and OC125 . While OC125 was relatively highly specific for serous tumours, SH-9 was characterized by marked reactivity
To compare the reactivities of antibodies obtained in this study and that of OC125 antibody, fraction II was electrophoresed with and without 2ME and subjected to immunoblotting (Fig . 2) . With 2ME, while OC125 reacted with both high molecular bands around 200,000 and low molecular bands around 49,000, the two antibodies of Group I
A
with mucinous tumours . Although OC125 stained normal endometrial glands and tissues with endometriosis, SH-9 did not react with these tissues at all .
C
B
a
b
a
b
a
b
Fig. 2. Immunoblotting analysis of fraction II with A ; IgM antibody (1), B; SH-9 and C; OC125. (a) 2ME(+),(b) 2ME(-) .
Mucinous ovarian tumours and lung cancer Table 1 .
MAb SH-9 reactivity with various organs
Tissues
Positive/total
Table 2. %
Female genital organ Ovary Fallopian tube Endometrium (secretory and proliferative) Endometrium (gestational) Endocervix Uterus cervical gland
0/10 0/2 2/2 6/7
0 0 100 86
Other tissues Brain Breast Bone marrow Artery Heart Lung-bronchus -pleura Thyroid Skin Oesophagus Stomach (foveolar epithelium) Duodenum Rectum Gallbladder Liver Adrenal Kidney Lymphnode Spleen Pancreas
0/3 0/5 0/3 0/3 0/3 5/5 0/5 0/3 0/3 0/2 1/1 0/2 0/3 0/1 0/3 0/2 0/2 0/3 0/3 0/2
0 0 0 0 0 100 0 0 0 0 100 0 0 0 0 0 0 0 0 0
0/20 0/5
0 0
Competitive inhibition study of SH-9 compared with those of OC125 and CEA Figure 4 shows the competitive inhibition study of SH-9 by use of labelled SH-9 and OC125 or unlabelled CEA . The curves obtained did not compete, showing that the epitopes of SH-9 are different from those of OC125 or CEA .
59
MAb SH-9 reactivity with various tumours
Tumours Female genital organ Ovarian tumour serous cystadenoma serous cystadenoma of borderline malignancy papillary serous cystadenocarcinoma mucinous cystadenoma mucinous cystadenoma of borderline malignancy mucinous cystadenocarcinoma endometrioid adenocarcinoma Brenner tumour granulosa cell tumour dermoid cyst (bronchial glands) Ovarian non-tumour region chocolate cyst paraovarian cyst simple cyst Cervix uteri condyloma dysplasia carcinoma in situ squamous cell carcinoma sarcoma Uterus endometrial hyperplasia well diff . adenocarcinoma adenomyosis leiomyoma Metastatic tumour Krukenberg tumour Other tissues Mammary carcinoma mastopathy gynecomastia Lymphoma Lung: squamous cell carcinoma adenocarcinoma Hepato-cellular carcinoma Gallbladder adenocarcinoma Rectal adenocarcinoma sarcoma
Positive/total
0/3
0
0/3
0
0/8 5/5
0 100
3/3 3/3 0/2 1/1 0/1 1/9
100 100 0 100 0 11
0/3 0/2 0/1
0 0 0
0/3 0/4 0/2 0/3 1/2
0 0 0 0 50
0/25 0/5 0/16 0/5
0 0 0 0
012
0
0/2 0/2 0/1 0/3 1/3 5/5 0/1 0/1 0/3 0/1
0 0 0 0 33 100 0 0 0 0
Dot-blot immunoassay using SH-9 in the sera from the patient with lung cancer The positive rates for adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma in the lung were 5/5 (100%), 9/10 (90%), 2/2 (100%), and 1/2 (50%) respectively (Table 4) .
DISCUSSION A cancer related-antigen, CA125, is reported to be a glycoprotein with molecular weight over 200 kDa which is recognized by a mouse monoclonal antibody, OC125, obtained by immunization using
human ovarian serous cystadenocarcinoma cells, OVCA 433 .' 5 The immunoradiometric assay (IRMA) with OC125 antibody is widely used for serological diagnosis of gynaecological malignant tumours . This antibody, however, is not necessarily specific to cancer, and positive reactions are also observed in the early stage of pregnancy, in some cases with normal endometrium and cases with benign diseases including cystic endometrial hyperplasia . Recently, Matsuoka et al ." reported that a monoclonal antibody, anti-CA130, derived from pulmonary adenocarcinoma cross-reacts with the CA125 antigen . They noted that their antibody specifically
60
S. Imai et al.
Fig. 3. (a) Immunoperoxidase staining shows strong positivity in the mucous as well as mucinous cystadenoma cells (-a) (ABC assay, original magnification x 120) . (b) Immunoperoxidase staining shows strong positivity in the mucous as well as mucinous cystadenocarcinoma cells (+) (ABC assay, original magnification x 200) . (c) Immunoperoxidase staining shows strong positivity in the luminar pulmonary adenocarcinoma cells ( .4) (ABC assay, original magnification x 200) .
Mucinous ovarian tumours and lung cancer Table 3 .
Comparison of MAb reactivity of SH-9 vs .
OC125
Tumours Serous tumours Benign Borderline Malignant Mucinous tumour Benign Borderline Malignant Endometrioid adenocarcinomas Endometrial hyperplasia Adenomyosis Krukenberg tumour
SH-9
OC125
Positive/ total %
Positive/ total %
0/3 0/3 0/9
(0) (0) (0)
3/3 3/3 6/9
(100) (100) (67)
5/5 3/3 3/3
(100) (100) (100)
0/5 0/3 0/3
(0) (0) (0)
0/2 0/25 0/16 0/2
(0) (0) (0) (0)
2/2 (100) 10/19 (53) 3/16 (19) 2/2 (100)
reacted with not only ovarian cancer (serous cystadenocarcinoma, in particular) as previously reported but also pulmonary cancer . It is thought that the feature of anti-CA130 antibody is essentially similar to that of the OC125 antibody . We have established a cell strain (SHIN-3) constantly producing CA125 from human ovarian serous cystadenocarcinoma and have studied the characteristics of CA125 . We previously reported that a low molecular component of molecular weight of about 49,000 was important for its antigenic activity .' We have focused our study on this peptide and attempted to prepare a new antibody which was more highly specific than currently reported monoclonal antibodies . In the present study, a purified low molecular protein fraction (Group I) and a crude high molecular protein fraction (Group II) were adsorbed by nitrocellulose membranes, and directly injected into the spleens of mice for immunization ." Of antibodies produced by obtained hybridoma, only IgG antibodies were immunohistologically studied because
61
Since antigens recognized by OC125 and antiCA130 are unstable at warmer temperatures, frozen sliced specimens have been used in immunohistological studies ."', " We used paraffin-embedded sliced specimens in the present study and obtained very favourable staining results by the ABC method . Application of paraffin-embedded specimens is advantageous from the viewpoints of permanent preservation and ease of handling . The antigen recognized by SH-9 antibody was stable under heating . This finding also suggests that the antibody is distinct from the currently used antibodies . When immunohistological characteristics were compared between SH-9 and OC125 by staining various tissues with the antibodies, marked differences were observed . Of normal tissues, SH-9 strongly reacted with the bronchial epithelium of the lung and the cervical glands of the uterus, as did OC125 . However, while OC125 stained the endometrium in the pregnant period or proliferation phase and tissues from cases with endometritis, endometrial cancer or ovarian serous cystadenocarcin."'" SH-9 did not react with these tissues . On the oma other hand, 100% of the reaction rate was obtained when tissues from cases with benign, intermediate and malignant mucinous ovarian tumour were treated with SH-9 . These findings indicate that the tissue staining specificity is definitely different between OC125 and SH-9 . Anti-CA130 antibody was reported to react strongly with pulmonary adenocarcinoma . 16 Positive reaction for SH-9 was also observed in many cases with lung cancer in the present study,' ,' and they were adenocarcinoma and squamous cell carcinoma . Moreover, in sera from patient with lung cancer (adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma), about 90%
non-specific reactions were marked in IgM antibodies . Analysis by immunoblotting revealed that anti-
was positive by dot-blot assay by use of SH-9 antibody . Therefore, SH-9 antibody is very useful to detect lung cancer patients by just applying it in the sera . Further studies are needed to differentiate between anti-CA130 and SH-9 antibodies in this respect. The antibodies prepared in the present study were established by using antigens derived from an ovarian
bodies of Group I recognized several bands around 50,000 although intensity of reaction varied between antibodies . Similar results were obtained with IgG antibodies of Group II . On the other hand, OC125 recognized both high and low molecular fractions (over 200,000 and about 50,000) . Without a reducing agent, our IgG antibodies reacted with very high molecular proteins . It is judged from these results that the antigen molecule recognized by our antibodies is apparently different from that for OC125 .
cystadenocarcinoma cell strain, but they were highly specific to ovarian mucinous tumour and lung cancers . In immunoblotting analysis, recognition of the low molecular component by the present antibodies was similar to that by OC125 . It is presumed from these results that high molecular components presenting antigens which histologically characterize ovarian tumours are essentially very similar to one another. Detailed analysis into this problem is projected for further studies .
S. Imai et al.
62
100
75
50
25
0 0 M0 M
100 75 50 25 0 0. 01
0.1
10 I SH-9(µg ml l )
100
1000
Fig. 4. (a) Competitive inhibition studies using (NH ,),SO, Ppt from SHIN-3 supernatant . Radioiodinated SH-9 antibody was incubated with increasing amounts of unlabelled SH-9 (0) and CEA ( •) antibodies . (b) Competitive inhibition studies using (NH,),SO, Ppt from SHIN-3 supernatant . Radioiodinated OC125 ( •) and SH-9 (0) antibodies were incubated with increasing amounts of unlabelled SH-9 antibody . The results were expressed as the percentage of radioactivity bound (B) in the absence of unlabelled SH-9.
ACKNOWLEDGEMENTS
Table 4 . Positive ratio of SH-9 using dot-blot assay in the sera with lung cancer patients Histological type Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Small cell carcinoma Normal human sera
This work was supported in part by a Grant-in-Aid from the Terumo Life Science.
Positive/total 5/5 9/10 1/2 2/2 0/15
100 90 50 100 0
REFERENCES 1 . Bast, R . C ., Feeney, M., Lazarus, H ., Nadler, L. M., Colvin, R. B . & Knapp, R . C. (1981). Reactivity of a monoclonal antibody with human ovarian carcinoma . Journal of Clinical Investigation 68, 1331-7 .
Mucinous ovarian tumours and lung cancer 2 . Bast, R . C . Jr ., Klug, T . L ., St . John, E. et al. (1983) . A radioimmunoassay using a monoclonal antibody to monitor the course of epithelial ovarian cancer . New England Journal of Medicine 309, 883-7 . 3 . Schilthus, M . S ., Aalders, J . G ., Bouma, J . et al. (1987) . Serum CA125 levels in epithelial ovarian cancer : Relation with findings at second-look operations and their role in the detection of tumour recurrence. British Journal of Obstetrics and Gynecology 94, 202-7 . 4. Kuzuya, K., Nozaki, M . & Chihara, T. (1986). Evaluation of CA125 as a circulating tumor marker for ovarian cancer . Acta Obstetrica et Gynaecologica japonica 38, 949-57. 5 . Takahashi, K., Kijima, S ., Yoshino, K., Shibukawa, T ., Maruo, T . & Kitao . M . (1986). Differential diagnosis between uterine myoma and endometriosis using CA125 as a new tumor marker of ovarian carcinoma . Asia-Oceania Journal of Obstetrics and Gynaecology 11,99-103 . 6 . Imai, S ., Maeda, H ., Kiyozuka, Y ., Noda, T ., Moriyama, I . & Ichijo, M . (1989) . Characterization of CA125 antigen secreted from a newly established human ovarian cancer cell line (SHIN-3). Acta Pathologica japonica 39, 43-9 . 7 . Imai, S ., Kiyozuka, Y ., Maeda, H ., Noda, T., Ichijo, M . & Hosick, H . L . (1990) . Establishment and characterization of a human ovarian serous cystadenocarcinoma cell line that produces the tumor marker, CA125 and TPA . Oncology 47, 177-84 . 8 . Laemmli U .K . (1970) . Cleavage of structure proteins during the assembly of the head of bacteriophage T4 . Nature 227, 680-5 . 9 . Burnette W . N . (1981) . Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfatepolyacrylamide gells to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated proteins . Analytical Biochemistry 112, 195-203 .
63
10 . Spitz, M ., Spitz, L ., Thorpe, R . & Eugui, E . (1984) . Intrasplenic primary immunization for the production of monoclonal antibodies . journal of Immunological Methods 70, 39-43. 11 . Kohler, G . & Milstein, C . (1975). Continuous cultures of fused cells secreting antibody of predifined specificity. Nature 256, 495-7. 12 . Hsu, J . M ., Raine, L., & Fanger, H . (1981). Use of avidinbiotin- peroxidase complex (ABC) in immunoperoxidase techniques : a comparison between ABC and unlabeled antibody (PAP) procedures. journal of Histochemistry and Cytochemistry 29, 577 . 13. Hawkes, R ., Niday, E . & Gordon, J . (1982) . A dotimmunobinding assay for monoclonal and other antibodies . Analytical Biochemistry 119, 142-7. 14. Bio-dot microfiltration apparatus instruction manual . Bio-Rad Laboratories, 32nd & Griffin Ave., Richmond, CA 94804 . 15 . Davis, H . M ., Zuzauski, V . R ., Bast, R . C ., Jr ., & Klug, T. L . (1986) . Characterization of the CA125 antigen association with human epithelial ovarian carcinomas . Cancer Research 46, 6143-8 . 16 . Matsuoka, Y ., Nakashima, T ., Endo, K . et al . (1987) . Recognition of ovarian cancer antigen CA125 by murine monoclonal antibody produced by immunization of lung cancer cells . Cancer Research 47, 6335-40 . 17 . Kabawat, S . E., Bast, R . C ., Jr ., Bhan, A . K ., Werch, W . R ., Knapp, R . C ., & Colvin, R . B. (1983). Tissue distribution of a coelomic epithelium related antigen recognized by the monoclonal antibody OC125. Gynecological Pathology 2, 275-85 . 18 . Kabawat, S . E ., Bast, R . C ., Welch, W . R ., Knapp, R. C ., & Colvin, R . B. (1983) . Immunopathologic characterization of a monoclonal antibody that recognizes common surface antigens of human ovarian tumors of serous, endometrioid, and clear cell types . American Society of Clinical Pathology 79, 98-104 .
