Monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells

Cell, Vol. 30, 697-705,

October

1982,

Copyright

0 1982

by MIT

Monoclonal Antibody to Murine Embryos Defines a Stage-Specific Embryonic Antigen Expressed on Mouse Embryos and Human Teratocarcinoma Cells Lynne Hamburger Shevinsky, Barbara 6. Knowles, Ivan Damjanov* and Davor Solter The Wistar Institute of Anatomy and Biology 36th Street at Spruce Philadelphia, Pennsylvania 19104 * and Department of Pathology and Laboratory Medicine The Hahnemann Medical College and Hospital Philadelphia, Pennsylvania 19104

Summary A murine stage-specific embryonic antigen (SSEA3) is defined by reactivity with a monoclonal antibody prepared by immunization of a rat with 4-to S-cellstage mouse embryos. This antigenic determinant, present on oocytes, becomes restricted first to the inner cell mass at the blastocyst stage, and later to the primitive endoderm. Murine teratocarcinoma stem cells do not react with this antibody, whereas human teratocarcinoma stem cells are SSEA3-positive. This antigenic determinant is not expressed on a variety of other human and murine cell lines, but is found on the surface of human erythrocytes. It is a carbohydrate and is present on both cellsurface glycolipids and glycopeptides. These results demonstrate the feasibility of identifying stage-specific antigenic determinants with monoclonal antibody prepared against embryos. The need for thorough screening on a variety of cell types to establish developmentally important crossreactivities is also emphasized. Introduction It has been proposed that the cell surface plays an important role during embryogenesis. Each blastomere of the S-cell-stage embryo appears to have the genetic potential to develop into a complete organism (Kelly, 1977); yet even at the 8-cell stage, the position of the individual blastomere within the embryo is an indication of its future developmental fate (Graham and Lehtonen, 1979; Johnson and Ziomek, 1981). Alteration of the blastomere cell surface occurring at the S-cell stage results in incipient interblastomere communication at the level of the cell surface. This communication, if experimentally unaltered, would shape subsequent morphogenesis and cell fate. Starting with fertilization, each subsequent division is accompanied by distinct biochemical changes in the pattern of biosynthetically radiolabeled spots on two-dimensional gels, which have been monitored by qualitative and semi-quantitative techniques (Levinson et al., 1978; Howe and Solter, 1979; Schultz et al., 1979; Cullen et al., 1980; Van Blerkom, 1981). Some of these changes do occur at the cell surface (Johnson and Calarco, 1980a; Magnuson and Epstein, 1981).

More recently it has been suggested that at least some of the protein changes are due to posttranslational modification of previously existing polypeptides (Van Blerkom, 1982). This indicates that there are stage-specific polypeptides synthesized by the embryo, some of which are expressed on the cell surface, and that they may be important in the regulation of the orderly development and differentiation of the preimplantation embryo. One approach to the identification and eventual isolation and chemical characterization of developmentally regulated molecules is the use of antibody probes that are reactive with antigenic determinants expressed on the surface of embryos in a stage-specific manner. Several such molecules, for example stage-specific embryonic antigen 2 (SSEA2; Shevinsky et al., 1981), have been identified with conventional antisera raised against cells or embryos. However, such work has been hampered by the complexity and multiple reactivities found in such antisera (for review, see Jacob, 1979; Solter and Knowles, 1979; Wiley, 1979; Johnson and Calarco, 1980b). With the advent of the monoclonal antibody technique (Kohler and M&stein, 1975), this particular drawback to antibody analysis of the developing embryo was eliminated. Indeed, several monoclonal antibodies made against various cells but cross-reactive with preimplantation embryos, have been described (Knowles et al., 1978; Solter and Knowles, 1978; Willison and Stern, 1978; Kemler et al., 1979). One of the problems with the use of cells rather than embryos as immunogens is that developmentally important molecules expressed only on embryos remain undetected. We therefore immunized mice and rats with mouse embryos a different stages in an attempt to detect SSEAs (Salter and Knowles, 1979). One monoclonal antibody derived from a rat immunized with 4- to 8-cell-stage mouse embryos reacts with all preimplantation mouse embryos up to early blastocyst stage. In early postimplantation embryos reactivity is restricted to endoderm, and in the adult mouse, to the kidney. Murine tumor cell lines, including embryonal carcinoma cell (ECC) lines, do not express this antigenic determinant, which is expressed on the stem cells of human teratocarcinoma. Analysis of immunoprecipitates from murine embryos and human ECC lines indicates that molecules bearing this stage-specific embryonic antigenic determinant (SSEAB) are both glycolipid and glycoprotein, which suggests a carbohydrate antigenie determinant. Results As shown by the apparent molecular weight in twodimensional SDS-polyacrylamide gels (Figure 11, and by cross-reactivity with mouse immunoglobulin classspecific reagents, this monoclonal antibody is an IgM. Analysis the

antibody

by indirect reacts

immunofluorescence with

ovarian

oocytes

reveals

that

(Figure

2A),

Cell 698

noma and containing ECCs were positive (Tables 1 and 2). Human erythrocytes from blood groups A, B and 0 were found positive when separated peripheral blood elements were tested (Table 2). Quantitative absorption showed that 5 X 1 O6 human erythrocytes were capable of removing 50% of the cytotoxic activity of the antibody on 4- to 8-cell-stage embryos. For comparison, 1 O6 human teratocarcinoma stem cells removed all cytotoxic activity, whereas absorption with 1 O7 cells from all of the other human and murine cell lines and human lymphocytes and granulocytes did not affect the antibody reactivity. Tumors arising in athymic (nu/nu) mice following injection of the human teratocarcinoma cell line 2102E displayed the typical histology of embryonal carcinomas. When cryostat sections of these tumors were reacted with antibody to SSEAS, areas of ECCs were found to express this determinant. The stromal tissue surrounding the nests of tumor cells did not react with the antibody (Figure 4). Extracts of cell-surface-radioiodinated human cells were immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. The immunoprecipitate of the human teratocarcinoma cell line 833KE with anti-SSEAS revealed several labeled polypeptides. A predominant polypeptide had an apparent molecular weight of 72 kilodaltons (kd), and several other labeled polypeptides were seen (Figure 5, lane 1). In addition, a radiolabled band running at the gel front was present. No labeled bands were observed when 833KE/LC, a lymphoid cell line derived from the same patient, was reacted with anti-SSEA3 (Figure 5, lane 3). Analysis of immunoprecipitates of another human teratocarcinoma-derived cell line, 2102E, metabolically labeled with 3H-fucose (Figure 5, lanes 58) revealed that the major 72 kd polypeptide is a

unfertilized eggs, zygotes (Figure 2B), cleavage-stage embryos (Figures 2C and 2D) and early blastocysts (Figures 2E and 2F). The trophectoderm of the expanded blastocyst (Figure 2G) is negative, although immunosurgically isolated inner cell masses (Figure 2H) are positive. Inner cell masses grown for 48 hr in vitro develop into embryonic bodies with an endodermal layer on the outside and an inner core of ectoderm (Salter and Knowles, 1975). Endoderm remains positive (data not shown), while ectoderm is now negative (Figure 21). The same stage-specific distribution of reactivity was shown on embryos by complement-mediated cytotoxicity (Figure 3). From this distribution (Figures 2 and 3) we conclude that the antigenic determinant recognized by this monoclonal antibody is expressed beginning with the oocyte, and that its expression becomes restricted at the early blastocyst stage. Because of this stagerelated expression, we designate the antigenic determinant SSEA3. We probed the specific distribution of the antigenic determinant by quantitatively absorbing supernatant from anti-SSEA3-producing cultures with cells from mechanically disrupted fetal and adult murine organs and testing the absorbed supernatant on 4- to 8-cellstage embryos in complement-dependent lysis. Significant reduction in activity (>50%) was seen following absorption with 7 x IO6 adult kidney cells, whereas IO7 cells derived from adult liver, brain, spleen, peripheral blood elements or sperm, or from fetal kidney, liver or brain, did not alter the reactivity of the supernatant with embryos. The antibody did not react with murine teratocarcinoma or other murine cell lines (Table 1). Expression of SSEA3 was tested on 54 different human cell lines derived from a variety of sources; only those derived from human teratocarci-

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Figure 1. Autoradiographs of a Two-Dimensional Polyacrylamide Gel of the Monoclonal Antibody Antibody-producing cultures were incubated with “S-methionine, and supernatant was removed after 5 hr. An aliquot yielding 5 X 1 O6 trichloroacetic acid-precipitable (TCA) cpm was incubated with 100 pl rabbit antimouse IgM (19 hr at 4°C). The resultant precipitate was washed as described in the Experimental Procedures, and run first on an isoelectric focusing gel and then on an SDS-polyacrylamide gel. The basic end of the isoelectric focused gel is on the left; the direction of SDS electrophoresis is from top to bottom. The glycosylated densities of the heavy chain &) of IgM are found at molecular weight 68,000 and increasing in molecular weight (with glycosylation) from pl 7.0 to 4.5.

Stage-Specific 699

Embryonic

Antigen

3

Figure 2. Reactivity of Monoclonal Antibody with Ovarian Oocytes and Preimplantation Mouse Embryos as Detected by indirect Immunofluorescence (a) Ovarian oocyte; (b) zygote; (c) 2-cell stages; (d) 4- to B-cell stages; (e) early blastocyst; (f) same blastocyst (light microscopy); (g) expanded blastocyst; (h) inner cell mass: (i) inner cell mass grown 2 days in vitro, endoderm removed by immunosurgery (Salter and Knowles, 1975).

EMBRYONIC

STAGE

Figure 3. Antibody-Dependent Complement-Mediated on Preimplantation-Stage Embryos

Cytotoxicity

Embryos of the stages indicated were incubated (for 1 hr at 37°C) in increasing dilutions of supernatant from antibody-producing cell cultures. Embryos were washed once in HEPES-buffered Whitten’s medium (HWM) and incubated in guinea pig serum as the complement source (1 hr at 37°C).

fucose-containing cell-surface glycoprotein without apparent subunit structure (Figure 5, lanes 5 and 7); a slight change in molecular weight following reduction was seen, which could be the result of distortion due to the heavy chain of immunoglobulin migrating

at approximately the same molecular weight as the 72 kd polypeptide. Other 3H-fucose-labeled molecules were discernible, including one that migrates at approximately 45 kd, a series migrating at about 29 kd and one at the running front of the gels (Figure 5, lanes 5 and 7). When N-linked glycosylation was inhibited by pretreatment of 2102E cells with tunicamycin prior to labeling with 3H-fucose and immunoprecipitation, the 72 kd polypeptide was not observed (Figure 6A, lane 1). Although the bands at 45 kd and at 29 kd were reduced in intensity they were still present, and the intensity of the band at the running front was comparatively increased in the tunicamycintreated sample. Treatment of 2102E cells and human erythrocytes with galactose oxidase, followed by 3Hlabeled sodium borohydride, resulted in labeling in the glycolipid region of the gel (Figure 6B, lanes 2 and 4). A 45 kd polypeptide was apparent with both control and specific antibody (Figure 6B, lanes 3 and 4). These results indicate that the SSEA3 antigenic determinant is present on several distinct cell-surface polypeptides and on glycolipid found at the running front of these gels, and that SSEAB is probably carbohydrate in nature. Tunicamycin pretreatment does not affect incorporation of the SSEA3 antigenic determinant into glycolipid and several of the other polypeptides, although it appears to have eliminated its incorporation into the 72 kd polypeptide. However, it is possible that the absence of the 72 kd polypeptide is due to inhibition of synthesis of the polypeptide itself, or alternatively, to inhibition of the addition to

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Table 1. Reactivity Defining SSEAB

of Mouse

Cell Lines Murine teratocarcinoma-derived MH-I 5b FA-25b F9’ PYS-2” Murine transformed OTT6050fb KD>SVe KF,A”

P3x63Ag6’ Human teratocarcinoma-derived Tera-I g SuSa” 833KEp31’ 2102E’ 577MF’ Human transformed 833KILC’ LNSV’ WI 8Va2” HeLa (D-98)’

and Human

Cell Lines

Characteristics

of SSEAB on Human

Cell lines Teratocarcinoma Choriocarcinoma Hepatoma Colorectal carcinoma Melanoma Transformed B-cell lymphoma T-cell lymphoma Myeloid leukemia

250 320 400 (129/ 170

Fibroblast (129/S+ SIJCP) SV40-transformed fibroblast (Bl O.D2) Adenovirus+transformed fibroblasts (C57Bl/6 x BALB/c) Myeloma (BALB/c)

Cell Lines and Peripheral

Positive

cpma

ECC (BALB/c) ECC (AKR/J) ECC (129/SV-SICP) Parietal endoderm sv-SIJCP)

Mixed culture ECCs Mixed culture ECCs ECCs ECCs Transformed fibroblastic-no

Table 2. Expression Blood Elements

with Antibody

60 60

Normal peripheral Erythrocytes Lymphocytes Granulocytes

70 80

a Peripheral

Tested

9 2 3 5 10 5 14 3 3

blood cellsa

blood from four different

4 0 0

4 4 4

donors.

containing 2770 containing 2630 3130 4260 ECC

Lymphocytes transformed by Epstein-Barr virus SV40-transformed fibroblast SV40-transformed fibroblast Carcinoma-derived

with supernatant a Bound in indirect “sl-radioimmunoassay SSEA3 producing cultures. b Data are from Solter and Knowles (1978). ’ Data are from Bernstine et al. (1973). d Data are from Lehman et al. (1974). e Data are from Knowles et al. (1979). ‘ Data are from Kohler and Milstein (1975). g Data are from Fogh and Trempe (1975). h Data are from Hogan et al. (1977). ’ Data are from Andrews et al. (1980). ’ Data are from Croce et al. (1973). k Data are from Weiss et al. (1968). ’ Data are from NelsonBees and Flandermeyer (1976).

200

130 100 170 110 from

01-

the polypeptides of another carbohydrate chain that contains fucose but not the SSEA3 determinant. To determine the molecules bearing the SSEA3 antigenic determinant on the murine embryo cell surface, we analyzed immunoprecipitates of radioiodinated 4-to 8-cell-stage embryos. Again, several polypeptide bands were observed that generally corresponded to those from the human teratocarcinoma cell surface. The major polypeptide migrated at approximately 72 kd. A band at 45 kd was seen, and a very pronounced band migrating at the gel front was present (Figure 7, lane 1).

Figure 4. Reactivity of Monoclonal bryonal Carcinoma Cells

Antibody

with

Islands

of Em-

A tumor derived by injection of the 2102E cell line into a nude mouse was reacted with monoclonal antibody, which was detected by indirect immunofluorescence. 220X.

Discussion Murine embryos appear to be rather poor immunogens; hyperimmunizations with syngeneic, allogeneic or xenogeneic embryos often do not yield antiserum with specific, strong activity (Salter and Knowles, 1979). This finding can be interpreted in several ways: the embryo itself lacks molecuies of proper antigenicity; the amount of embryos used for immunization is too low; or the screening methods for detection of

Stage-Specific 701

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Antigen

3

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111,

B

i

6845-,,

W

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29-

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.

Y

I Figure

I234 Figure

5.

lmmunoprecipitation

5678 of the SSEA3

Antigenic

Determinant

Cells were labeled with ‘251 by the lactoperoxidase-glucose oxidase method. (Lane 1) 833KEp31 cells reacted with anti-SSEAS nude mouse ascites; 1 X 1 O4 cpm; 10 day exposure. (Lane 2) 833KEp31 cells reacted with rat IgM ascites, 1 X 1 O3 cpm. (Lane 3) 833K/LC ceils reacted with antiSSEA nude mouse ascites; 1.2 X 1 O3 cpm. (Lane 4) 833K/LC cells reacted with rat IgM ascites; 1.6 x 1 O3 cpm. Human teratocarcinoma-derived 2102E cells were labeled with ‘Hfucose. (Lane 5) 2102Ep cells immunoprecipitated with.anti-SSEAB nude mouse ascites antigen-antibody complexes not reduced prior to SDS-polyacrylamide gel electrophoresis; 8 X 1 O3 cpm; 3 month exposure. (Lane 6) 2102Ep cells immunoprecipitated with rat IgM ascites, not reduced: 1 X lo3 cpm; 3 month exposure. (Lane 7) Same as lane 5, but reduced prior to SDS-polyacrylamide gel electrophoresis. (Lane 8) Same as lane 6, but reduced prior to SDSpolyacrylamide gel electrophoresis. Molecular weight markers were myosin (212 kd): phosphorylase A (92 kd): bovine serum albumin (68 kd); actin (45 kd); carbonic anhydrase (29 kd); and cytochrome C (12 kd).

these antibodies is of insufficient sensitivity. However, some antisera have been obtained that react specifically to embryos (Wiley and Calarco, 1975; Solter and Knowles, 1979; Johnson and Calarco, 1980~) so it appears that the embryo can be immunogenic. Despite our numerous attempts to obtain hybridomas with embryo-sensitized mouse or rat spleens, only one embryo-specific monoclonal antibody has been obtained (one of 2000 clones screened from 14 different fusions). This result might indicate that few B cells respond specifically to embryo immunization; these immunizations often yielded hybrid clones, excluded from further investigation, that reacted with both embryos and cell lines. The expression of SSEA3 is developmentally regulated in murine embryos. This antigenic determinant, expressed on all preimplantation mouse embryos, is progressively restricted in the preimplantation period, first to inner cell mass and subsequently to primitive endoderm. In the adult mouse, the antigen is restricted to the tubules of the kidney (Fox et al., 1982). Human teratocarcinoma-derived cell lines containing ECCs and human erythrocytes were the only cell types found

6. A Further

2

I

Analysis

2

of the SSEA3

3 Antigenic

4 Determinant

(a) 2102Ep cells labeled with ‘H-fucose. (Lane 1) Cells were treated with 2.5 (Lg tunicamycin (4.5 hr), then labeled with 3H-fucose and immunoprecipitated with anti-SSEAB nude mouse ascites; 1.6 X lo4 cpm. (Lane 2) Cells were not incubated with tunicamycin, but were immunoprecipitated with anti-SSEAS nude mouse ascites; 3.2 x 1 O4 cpm; 9 week exposure. (b) Cells labeled by the galactose oxidase‘H-labeled sodium borohydride technique. (Lane 1) LNSV cells immunoprecipitated with anti-SSEAS nude mouse ascites; 6 x 10’ cpm. (Lane 2) 2102Ep cells immunoprecipitated with anti-SSEAS; 2 X 1 O3 cpm. (Lane 3) Human red blood cells immunoprecipitated with rat IgM ascites; 1 X 1 O3 cpm. (Lane 4) Human red blood cells immunoprecipitated with anti-SSEAS; 1 x lo4 cpm. Exposure for lanes l-4 was for 1 month.

I

2

Figure 7. Four- to Eight-cell-Stage Lactoperoxidase-Glucose Oxidase

Embryos Method

Labeled

with “?

by the

(Lane 1) Embryos were immunoprecipitated with antiSSEA nude mouse ascites; 9 X 1 O3 cpm. (Lane 2) Embryos were immunoprecipitated with rat IgM ascites: 2 x lo3 cpm. Exposure for both lanes was for 3 weeks.

Cell 702

reactive with this antibody in our survey. Comparison of the distribution of SSEA3 with other antigens previously described as expressed on murine preimplantation embryos indicates that SSEA3 is a new SSEA. With one exception (Johnson and Calarco, 1980~) the previously described antigens have been expressed on both preimplantation embryos and murine ECCs (Solter and Knowles, 1979); SSEA3 does not correspond to the antigen defined by Johnson and Calarco (198Oc), who used rabbit anti-murine-blastocyst serum, since their antigen is absent from unfertilized eggs, zygotes and 2-cell stage embryos. The existence of SSEA3 confirms our previous notion that immunization with embryos rather than teratocarcinomas is required to define embryo-specific antigens. The interspecies distribution of SSEA3 places it in the category of the heterogenetic antigens (Landsteiner, 1936), which have been previously described as present on mouse embryos and on tissues or cells from other species; these include the Forssman antigen (Willison and Stern, 1978) SSEAl (Knowles et al., 1982) ABH blood group antigens (Szulman, 1980) human blood group antigen I (Kapadia et al., 1981; Knowles et al., 1982) and globoside (K. Willison, personal communication). The antigenic determinant of each of these molecules is carbohydrate in nature and can be expressed on glycoprotein or glycolipid molecules, or both (Watkins, 1980). Our results indicate that the same is true for SSEA3. The exact nature of the carbohydrate antigenic determinant is not known, although it appears to bear some relationship to the globoside molecule (S.-i. Hakomori, personal communication). Because the antibody reacts with all stages of preimplantation embryos, including unfertilized eggs, the present data do not indicate whether the embryo actively synthesizes SSEA3 or whether the antigen is manufactured during oogenesis and is carried only on the membrane until it is diluted out or replaced at the blastocyst stage. While both SSEA3containing glycoproteins and glycolipids of human teratocarcinoma cell lines become metabolically labeled with 3H-fucose, no detectable immunoprecipitate of any glycoprotein molecule was found after a 5 hr labeling period with either 35S-methionine or a 3Hlabeled amino acid mixture (data not shown). This could indicate that the turnover of the protein components is extremely slow or that these proteins are present in relatively low abundance in the cell as a whole. Determination of a functional role for cell-surface components in preimplantation embryos is extremely difficult; the only positive results have been the reported use of conventional, xenogeneic antisera, which affect compaction (Kemler et al., 1977; Johnson et al., 1979; Ducibella, 1980). These experiments led to the identification of a polypeptide, uveomorulin, which interacts with Ca2+ ions in regulating cell contact (Hyafil et al., 1980, 1981). Monoclonal antibody to SSEA3 does not appear to affect normal develop-

ment of preimplantation embryos in vitro (L. Shevinsky, unpublished results). Without precise definition of the carbohydrate structure of SSEAS, it is not yet possible to remove this antigenic determinant specifically from the embryo cell surface and thus test for its role in development. It may be that subtle changes in carbohydrate structure, as mediated, perhaps, by different glycosyl transferases, are very important in directing preimplantation development. While the role of SSEA3 in development awaits further clarification, the anti-SSEA3 monoclonal antibody has proven very useful in defining the stem cells in human teratocarcinoma cultures (Andrews et al., 1982) providing for the first time a specific marker of this previously undefinable cell type. The ECCs in tumors derived from injection of these same cell lines also express SSEA3. Antibody to SSEAB has also been used to detect the presence of stem cells in frozen sections of human germ cell tumors (Damjanov et al., 1982) and will likely prove useful in the clinical immunodetection of human metastatic teratocarcinoma, as previously described for murine teratocarcinema and antibody to SSEAl (Ballou et al., 1979). Experimental

Procedures

Preparation of Hybridomas A Fisher rat was immunized with 4-to 8-cell-stage, zonae pellucidaefree mouse embryos isolated from random-bred ICR mice. The embryos were isolated 2 days after the presence of a vaginal plug, in Dulbecco’s modified phosphate-buffered saline (PBS), treated with 0.5% pronase to digest zonae pellucidae and allowed to recover for 1 hr in 0.1 M HEPES-buffered Whitten’s medium (HWM) (Whitten, 1971) without bovine serum albumin, but with the addition of 2% normal mouse serum. The embryos were frozen in solubilization buffer containing 0.1% Nonidet-P40 (NP40) and 2 mM phenylmethylsulfonyl fluoride. The first immunization contained 1000 embryos, the second immunization contained 1200 embryos and the third and final immunization contained 2300 embryos; all immunizations were intraperitoneal. The rat was bled 7 days after the second immunization, and the serum was found to have a 50% cytotoxic endpoint at a dilution of 1:400 on 4- to 8-cell stage embryos. The rat was immunized again and killed 3 days later, and its spleen was removed for fusion: 3 X 10’ spleen cells were fused with 3 X IO’ SP2/0, P3X63Ag, or P3x63Ag8 variant 653 (mouse myeloma) cells (Kohler and Milstein, 1975). distributed in 300 wells (Linbro 24-well plates) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) (DMEM with 10% FBS) supplemented with 2 mM glutamine and the hypoxanthine, aminopterin and thymidine (HAT) components (Littlefield, 1964). Supernatants from wells containing growing colonies were tested for reactivity on 4- to 8-cell-stage embryos by indirect immunofluorescence and indirect radioimmunoassay. One positive colony out of 220 tested was transferred to mass culture and cloned: this colony derived from a fusion with SP2/0. The cloned hybrid cells were removed from HAT-selective media by successive plating in DMEM, and injected into pristane-primed nude mice and nude rats for ascites production. The ascites fluid was removed from animals with visible tumors, clarified by centrifugation and frozen at -70°C until further use. Reagents Rabbit antirat IgG (heavy- and light-chain-specific), uncoupled or conjugated with fluorescein isothiocyanate (FITC). and goat antirat IgG (heavyand light-chain-specific), conjugated with tetramethyl rhodamine (TMR) were purchased from Cappel Laboratories. Rabbit antimouse IgM and rabbit antimouse IgG were prepared as described by Aden and Knowles (1976) Guinea pig sera were collected, ali-

Stage-Specific 703

Embryonic

Antigen

3

quoted and stored at -70°C until used as a complement source: these sara were screened to eliminate those with spontaneous cytotoxicity for embryos. Rabbit antimouse IgM and rabbit antimouse IgG were iodinated by the chloramine-T method (Hunter, 1978). Tunicamycin was obtained from Calbiochem. Absorption Cell lines used for absorption or radioimmunoassay were harvested by a brief exposure to 0.25% trypsin in 0.1 M EDTA and pipetted to obtain single-cell suspensions in 0.01 M HEPES-buffered DMEM with 10% FBS. Quantitative absorptions were performed with supernatant diluted 1:25 or ascites fluid diluted 1 :lO,OOO (approximately 80% of maximum activity on embryos), incubated with 1 O’, 5 x 1 06, 2 x 1 O6 or 1 O6 cells for 1 hr at 4°C. Absorption with cells derived from organs was similarly performed with cells liberated from the organs by gentle homogenization with a loose-fitting pestle in a test tube; cells were separated from debris by several washings in DMEM with 10% FBS, counted and resuspended for absorption at the above concentrations. Leukocytes and erythrocytes were separated from normal human peripheral blood by sedimentation at 37°C after addition of 20% PLASMAGEL (R. Bellon Labs). Granulocytes were separated from mononuclear cells on a Ficoll-Hypaque (Pharmacia) discontinuous density gradient. Cells were washed three times in PBS without calcium and magnesium salts, prior to resuspension and use. The absorbed antibody was clarified by centrifugation and tested on 4-to 8-cell-stage embryos by complement-mediated cytotoxicity. Radioimmunoassay To screen for embryo-reactive hybridomas. we incubated ten embryos (4- to 8-cell-stage) without zonae pellucidae in undiluted culture supernatants (1 hr at 37”C), washed them three times in HWM, transferred them to a mixture of ‘251-labeled rabbit antimouse IgM and rabbit antimouse IgG (1 pl drops under paraffin oil) and incubated them for 1 hr at 37°C. Embryos were then washed six times in HWM, transferred to tubes and assayed for radioactivity in a Packard gamma counter. To test for reactivity of the hybridoma on different cell lines, we incubated lo5 cells with hybridoma supernatant for 1 hr at 4°C or with SP2/0 supernatant, washed them three times with PBS with 2% FBS and 0.02 M sodium azide, then incubated them with ‘251-labeled rabbit antimouse IgM diluted to give 50,000 input cpm per sample. Cells were again washed three times, transferred to tubes and assayed for reactivity in a Packard gamma counter. Reactivity with cell lines was considered positive if the average cpm of triplicate samples was three times the control value. lmmunofluorescence and Complement-Mediated Cytotoxicity on Embryos For immunofluorescent assays, unfertilized eggs and preimplantation embryos were isolated from randomized ICR mice as described by Solter and Schachner (1976). Inner cell masses were isolated from blastocysts by immunosurgery (Salter and Knowles, 1975). Endoderm was removed from inner cell masses, after growth in vitro for 3 days, by repeated immunosurgery (Strickland et al., 1976). Embryos without zonae pellucidae were incubated in 20 gl drops of either undiluted culture supernatant or nude mouse ascites diluted 1 :I 00 for 30 min at 37°C. Embryos were washed three times in HWM and then incubated in FITC- or TMR-conjugated IgG (diluted 1 :lO, for 30 min at 37’C). Embryos were washed three times in HWM. put in individual drops on a microscope slide under paraffin oil and examined with a Leitz Microscope equipped with fluorescent epi-illumination and a 50x water immersion lens. For complement-mediated cytotoxicity, embryos were incubated in 20 pl drops of either undiluted culture supernatant or nude mouse ascites diluted 1 :lOO for 1 hr at 37°C washed once with HWM and transferred to 20 ~1 drops of guinea pig complement (1 hr at 37’0. Complement-mediated cytotoxicity was scored as described by Solter and Schachner (1976). lmmunofluorescence of Ovarian Oocytes and Tumors Ovaries were quick-frozen in O.C.T. compound (Miles Laboratories), sectioned on a cryostat (7 pm thickness), placed on albuminized

slides and fixed in acetone. Sections were incubated in monoclonal antibody (nude mouse ascites 150) or control rat IgM for 2 hr at 22°C in a humidified chamber, washed three times in PBS, incubated with FITC-conjugated rabbit antimouse IgM diluted 1 :lO, for 1 hr, washed three times in PBS, covered with a coverslip after adding a drop of 50% glycerol, 50% PBS and examined immediately. This same procedure was utilized on tumors derived from subcutaneous injection of 1 O7 2102E cells into the right flank of athymic (nu/nu) mice. Radioisotope Labeling of Cells and Antibody The human teratocarcinoma cell lines 833KE (Bronson et al., 1980) and 2102E (Andrews et al., 1980) were surface-labeled with ‘*? by the lactoperoxidase-glucose oxidase method (Hynes, 1973) or with ‘H-labeled sodium borohydride by the galactose oxidase technique (Gahmberg and Hakomori. 1973). or metabolically labeled with 3HL-5,6-fucose. Cells were seeded in 100 mm petri dishes at a density of 5 x lo6 cells per dish. They were grown for 2 days and then labeled either with 0.6 mCi ‘Z51-labeled sodium iodide (New England Nuclear; NEZ-033H) per petri dish, or with 0.5 mCi 3H-fucose New England Nuclear; NET-5761 for 6 hr. After labeling, cells were washed and scraped. For labeling with 3H-labeled sodium borohydride, the cells were scraped, washed in PBS and incubated with galactose oxidase (Sigma), followed by 1 mCi freshly prepared 3H-labeled sodium borohydride (New England Nuclear: NET-023). Following washing and centrifugation, cells were resuspended in 0.1 M Tris (pH 6.8). 2 mM EDTA, 15% glycerol and 2 mM phenylmethylsulfonyl fluoride (solubilization buffer) and extracted for 1 hr at 4*C with 0.1% NP40. Following 30 min of centrifugation at 18,000 rpm, 5 ~1 samples were removed to determine the counts precipitable by trichloroacetic acid (TCA). The hybridoma cells were labeled with ??,-methionine. A culture of the hybridoma yielding 2.3 x 1 O7 cells was incubated with 2.0 mCi ?S-methionine (New England Nuclear: NEG-OOST) in methionine-free media for 5 hr. Cells were removed by centrifugation, and a 5 pl sample of supernatant was taken for TCA counts. Surface-Labeled Embryos Approximately 200 4- to 8-cell-stage zonae-pellucidae-free embryos were placed in a 50 ~1 drop of Whitten’s medium (without bovine serum albumin and phenol red) supplemented with 0.5% Ficoll. Five microliters of 50 mM glucose, 7 gl containing 35 PCi ‘251-labeled sodium iodide and 2 gl containing enzymes (1 mg lactoperoxidase. 5 U glucose oxidase. 450 gl PBS) was added and mixed every 5 min for a total reaction time of 30 min. Fifty pl of PBI (PBS with Nal substituted for NaCI) was then added to the reaction mixture, and embryos were washed by transfer seven times through Whitten’s medium with Ficoll. Labeling and washings were carried out under the dissecting microscope. Live embryos were then placed in 50 ~1 solubilization buffer with 0.1% NP40 for 1 hr at 40°C 5 pl was removed to determine TCA-precipitable counts and the remaining suspension was quick-frozen and stored at -2O’C. Eight different iodinations were pooled (total 1 .5 X 10” cpm). For immunoprecipitation, the iodinated sample was divided in half; one portion was reacted with the monoclonal antibody, and one portion with the control IgM antibody. lmmunoprecipitation 35S-labeled supernatant from the hybridoma cells was immunoprecipitated with rabbit antirat IgG and a cross-reactive rabbit antimouse IgM. The resultant precipitate was washed in 0.1 M NaCI, 0.1 M Tris (pH 7.4), 0.25% NP40 until the counts remaining in the supernatant wash were less than 1000 cpm. The pellet was resuspended in lysis buffer and analyzed by two-dimensional electrophoresis (O’Farrell, 1975). Cell extracts yielding a minimum of 2 x 10’ cpm, or embryo extracts, were incubated for 1 hr at 4OC with 5 PI nude mouse ascites containing antiSSEA monoclonal antibody or with 5 fil of a control ascites containing an unrelated monoclonal rat antibody of the IgM class (rat IgM ascites, supplied by T. McKearn). Rabbit antimouse IgM (25 pl) was added, and incubation was continued for 18 hr. Precipitates were washed as described above until less than 1000

Cell 704

cpm remained in the wash. Pellets were resuspended in solubilization buffer containing 2% SDS. Dithiothreitoi (0.1 M) was added as a reducing agent where required, and samples were boiled for 2 min and analyzed on 7.5-l 5% SDS-polyacrylamide gels (Laemmli. 1970). Acknowledgments This work was supported in part by grants from the National Institutes of Health, the National Science Foundation and by the American Cancer Society. L. H. S. was supported by a grant from the National Institutes of Health in partial fulfillment of the requirements for the Ph.D. degree. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

March

15, 1982;

revised

July 20, 1982

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