Proc. Natl. Acad. Sci. USA Vol. 76, No. 1, pp. 377-380, January 1979
Developmental Biology
Monoclonal antibodies reacting with murine teratocarcinoma cells (radioimmunoassay/cultured cells/adult tissues/preimplantation embryos)
PETER N. GOODFELLOW, JOHN R. LEVINSON, VIRGINIA E. WILLIAMS II, AND HUGH 0. MCDEVITT Division of Immunology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
Contributed by Hugh 0. McDevitt, October 16, 1978
Monoclonal antibodies were produced in vitro ABSTRACT by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000.
The cell surface has been shown to play an important role in several cell-cell interaction systems (for several examples see ref. 1). It has been postulated that cell-cell interaction in the developing mammalian embryo is also mediated by cell surface-expressed molecules (2). Consistent with this hypothesis is the production of antisera that can block the development of preimplantation embryos cultured in vitro (3, 4). Unfortunately these antisera, which are produced by immunization with embryos (3) or with teratocarcinoma cells (4), are complex and often difficult to analyze. This complexity makes it particularly difficult to interpret functional experiments. Recent progress in the in vitro production of monoclonal antibody (5, 6) has prompted us to use this technique for investigating cell surface antigens expressed on cultured murine teratocarcinomas and embryos. Cultured teratocarcinoma stem cells were used for the immunogen because of the close relationship of these cells to embryonic cells (7) and because of the ease of producing in vitro the large amounts of tissue needed for immunization and screening. We have produced several monoclonal antibodies that react with cultured teratocarcinomas. In this communication we describe the analysis of the first two of these monoclonal antibodies. The first antibody recognizes an antigen that is restricted to teratocarcinoma stem cells, the endodermal cell line PYS-2 (8), and the neuroblastoma C1300 (9). The second antibody recognizes an antigen expressed on adult mouse brain and ubiquitously on cultured murine cells. MATERIALS AND METHODS Cell Fusion to Produce Monoclonal Antibodies. A female Lewis rat was immunized with the embryo-derived C3H mouse teratocarcinoma cell line C86.S1 (10). Under the conditions used to maintain C86.S1 in vitro it does not undergo significant differentiation (10). The rat was primed by a single intraperitoneal injection of 108 C86-S1 cells homogenized in 0.5 ml of complete Freund's adjuvant. After 7 days the rat was boosted intravenously with 5 X 107 cells suspended in saline. Three days later the animal was bled and sacrificed, and the spleen was used as a source of immune cells for fusion to the BALB/c The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
mouse myeloma P3-X63-Ag8 (5). The fusion was performed as described by Kohler and Milstein (5), using the enriched medium described by Kennett et al. (11). P3-X63-Ag8 cells (3 X 107) were fused with 3 X 108 rat spleen cells; no attempt was made to remove erythrocytes. The fused cells were suspended in hypoxanthine/aminopterin/thymidine (HAT) selective medium (which selects against the myeloma parent) and divided equally amongl96 1-ml cultures. Cultures were fed by replacing half the medium in each well every 3 or 4 days. Cloning was achieved by the agar overlay method (12) or by limiting dilution. Cloning was performed in the absence of HAT selection. Cell Culture. All cells except PCC4/aza were grown in Dulbecco's modified Eagle's medium with high glucose (purchased from GIBCO) supplemented with 15% fetal calf serum (GIBCO), 100 international units of penicillin per ml, and 100 ,ug of streptomycin (GIBCO) per ml. PCC4/aza was grown in Dulbecco's modified Eagle's medium with low glucose (GIBCO). F9 was grown on gelatin-coated dishes (13). Attached cells were removed for testing by a combination of scraping and treatment with 2 mM EDTA in phosphate-buffered saline. References to the cell lines used are given in Table 1. Serological Techniques. Radioimmunoassay (RIA). All manipulations were performed in RIA buffer, which consisted of Dulbecco's modified phosphate-buffered saline supplemented with 5% fetal calf serum and 0.1% sodium azide. Staphylococcal protein A (purchased from Pharmacia) and purified rabbit F(ab')2 anti-rat Fab fragment of IgG (a gift from A. Williams, Medical Research Council Cellular Immunology Group, Oxford) were labeled by the method of Jensenius and Williams (14) and used at a specific activity of 20-30 ,uCi/,g (1 Ci = 3.7 X 1010 becquerels). C86*S1 target cells (2 X 105) in 10 Al of RIA buffer were added to round-bottomed multiwell plates and 50 ,u of culture supernatants was added. The cells and supernatants were incubated for 1 hr at room temperature and then washed twice. Labeled detecting reagent (105 cpm) in 50 ,u of RIA buffer was added and the mixture was incubated for 2 hr at room temperature or overnight at 4°C. After the incubation the cells were washed four times and assayed for radioactivity. Maximal binding with the immune serum from the rat spleen donor was about 4 X 104 cpm. The background binding to C86-S1 by supernatants from P3-X63-Ag8 cells was about 2 X 102 cpm. All tests were performed in duplicate. Testing cell lines by RIA. Supernatants were collected from cultures of clones 4A1-9 and 3C4-10 and from P3-X63-Ag8. The supernatants were concentrated 5-fold by using an Amicon ultrafiltration unit fitted with a PM30 filter. All cells were tested at a concentration of 2 X 105 cells per well (2 X 107/ml) with five serial doubling dilutions of the three concentrated supernatants. E/C values (see Fig. 1) given in Tables 1 and 2 represent the maximal value in the titration curve. Testing cells from adult mice. Thymus and spleen cells were prepared by mincing and teasing in RIA buffer, followed by Abbreviation: RIA, radioimmunoassay.
377
Developmental Biology: Goodfellow et al.
378
25
Proc. Nati. Acad. Sci. USA 76 (1979)
I ~~I ~I ~ ~I ~
IJ
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4A1
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10
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0
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-a
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WELL NUMBER FIG. 1. The open bars represent the test with radiolabeled anti-rat Fab. The solid bars represent the test with radiolabeled protein A.
E/C
=
cpm bound by cells + hybrid supernatant bound by cells + P3-X63-Ag8 supernatant'
cpm
filtration through muslin. Sperm cells were prepared by the method of Goldberg et al. (15). Testicular cells were prepared by repeated pipetting of seminiferous tubules followed by filtration through muslin. Absorption analysis was performed on crude membrane fractions prepared according to Acton et al. (16). Culture supernatants were diluted to give 80% maximal binding. A 15-Mul sample of diluted culture supernatant was absorbed with an equal volume of packed membranes for 1 hr at 4VC. Dilution errors were controlled. The results presented are the average of two independent determinations. The error is estimated to be +15%. All cells and tissues were taken from C3H/DiSn mice.
RESULTS On the 17th day after the fusion, 50 of 96 cultures showed growth. These 50 cultures were tested for the production of antibody recognizing the immunogen by RIA using both 125I-labeled protein A and 125I-labeled rabbit anti-rat Fab. The results are presented as a histogram in Fig. 1. Arbitrarily, an E/C ratio of greater than 4 was taken as a positive result; thus 12/96 wells were positive with the polyvalent reagent and 5/96 with the iodinated protein A. The two cultures with the highest E/C ratios (3C4 and 4A1) were chosen for cloning and further analysis. Initial cloning was achieved by the agar overlay method (12). Of 12 subclones of 3C4, 8 were producers of specific antibody; and 12 of 12 subclones for 4A1 were producers. The two subclones 3C4-10 and 4A1-9 were further cloned by the limiting dilution method. All subclones (16/16 and 10/10, respectively) were positive for anti-C86.Sl antibody production, suggesting that these original clones are stable
producers of monoclonal antibody. This has been confirmed by growing 3C4-10 and 4A1-9 continuously in vitro for several months and by culturing in vivo in nude mice. Further serological analysis of 3C4-10 and 4A1-9 was performed with concentrated culture supernatants and radiolabeled protein A. Cells cultured in vitro offer clonal populations of cells antigenically related to the normal tissues from which they are derived. Preliminary screening of monoclonal antibodies on a diverse panel of cultured cells can be used to provide an indication of the specificity of the antibody being studied (Table 1). The monoclonal antibody 3C4-10 appears to react with an antigen restricted to teratocarcinoma stem cells, the endoderm related line PYS-2, and the neuroblastoma C1300. Other cell lines, including those derived from embryonic material [e.g., MB4-9 (18)], do not express the 3C4-10-defined antigen. In contrast, 4A1-9 reacts with all the cultured cell lines tested, including the BALB/c parent myeloma line. Of particular interest are the reactions with the three intraspecific somatic cell hybrids tested. C2a-2 and B6b-10 are independent hybrids formed between the teratocarcinoma stem cell line PCC4/aza and CSH thymocytes. These hybrids have the morphology of teratocarcinoma stem cells and are capable of differentiating when injected subcutaneously into (C3H X 129)F1 mice. (P. Andrewes and P. Goodfellow, unpublished observations). PHOiM is a hybrid between PCC4 and the oligomycin-resistant L cell mutant clone 1 (22); this hybrid has the morphology of the L cell parent. 3C4-10 reacts with C2a-2 and B6b-10 but not with PHOLM; 4A1-9 reacts with all three hybrids. These results demonstrate that somatic cell hybrids can be used to investigate the genetics and functional role of the 3C4-10-defined antigen.
Developmental Biology: Goodfellow et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
379
Table 1. Testing monoclonal antibodies 4A1-9 and 3C4-10 on cultured cells and sheep erythrocytes Mouse strain E/C Cell Ref. Cell type of origin 3C4-10 4A1-9 10 Teratocarcinoma C86.S1 C3H 33 28 17 Teratocarcinoma 129 41 PCC4 25 129 13 8 F9 Nullipotent teratocarcinoma 41 8 Teratocarcinoma-derived endoderm line 129 PYS-2 60 40 C2a-2 * 129-C3H 28 PCC4-thymocyte hybrid 25 B6b-10 * PCC4-thymocyte hybrid 129-C3H 10 10 PCC4-L cell hybrid * PHO0M 129-C3H 1 9 9 Neuroblastoma C1300 A.J 21 10 MB2M SWR 18 Embryo-derived cell line 1 18 SWR 1 MB4-9 18 Embryo-derived cell line 15 18 MB4-11 SWR 1 Embryo-derived cell line 9 3 PG19 19 Melanoma 25 C57BL/6 t H6-1 A Hepatoma 2 10 t STO Transformed fibroblast Sim 2 15 AKR 1 BW5147 20 Thymoma 33 1 5 P3-X63-Ag8 Myeloma BALB/c 12 21 2 FBU Friend cell leukemia 45 DBA/2 SRBC 1 Erythrocytes (Sheep) 1 Sc-TA § Lymphoblastoid cell line 1 (Human) 1 All E/C values are the average of at least two independent experiments. * These somatic cell hybrids were made in collaboration with P. W. Andrewes (Wistar Institute, Philadelphia) and are described in the text. t H-6-1 was obtained from the Jackson Laboratory. STO is a fibroblast-like cell line isolated by A. Bernstein (Ontario Cancer Institute, Toronto, ON, Canada). § Sc-TA is a human lymphoblastoid cell line transformed by Epstein-Barr virus.
The specificity of the monoclonal antibodies was further studied on adult tissues both by direct testing and absorption (Table 2) and on preimplantation embryos by indirect immunofluorescence. 3C4-10 does not react with any adult tissues; the A41-9 antigen is expressed on brain and on testicular cells but not sperm. It should be stressed that the experiments performed might not detect a small population of antigen-positive cells. Staining of preimplantation embryos was attempted by using a sensitive "double sandwich" technique. Embryos were incubated successively in the test antibody, polyvalent rabbit anti-rat immunoglobulin (1 mg/ml) that had been columnpurified on rat immunoglobulin, and fluorescein-labeled goat anti-rabbit immunoglobulin (the last two reagents were generous gifts from J. Rozing, Stanford Medical Center). Each incubation was for 15 min at 37°C in the presence of 0.2% azide. Although the immune serum from the rat spleen donor specifically stained all stages of preimplantation embryos at a dilution of 1:50 and the monoclonal antibodies strongly stained PCC4/aza under identical conditions, no reaction was observed when 3C4-10 and 4A1-9 were used to stain preimplantation
embryos. Expression on early postimplantation embryos has not yet been tested. DISCUSSION It is difficult to compare monoclonal antibodies directly with conventional sera because of the obvious complexity of the latter. However, it is clear that the major components of the previously described antisera produced against teratocarcinomas and embryos recognize specificities different from those recognized by the two monoclonal antibodies we have described. The "F9" antiserum produced by syngeneic immunizations reacts with sperm and preimplantation embryos and does not react with PYS-2 (23). The "PCC4" antiserum also reacts with sperm and fails to react with PYS (24). Similar discrepancies exist between the 3C4-10 and 4A1-9 antigens when compared with ENDO (25), and several antigens recognized by sera prepared against teratocarcinomas (4, 26-28) and embryos (3, 29), Similarly, NS-4 and NS-5 antigens, which are expressed on F9 (30), can also be distinguished from the 3C4-10 and 4A1-9 antigens. Recently two other groups have independently produced monoclonal antibodies that react with teratocarcinomas. Stern
Table 2. Testing monoclonal antibodies 4A1-9 and 3C4-10 on normal tissues Direct testing Absorption E/C % absorption* Cell 3C4-10 4A1-9 Tissue 3C4-10 4A1-9 1 Thymus 2 Kidney 4 2 1 Spleen 1.5 Lung 7 4 1 Sperm 2 Liver 7 10 Testicular cells 3 12 Heart 5 11 Brain 1 85 *
abs
tio
100
serum - cpm bound by absorbed serum x 100. _(cpm bound by unabsorbed cpm bound by unabsorbed serum
380
Developmental Biology: Goodfellow et al.
and colleagues (31, 32) serendipitously produced a monoclonal antibody reacting with teratocarcinoma cells when immunizing rats with mouse spleen cells. The antibody recognizes a Forsman antigen expressed on trophectoderm, teratocarcinoma stem cells, sheep erythrocytes, and a small subpopulation of spleen cells. Neither the 3C4-10 antigen nor the 4AI-9 antigen is expressed on sheep erythrocytes. A syngeneic immunization was used by Knowles et al. (33) to produce a monoclonal antibody reacting with F9. This monoclonal antibody reacts with preimplantation embryos and sperm and fails to react with PYS-2. The 3C4-10 antigen may be a useful marker or functionally involved in either development in nitro or "postimplantation" differentiation in vivo. The 4A1-9-defined antigen, although not likely to be helpful in studies of differentiation, may be useful in studying oncofetal antigens and tumor antigens in general. In this regard it is particularly noteworthy that teratocarcinoma stem cells have been reported to lack C type virus antigens (34). Although detailed chemical analysis of the antigens defined by the monoclonal antibodies 3C4-10 and 4A1-9 has not been completed, preliminary studies have shown that the 3C4-10defined antigen can be solubilized by papain treatment and both antigens are Pronase sensitive. Neither antigen can be extracted from glutaraldehyde-fixed cells by either detergents or methanol. These results strongly suggest that both antigens and surface expressed proteins are glycoproteins. This has been confirmed for 3C4-10 by immunoprecipitation of [35S]methionine-labeled cell lysates followed by sodium dodecyl sulfate gel analysis. The 3C4-10 monoclonal antibody specifically precipitates a polypeptide with an apparent molecular weight greater than 100,000. This report demonstrates that monoclonal antibodies can be produced against teratocarcinoma cells. Undoubtedly these antibodies and others will define antigenic markers for developing embryonic cells and will facilitate investigation of the role of the cell surface in development. We thank Drs. R. Kennett, W. F. Bodmer, I. Melchers, and P. W. Andrewes for many helpful comments. P.N.G. is the grateful recipient of a Senior California Division, American Cancer Society Fellowship. J.R.L. is a Predoctoral Fellow of the Medical Scientist Training Program, National Institutes of Health (GM 1922). This work was supported in part by National Institutes of Health Grant HD 09410. 1. Lerner, R. A. & Bergsma, D., eds. (1978) The Molecular Basis of Cell-Cell Interaction (Alan R. Liss, New York). 2. Bennett, D., Boyse, E. A. & Old, L. J. (1971) in Proceedings of the Third Lepetit Colloquium, London, Nov. 1971 (NorthHolland, Amsterdam), pp. 247-263. 3. Wiley, L. M. & Calarco, P. G. (1975) Dev. Biol. 47,407-418.
Proc. Natl. Acad. Sci. USA 76 (1979) 4. Kemler, R., Babinet, C., Eisen, H. & Jacob,-F. (1977) Proc. Natl. Acad. Sd. USA 74,4449-4452. 5. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495497. 6. Galfre, G., Howe, S. C., Milstein, C., Butcher, G. W. & Howard, J. C. (1977) Nature (London) 266,550-552. 7. Martin, G. R. (1975) Cell 5,229-243. 8. Lehman, J. M., Speers, W. C., Swartzendruber, D. E. & Pierce, G. B. (1974) J. Cell Physiol. 84, 13-28. 9. Augusti-Tocco, G. & Sato, G. (1969) Proc. Natl. Acad. Sci. USA 64,311-315. 10. McBurney, M. W. (1976) J. Cell Physiol. 89,441-445. 11. Kennett, R. H., Denis, K. A., Tung, A. S. & Klinman, N. R. (1978) Curr. Top. Microbiol. Immunol. 81, 77-91. 12. Cotton, R. G. H., Secher, D. S. & Milstein, C. (1973) Eur. J. Immunol. 3, 136-140. 13. Bernstine, E. G., Hooper, M. L., Grandchamp, S. & Ephrussi, B. (1973) Proc. Natl. Acad. Sci. USA 70,2899-3903. 14. Jensenius, J. R. & Williams, A. F. (1974) Eur. J. Immunol. 4, 91-97. 15. Goldberg, E. H., Aoki, T., Boyse, E. A. & Bennett, D. (1970) Nature (London) 228,570-577. 16. Acton, R. T., Morris, R. J. & Williams, A. F. (1974) Eur. J. Immunol. 4, 498-603. 17. Jakob, H., Boon, T., Gaillard, J., Nicolas, J. F. & Jacob, F. (1973) Ann. Microbiol. (Paris) 124B, 269-282. 18. Sherman, M. I. (1975) DIfferentiation 3,57-67. 19. Jones, E. A., Goodfellow, P. N., Kennett, R. H. & Bodmer, W. F. (1976) Somatic Cell Genet. 2, 483-496. 20. Hyman, R. & Stallings, V. (1974) J. Natl. Cancer Inst. 52, 429-436. 21. Miller, R. A. & Ruddle, F. H. (1977) Dev. Biol. 56, 157-173. 22. Lichtor, T. & Getz, G. S. (1978) Proc. Natl. Acad. Sci. USA 75, 324-328. 23. Artzt, K., Dubois, P., Bennett, D., Condamine, H., Babinet, C. & Jacob, F. (1973) Proc. Natl. Acad. Sci. USA 70,2988-2992. 24. Gachelin, G., Kemler, R., Kelly, F. & Jacob, F. (1977) Dev. Btol. 57, 199-209. 25. Artzt, K., Hamburger, L., Jacob, H. & Jacob, F. (1976) Dev. Biol. 51, 152-157. 26. Stern, P. L., Martin, G. R. & Evans, M. J. (1975) Cell 6, 455465. 27. Gooding, L. R. & Edidin, M. (1974) J. Exp. Med. 140,61-78. 28. Dewey, M. J., Gearhart, J. D. & Mintz, B. (1977) Dev. Biol. 55, 359-374. 29. Koprowski, H., Sawicki, W. & Koklovsky, P. (1971) J. Natl. Cancer Inst. 46, 1317-1323. 30. Gordis, C., Artzt, K., Wortham, K. A. & Schachner, M. (1978) Dev. Biol. 65,238-243. 31. Stern, P. L., Willison, K. R., Lennox, E., Galfre, G., Milstein, C., Secher, A., Ziegler, A. & Springer, T. (1978) Cell 14,775-783. 32. Willison, K. R. & Stern, P. L. (1978) Cell 14,783-785. 33. Knowles, B. B., Aden, D. P. & Solter, D. (1978) Curr. Top. Mi-
crobiol. Immunol. 81,51-53. 34. Teich, N. M., Weiss, R. A., Martin, G. R. & Lowy, A. R. (1977) Cell 12, 973-982.
Developmental Biology
Monoclonal antibodies reacting with murine teratocarcinoma cells (radioimmunoassay/cultured cells/adult tissues/preimplantation embryos)
PETER N. GOODFELLOW, JOHN R. LEVINSON, VIRGINIA E. WILLIAMS II, AND HUGH 0. MCDEVITT Division of Immunology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305
Contributed by Hugh 0. McDevitt, October 16, 1978
Monoclonal antibodies were produced in vitro ABSTRACT by fusing mouse myeloma cells with spleen cells from a rat immunized with the C3H mouse teratocarcinoma C86S1. After the fusion two clones were chosen for further analysis. The first clone, 3C4-10, produced an antibody recognizing an antigen with a distribution restricted to teratocarcinoma cell lines, an endoderm cell line, and a neuroblastoma. The second clone, 4A1-9, produced an antibody that reacted with all cultured murine cells tested and adult brain. Neither antibody reacted with preimplantation embryos. The 3C4-10 antibody recognized an antigen associated with proteins. The apparent molecular weight of the 3C4-10 antigen was greater than 100,000.
The cell surface has been shown to play an important role in several cell-cell interaction systems (for several examples see ref. 1). It has been postulated that cell-cell interaction in the developing mammalian embryo is also mediated by cell surface-expressed molecules (2). Consistent with this hypothesis is the production of antisera that can block the development of preimplantation embryos cultured in vitro (3, 4). Unfortunately these antisera, which are produced by immunization with embryos (3) or with teratocarcinoma cells (4), are complex and often difficult to analyze. This complexity makes it particularly difficult to interpret functional experiments. Recent progress in the in vitro production of monoclonal antibody (5, 6) has prompted us to use this technique for investigating cell surface antigens expressed on cultured murine teratocarcinomas and embryos. Cultured teratocarcinoma stem cells were used for the immunogen because of the close relationship of these cells to embryonic cells (7) and because of the ease of producing in vitro the large amounts of tissue needed for immunization and screening. We have produced several monoclonal antibodies that react with cultured teratocarcinomas. In this communication we describe the analysis of the first two of these monoclonal antibodies. The first antibody recognizes an antigen that is restricted to teratocarcinoma stem cells, the endodermal cell line PYS-2 (8), and the neuroblastoma C1300 (9). The second antibody recognizes an antigen expressed on adult mouse brain and ubiquitously on cultured murine cells. MATERIALS AND METHODS Cell Fusion to Produce Monoclonal Antibodies. A female Lewis rat was immunized with the embryo-derived C3H mouse teratocarcinoma cell line C86.S1 (10). Under the conditions used to maintain C86.S1 in vitro it does not undergo significant differentiation (10). The rat was primed by a single intraperitoneal injection of 108 C86-S1 cells homogenized in 0.5 ml of complete Freund's adjuvant. After 7 days the rat was boosted intravenously with 5 X 107 cells suspended in saline. Three days later the animal was bled and sacrificed, and the spleen was used as a source of immune cells for fusion to the BALB/c The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
mouse myeloma P3-X63-Ag8 (5). The fusion was performed as described by Kohler and Milstein (5), using the enriched medium described by Kennett et al. (11). P3-X63-Ag8 cells (3 X 107) were fused with 3 X 108 rat spleen cells; no attempt was made to remove erythrocytes. The fused cells were suspended in hypoxanthine/aminopterin/thymidine (HAT) selective medium (which selects against the myeloma parent) and divided equally amongl96 1-ml cultures. Cultures were fed by replacing half the medium in each well every 3 or 4 days. Cloning was achieved by the agar overlay method (12) or by limiting dilution. Cloning was performed in the absence of HAT selection. Cell Culture. All cells except PCC4/aza were grown in Dulbecco's modified Eagle's medium with high glucose (purchased from GIBCO) supplemented with 15% fetal calf serum (GIBCO), 100 international units of penicillin per ml, and 100 ,ug of streptomycin (GIBCO) per ml. PCC4/aza was grown in Dulbecco's modified Eagle's medium with low glucose (GIBCO). F9 was grown on gelatin-coated dishes (13). Attached cells were removed for testing by a combination of scraping and treatment with 2 mM EDTA in phosphate-buffered saline. References to the cell lines used are given in Table 1. Serological Techniques. Radioimmunoassay (RIA). All manipulations were performed in RIA buffer, which consisted of Dulbecco's modified phosphate-buffered saline supplemented with 5% fetal calf serum and 0.1% sodium azide. Staphylococcal protein A (purchased from Pharmacia) and purified rabbit F(ab')2 anti-rat Fab fragment of IgG (a gift from A. Williams, Medical Research Council Cellular Immunology Group, Oxford) were labeled by the method of Jensenius and Williams (14) and used at a specific activity of 20-30 ,uCi/,g (1 Ci = 3.7 X 1010 becquerels). C86*S1 target cells (2 X 105) in 10 Al of RIA buffer were added to round-bottomed multiwell plates and 50 ,u of culture supernatants was added. The cells and supernatants were incubated for 1 hr at room temperature and then washed twice. Labeled detecting reagent (105 cpm) in 50 ,u of RIA buffer was added and the mixture was incubated for 2 hr at room temperature or overnight at 4°C. After the incubation the cells were washed four times and assayed for radioactivity. Maximal binding with the immune serum from the rat spleen donor was about 4 X 104 cpm. The background binding to C86-S1 by supernatants from P3-X63-Ag8 cells was about 2 X 102 cpm. All tests were performed in duplicate. Testing cell lines by RIA. Supernatants were collected from cultures of clones 4A1-9 and 3C4-10 and from P3-X63-Ag8. The supernatants were concentrated 5-fold by using an Amicon ultrafiltration unit fitted with a PM30 filter. All cells were tested at a concentration of 2 X 105 cells per well (2 X 107/ml) with five serial doubling dilutions of the three concentrated supernatants. E/C values (see Fig. 1) given in Tables 1 and 2 represent the maximal value in the titration curve. Testing cells from adult mice. Thymus and spleen cells were prepared by mincing and teasing in RIA buffer, followed by Abbreviation: RIA, radioimmunoassay.
377
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25
Proc. Nati. Acad. Sci. USA 76 (1979)
I ~~I ~I ~ ~I ~
IJ
~
F
l
15
4A1
3C4
10
E/C
5
11nIIIlBHI1nGGB11DgnGBNNNiUij0HDDIdji AL-
0
AL
L.
ALL
20
10
30ELI
-a
30
40
AL
50
WELL NUMBER FIG. 1. The open bars represent the test with radiolabeled anti-rat Fab. The solid bars represent the test with radiolabeled protein A.
E/C
=
cpm bound by cells + hybrid supernatant bound by cells + P3-X63-Ag8 supernatant'
cpm
filtration through muslin. Sperm cells were prepared by the method of Goldberg et al. (15). Testicular cells were prepared by repeated pipetting of seminiferous tubules followed by filtration through muslin. Absorption analysis was performed on crude membrane fractions prepared according to Acton et al. (16). Culture supernatants were diluted to give 80% maximal binding. A 15-Mul sample of diluted culture supernatant was absorbed with an equal volume of packed membranes for 1 hr at 4VC. Dilution errors were controlled. The results presented are the average of two independent determinations. The error is estimated to be +15%. All cells and tissues were taken from C3H/DiSn mice.
RESULTS On the 17th day after the fusion, 50 of 96 cultures showed growth. These 50 cultures were tested for the production of antibody recognizing the immunogen by RIA using both 125I-labeled protein A and 125I-labeled rabbit anti-rat Fab. The results are presented as a histogram in Fig. 1. Arbitrarily, an E/C ratio of greater than 4 was taken as a positive result; thus 12/96 wells were positive with the polyvalent reagent and 5/96 with the iodinated protein A. The two cultures with the highest E/C ratios (3C4 and 4A1) were chosen for cloning and further analysis. Initial cloning was achieved by the agar overlay method (12). Of 12 subclones of 3C4, 8 were producers of specific antibody; and 12 of 12 subclones for 4A1 were producers. The two subclones 3C4-10 and 4A1-9 were further cloned by the limiting dilution method. All subclones (16/16 and 10/10, respectively) were positive for anti-C86.Sl antibody production, suggesting that these original clones are stable
producers of monoclonal antibody. This has been confirmed by growing 3C4-10 and 4A1-9 continuously in vitro for several months and by culturing in vivo in nude mice. Further serological analysis of 3C4-10 and 4A1-9 was performed with concentrated culture supernatants and radiolabeled protein A. Cells cultured in vitro offer clonal populations of cells antigenically related to the normal tissues from which they are derived. Preliminary screening of monoclonal antibodies on a diverse panel of cultured cells can be used to provide an indication of the specificity of the antibody being studied (Table 1). The monoclonal antibody 3C4-10 appears to react with an antigen restricted to teratocarcinoma stem cells, the endoderm related line PYS-2, and the neuroblastoma C1300. Other cell lines, including those derived from embryonic material [e.g., MB4-9 (18)], do not express the 3C4-10-defined antigen. In contrast, 4A1-9 reacts with all the cultured cell lines tested, including the BALB/c parent myeloma line. Of particular interest are the reactions with the three intraspecific somatic cell hybrids tested. C2a-2 and B6b-10 are independent hybrids formed between the teratocarcinoma stem cell line PCC4/aza and CSH thymocytes. These hybrids have the morphology of teratocarcinoma stem cells and are capable of differentiating when injected subcutaneously into (C3H X 129)F1 mice. (P. Andrewes and P. Goodfellow, unpublished observations). PHOiM is a hybrid between PCC4 and the oligomycin-resistant L cell mutant clone 1 (22); this hybrid has the morphology of the L cell parent. 3C4-10 reacts with C2a-2 and B6b-10 but not with PHOLM; 4A1-9 reacts with all three hybrids. These results demonstrate that somatic cell hybrids can be used to investigate the genetics and functional role of the 3C4-10-defined antigen.
Developmental Biology: Goodfellow et al.
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Table 1. Testing monoclonal antibodies 4A1-9 and 3C4-10 on cultured cells and sheep erythrocytes Mouse strain E/C Cell Ref. Cell type of origin 3C4-10 4A1-9 10 Teratocarcinoma C86.S1 C3H 33 28 17 Teratocarcinoma 129 41 PCC4 25 129 13 8 F9 Nullipotent teratocarcinoma 41 8 Teratocarcinoma-derived endoderm line 129 PYS-2 60 40 C2a-2 * 129-C3H 28 PCC4-thymocyte hybrid 25 B6b-10 * PCC4-thymocyte hybrid 129-C3H 10 10 PCC4-L cell hybrid * PHO0M 129-C3H 1 9 9 Neuroblastoma C1300 A.J 21 10 MB2M SWR 18 Embryo-derived cell line 1 18 SWR 1 MB4-9 18 Embryo-derived cell line 15 18 MB4-11 SWR 1 Embryo-derived cell line 9 3 PG19 19 Melanoma 25 C57BL/6 t H6-1 A Hepatoma 2 10 t STO Transformed fibroblast Sim 2 15 AKR 1 BW5147 20 Thymoma 33 1 5 P3-X63-Ag8 Myeloma BALB/c 12 21 2 FBU Friend cell leukemia 45 DBA/2 SRBC 1 Erythrocytes (Sheep) 1 Sc-TA § Lymphoblastoid cell line 1 (Human) 1 All E/C values are the average of at least two independent experiments. * These somatic cell hybrids were made in collaboration with P. W. Andrewes (Wistar Institute, Philadelphia) and are described in the text. t H-6-1 was obtained from the Jackson Laboratory. STO is a fibroblast-like cell line isolated by A. Bernstein (Ontario Cancer Institute, Toronto, ON, Canada). § Sc-TA is a human lymphoblastoid cell line transformed by Epstein-Barr virus.
The specificity of the monoclonal antibodies was further studied on adult tissues both by direct testing and absorption (Table 2) and on preimplantation embryos by indirect immunofluorescence. 3C4-10 does not react with any adult tissues; the A41-9 antigen is expressed on brain and on testicular cells but not sperm. It should be stressed that the experiments performed might not detect a small population of antigen-positive cells. Staining of preimplantation embryos was attempted by using a sensitive "double sandwich" technique. Embryos were incubated successively in the test antibody, polyvalent rabbit anti-rat immunoglobulin (1 mg/ml) that had been columnpurified on rat immunoglobulin, and fluorescein-labeled goat anti-rabbit immunoglobulin (the last two reagents were generous gifts from J. Rozing, Stanford Medical Center). Each incubation was for 15 min at 37°C in the presence of 0.2% azide. Although the immune serum from the rat spleen donor specifically stained all stages of preimplantation embryos at a dilution of 1:50 and the monoclonal antibodies strongly stained PCC4/aza under identical conditions, no reaction was observed when 3C4-10 and 4A1-9 were used to stain preimplantation
embryos. Expression on early postimplantation embryos has not yet been tested. DISCUSSION It is difficult to compare monoclonal antibodies directly with conventional sera because of the obvious complexity of the latter. However, it is clear that the major components of the previously described antisera produced against teratocarcinomas and embryos recognize specificities different from those recognized by the two monoclonal antibodies we have described. The "F9" antiserum produced by syngeneic immunizations reacts with sperm and preimplantation embryos and does not react with PYS-2 (23). The "PCC4" antiserum also reacts with sperm and fails to react with PYS (24). Similar discrepancies exist between the 3C4-10 and 4A1-9 antigens when compared with ENDO (25), and several antigens recognized by sera prepared against teratocarcinomas (4, 26-28) and embryos (3, 29), Similarly, NS-4 and NS-5 antigens, which are expressed on F9 (30), can also be distinguished from the 3C4-10 and 4A1-9 antigens. Recently two other groups have independently produced monoclonal antibodies that react with teratocarcinomas. Stern
Table 2. Testing monoclonal antibodies 4A1-9 and 3C4-10 on normal tissues Direct testing Absorption E/C % absorption* Cell 3C4-10 4A1-9 Tissue 3C4-10 4A1-9 1 Thymus 2 Kidney 4 2 1 Spleen 1.5 Lung 7 4 1 Sperm 2 Liver 7 10 Testicular cells 3 12 Heart 5 11 Brain 1 85 *
abs
tio
100
serum - cpm bound by absorbed serum x 100. _(cpm bound by unabsorbed cpm bound by unabsorbed serum
380
Developmental Biology: Goodfellow et al.
and colleagues (31, 32) serendipitously produced a monoclonal antibody reacting with teratocarcinoma cells when immunizing rats with mouse spleen cells. The antibody recognizes a Forsman antigen expressed on trophectoderm, teratocarcinoma stem cells, sheep erythrocytes, and a small subpopulation of spleen cells. Neither the 3C4-10 antigen nor the 4AI-9 antigen is expressed on sheep erythrocytes. A syngeneic immunization was used by Knowles et al. (33) to produce a monoclonal antibody reacting with F9. This monoclonal antibody reacts with preimplantation embryos and sperm and fails to react with PYS-2. The 3C4-10 antigen may be a useful marker or functionally involved in either development in nitro or "postimplantation" differentiation in vivo. The 4A1-9-defined antigen, although not likely to be helpful in studies of differentiation, may be useful in studying oncofetal antigens and tumor antigens in general. In this regard it is particularly noteworthy that teratocarcinoma stem cells have been reported to lack C type virus antigens (34). Although detailed chemical analysis of the antigens defined by the monoclonal antibodies 3C4-10 and 4A1-9 has not been completed, preliminary studies have shown that the 3C4-10defined antigen can be solubilized by papain treatment and both antigens are Pronase sensitive. Neither antigen can be extracted from glutaraldehyde-fixed cells by either detergents or methanol. These results strongly suggest that both antigens and surface expressed proteins are glycoproteins. This has been confirmed for 3C4-10 by immunoprecipitation of [35S]methionine-labeled cell lysates followed by sodium dodecyl sulfate gel analysis. The 3C4-10 monoclonal antibody specifically precipitates a polypeptide with an apparent molecular weight greater than 100,000. This report demonstrates that monoclonal antibodies can be produced against teratocarcinoma cells. Undoubtedly these antibodies and others will define antigenic markers for developing embryonic cells and will facilitate investigation of the role of the cell surface in development. We thank Drs. R. Kennett, W. F. Bodmer, I. Melchers, and P. W. Andrewes for many helpful comments. P.N.G. is the grateful recipient of a Senior California Division, American Cancer Society Fellowship. J.R.L. is a Predoctoral Fellow of the Medical Scientist Training Program, National Institutes of Health (GM 1922). This work was supported in part by National Institutes of Health Grant HD 09410. 1. Lerner, R. A. & Bergsma, D., eds. (1978) The Molecular Basis of Cell-Cell Interaction (Alan R. Liss, New York). 2. Bennett, D., Boyse, E. A. & Old, L. J. (1971) in Proceedings of the Third Lepetit Colloquium, London, Nov. 1971 (NorthHolland, Amsterdam), pp. 247-263. 3. Wiley, L. M. & Calarco, P. G. (1975) Dev. Biol. 47,407-418.
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