Heterogeneity of circulating carcinoembryonic antigen analyzed by sandwich-enzyme immunoassays with different specificities

Nine monoclonal antibodies against carcinoembryonic antigen (CEA) were prepared and used to investigate the immunological and physicochemical heterogeneity of circulating CEA. Two of these, M221-73 and M272-11, recognized "CEA-distinctive" epitopes and they gave sandwich-enzyme immunoassays far less reactive with nonspecific cross-reacting antigen (NCA) and nonspecific cross-reacting antigen-2 (NCA-2). Sandwich-enzyme immunoassays selective for NCA-2 and NCA were also established using suitable monoclonal antibodies as competitive inhibitors against enzyme-labeled antibodies. The studies on the serum CEA levels determined by the "CEA-specific" assay indicated that CEA molecules recognized by M221-73 and M272-11 were generally found in the sera of both cancer patients and normal adults. CEA and related substances in sera were further analyzed by the sandwich-enzyme immunoassays after adsorption on M272-11-coupled immunosorbents and by gel filtration on an Ultrogel AcA-34 column. These studies revealed the presence of a CEA variant detected by the "NCA-2-selective" assay. The variant seemed to be closely related to NCA-2 because it lacked CEA-distinctive epitopes and had an apparent molecular weight similar to that of NCA-2. This variant appeared in the sera of some cancer patients and normal adults.     

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.     

Natural antibodies against the carcinoembryonic antigen (CEA) and a related antigen, NCA, in human sera

Natural antibodies directed against CEA and a related antigen, NCA, have been demonstrated in all normal and pathological sera using an EIA, although they have never been detected by RIA. These antibodies are not anti-blood group antibodies, as their titer was decreased only slightly by absorption with blood group substances. The study of their reactivity with deglycosylated antigens demonstrated that they were directed against peptidic epitopes. Antibodies against NCA or CEA, purified using specific immunosorbents, cross-reacted with all the antigens of the "CEA family" but not or only weakly with unrelated antigens.     

Proteolytic release of antigenic fragments corresponding to normal fecal antigen and non-specific cross-reacting antigen from carcinoembryonic antigen.

Three immunogenic parts have so far been identified in the carcinoembryonic antigen (CEA) molecule. These are: determinants cross-reactint with the normal fecal antigen (NFA) (NFA determinant); determinants cross-reacting antigen (NCA) (NCA determinant); and determinants which appear to be more cancer-specific (cancer determinant). The chemical nature of these parts of the CEA molecule was investigated by digestion with proteolytic enzymes together with anti-CEA preparations with which these three immunogenic parts of CEA molecule could be identified. The CEA digest obtained with pepsin did not react in immunodiffusion and radioimmunoassay, indicating that pepsin completely destroyed all the antigenic parts. Digestion by pronase E destroyed only the cancer determinant and liberated two antigenic fragments corresponding to the NFA determinant and the NCA determinant, respectively. These results suggest that the cancer determinant may reside in a protein or a peptide part of the molecule. The chemical nature of the NFA and NCA determinant remains to be clarified.     

Characterization of carcinoembryonic antigen-specific monoclonal antibodies and specific carcinoembryonic antigen assay in sera of patients

Six murine monoclonal antibodies (mAbs) reactive with carcinoembryonic antigen (CEA) were prepared. Four of these, CA204, CA205, CA206 and CA208, were specific to purified CEA whereas the other two, NA201 and NA203, were also reactive with the non-specific cross-reacting antigen (NCA). These mAbs were all IgG1, except one IgG2a (CA206), with high affinities for CEA. NA203, CA204 and CA208 appear to define three different epitopes on the CEA molecule as determined by competitive binding assay. These mAbs also reacted with CEA-producing cells. The treatment of the cells with tunicamycin did not affect the binding of the mAbs to CEA-producing cells. None of the above mAbs bound to CEA-related antigens, NCA-2, alpha 1-acid glycoprotein, or blood group antigens. The combination of CA208 (solid phase) and CA204 (tracer) was used to construct a sensitive CEA-specific sandwich ELISA to detect CEA in the sera of patients with various malignant and non-malignant diseases. Particularly when CEA values were low in sera from non-cancerous patients, the above mAbs sandwich assay showed reduced background reactivity with NCA-like substances and permitted the detection of CEA at a level as low as 1 ng/ml.     

Protein epitopes in carcinoembryonic antigen. Report of the ISOBM TD8 workshop.

To characterize antigenic sites in carcinoembryonic antigen (CEA) further and to investigate whether there are differences between colon tumor CEA and meconium CEA (NCA-2) that can be detected by anti-CEA monoclonal antibodies (MAb), 19 new anti-CEA MAb were analyzed with respect to specificity, epitope reactivity and affinity. Their reactivities were compared with 10 anti-CEA MAb with known CEA-domain binding specificity that have previously been classified into five nonoverlapping epitope groups, GOLD 1-5. Cross-inhibition assays with antigen-coated microtiter plates and immunoradiometric assays were performed in almost all combinations of MAbs, using conventionally purified CEA (domain structure: N-A1B1-A2B2-A3B3-C) from liver metastasis of colorectal carcinomas, recombinant CEA, meconium CEA (NCA-2), truncated forms of CEA and NCA (CEACAM6) as the antigens. The affinity of the MAbs for CEA was also determined. The new MAbs were generally of high affinity and suitable for immunoassays. Three new MAbs were assigned to GOLD epitope group 5 (N-domain binding), 3 MAbs to group 4 (A1B1 domain), 1 to group 3 (A3B3 domain), 3 to group 2 (A2B2 domain) and 3 to group 1 (also the A3B3 domain). Three MAbs formed a separate group related to group 4, they were classified as GOLD 4' (A1B1 domain binding). The remaining 3 MAbs appear to represent new subspecificities with some relationship to GOLD groups 1, 2 or 4, respectively. Five MAbs, all belonging to epitope group 1 and 3, reacted strongly with tumor CEA but only weakly or not at all with meconium CEA, demonstrating that the two products of the CEA gene differ from each other, probably due to different posttranslational modifications.     

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.     

Monkey antisera with increased specificity to carcino-embryonic antigen (CEA).

Three Macaca irus monkeys were immunized with purified human CEA. One received unmodified CEA and two were immunized with haptenated (4-hydroxy-5-iodo-3-nitrophenyl acetyl) CEA. All three monkeys formed precipitating antibodies to CEA. The antisera did not precipitate the CEA-related normal glycoprotein antigen (NCA). In contrast, CEA-immunized rabbits, sheep and goats invariably form antibodies against NCA. Radio-immunoassays showed the monkey antisera to contain trace amounts of antibodies to NCA but the titers were about 10-1,000 times lower than those in rabbit and sheep sera with comparable anti-CEA titers. These results show that monkeys produce antibodies against CEA, but respond weakly to a normal, CEA-related antigen. The weak response to NCA suggests that monkeys may possess an NCA-related antigen. Antisera lacking reactivity to normal CEA-related antigens may be helpful in establishing a more specific diagnostic test for CEA.     

Carcinoembryonic antigen immunocytochemistry in primary breast cancer

We studied the immunoreactivity by immunohistology of two carcinoembryonic antigens (CEA) with specific and two CEA antibodies with nonspecific cross-reacting antigen (NCA) cross reactivity (CEA/NCA) in 180 primary breast carcinomas. Positive tissue staining was found in more than 90% of the specimens with CEA/NCA antibodies, compared with less than 30% for both CEA-specific antibodies. There was no correlation between the positivity of CEA immunocytochemistry for any of the four antibodies and histologic grade, lymph node stage, locoregional recurrence, disease-free interval (DFI), or patient survival. This large study with a long follow-up period for patients has shown that CEA and CEA/NCA immunocytochemistry have no relation to prognosis in breast cancer. An extensive review of the literature that confirms these findings should end the controversy over the place of CEA and CEA/NCA immunocytochemistry in breast cancer.     

Determination of the specificities of monoclonal antibodies recognizing members of the CEA family using a panel of transfectants

Carcinoembryonic antigen (CEA), one of the most clinically important tumor markers, is mainly used in the post-surgical surveillance of patients with colorectal carcinomas. CEA belongs to a large protein family, which includes cross-reacting antigens, e.g., non-specific cross-reacting antigens (NCAs) and biliary glycoprotein (BGP) as well as pregnancy-specific glycoproteins (PSGs). The genes encoding these proteins can be subdivided into the CEA and PSG subgroups. The members of the subgroups share antigenic determinants and show high similarity in amino-acid sequences. Their derived secondary structures show them to belong to the Immunoglobulin superfamily. Due to the close relationship of the members of the CEA subgroup, it is very difficult to distinguish between the individual members with MAbs. Here we have used flow cytometric analysis of transfectants expressing individual members of the CEA subgroup as an alternative approach to determine the specificities of 13 MAbs. This allows us to examine the specificities of these antibodies for members of the CEA family, even of those which have not yet been characterized at the protein level. In addition, binding of the MAbs to NCAs expressed by polymorphonuclear cells (PMN) was tested by Western-blot analysis, immunoprecipitation and flow cytometry. Four antibodies bound exclusively to NCA-50/90 and one MAb (80H3) only to NCA-95. MAb 4/3/17 recognizes CEA and BGP on the surface of transfectants and NCA-160 from granulocytes. We assume that NCA-160 is a product of the BGP gene. On granulocytes, which do not express CEA, MAb 4/3/17 is specific for NCA-160 (BGP). Mutual inhibition of the MAbs binding to NCA-50/90 revealed 3 different epitope groups.     

Nonspecific crossreacting antigen (NCA) is a major member of the carcinoembryonic antigen (CEA)-related gene family expressed in lung cancer.

Carcinoembryonic antigen (CEA) is one of the most important tumour markers in the management of human carcinoma, including lung cancer. So far, however, because of the nonspecificity of anti-CEA antibodies, it remains unclear whether the experimental measurements of CEA expression really reflect genuine CEA. In normal lung, nonspecific cross reacting antigen (NCA) has been described as a major component of CEA-related antigens. Recently isolated CEA and NCA cDNA clones enabled us to analyse CEA and NCA expression of in vivo tumour specimens and tumour cell lines at mRNA levels. NCA-specific mRNA (but not CEA-specific mRNA) was detected in all normal lung tissues examined. Of 21 lung cancer tissue specimens, nine expressed both NCA and CEA and five expressed only NCA. Of 16 tumour cell lines, two expressed only NCA and one expressed both NCA and CEA, although its level of CEA mRNA was weaker than that of NCA mRNA. Therefore, CEA-related mRNA expression was always accompanied by NCA mRNA expression; there were no cases of CEA mRNA expression alone. These findings suggest that NCA is a major member of the CEA-related gene family expressed in lung cancer.     

Immunochemical differences among carcinoembryonic antigen in tumor tissues and related antigens in meconium and adult feces

We have isolated carcinoembryonic antigen (CEA)-related antigens from meconium and compared them with those in adult feces. Two CEA-related antigens were detected in meconium [nonspecific cross-reacting antigen 2 (NCA-2) and meconium nonspecific cross-reacting antigen] while four CEA-related antigens were found in adult feces [normal fecal antigen 1, normal fecal antigen 2 (NFA-2), normal fecal cross-reacting antigen, and fecal nonspecific cross-reacting antigen, respectively]. By conventional anti-CEA antisera, NCA-2 in meconium, NFA-2 in adult feces, and CEA in tumor tissues were indistinguishable from each other, but they could be distinguished by specific antibody preparations against a determinant unique to CEA (CEA-distinctive determinant) or to NFA-2 (NFA-2-distinctive determinant). Neither the CEA-distinctive determinant nor the NFA-2-distinctive determinant was detected on the NCA-2 molecule. No antigenic determinants unique to NCA-2 have been detected with the anti-NCA-2 antisera which we have prepared thus far. The molecular weight of purified NCA-2 was estimated to be 150,000 to 170,000 as compared to 160,000 to 170,000 for NFA-2 and 170,000 to 180,000 for CEA. NCA-2 had amino acid and carbohydrate compositions similar to those of CEA and NFA-2. All NFA-2 preparations and about one-half of the CEA preparations were sensitive to Pronase E digestion, which released two antigen fragments from these molecules, but NCA-2-preparations were resistant to such digestion.     

Definition of the expression of the human carcinoembryonic antigen and non-specific cross-reacting antigen in human breast and lung carcinomas

Mouse cell lines transfected with carcinoembryonic antigen (CEA) and with 2 other members of the human CEA gene family, non-specific cross-reacting antigen (NCA) and biliary glycoprotein (BGP), were used to analyze the specificity of several monoclonal antibodies (MAbs). MAbs COL-1 and COL-6 were shown to react with the transfected CEA gene product but not with NCA, confirming previous results. Cells expressing the transfected BGP gene product also failed to react with COL-1 and COL-6. The MAb B6.2 reacted with cells expressing the NCA gene product but not with those expressing CEA or BGP. The MAb B1.1 reacted strongly with the transfected CEA and BGP gene products but only weakly with the NCA gene product. These antibodies were then utilized in the histochemical analysis of a number of primary and secondary breast and lung tumors. The results indicate that a majority of breast and lung tumors express CEA, and nearly all breast and lung tumors express NCA. Fairly homogeneous expression of CEA and NCA was seen in the majority of both breast and lung tumors. Our results indicate that CEA may be an important target for immunotherapy in a large number of patients with breast and lung tumors.     

Investigation of nonspecific cross-reacting antigen 2 as a prognostic biomarker in bone marrow plasma from colorectal cancer patients

Carcinoembryonic antigen (CEA) is still the only routinely used biomarker in colorectal cancer (CRC), but its utility is hampered by poor specificity and sensitivity, and the search for novel biomarkers is highly warranted. The nonspecific cross-reacting antigen 2 (NCA-2), a truncated CEA species molecule which is transcribed from the same gene, has been suggested as an alternative biomarker to CEA. In the present work, specific immunofluorometric assays were used for detection of NCA-2 and full-length CEA in bone marrow plasma from 277 CRC patients to assess their value as prognostic biomarkers, and detection was also performed in tumor tissue and a CRC cell line. Elevated plasma CEA was associated with advanced tumor stage at diagnosis and adverse patient outcome, while for NCA-2, although the same trends were observed, no additional prognostic information was gained. While specific detection of NCA-2 was clearly achieved in plasma samples, cross-reactivity with full-length CEA was observed when the antigen was exposed to common fixation chemicals. The results from this study indicate that NCA-2 is probably not a prognostic biomarker in CRC and, furthermore, underline the issue of antibody specificity when investigating CEA species molecules.     

Sensitive detection of carbohydrate determinants on carcinoembryonic antigen preparations by lectin and antibody binding using polyethylene glycol

Blood group determinants have been found in five carcinoembryonic antigen (CEA) preparations with a lectin and antibody-binding assay using polyethylene glycol 6000 to separate free from bound radiolabeled antigen. The assay described gives excellent sensitivity for the binding or inhibition of binding of various lectins or antibodies to CEA. One of the CEA preparations investigated has an A1 determinant, another has a B determinant, and all have H, Lea, Leb, and MN blood group determinants. In addition, all of the preparations tested bound concanavalin A. These findings are consistent with the idea that incomplete or unexpected glycosylation patterns occur in glycoproteins produced by tumor cells. Since antibodies directed against blood group substances cross react with carbohydrate determinants on CEA, clinical determinations of CEA or anti-CEA levels in serum may be adversely affected.     

CEA-specificity of CEA-reactive monoclonal antibodies. Immunochemical and immunocytochemical studies

Four carcinoembryonic antigen (CEA) reactive monoclonal antibodies Parlam 1, 4, 5 and 6 were studied in respect to reactivity with CEA and its cross-reacting antigens (NCA-1, NCA-2, BGP). In immunochemical studies (ELISA and SDS-PAGE followed by immunoblotting) Parlam 4 did not react with these crossreacting antigens, whereas Parlam 1, 5, and 6 demonstrated variable reactivity with these antigens. As expected, by immunocytochemistry Parlam 1, 5, and 6 stained bile canaliculi in the liver (due to crossreactivity with BGP), pneumocytes and splenic tissue (NCA-1) to an extent comparable with the results in biochemical tests. In contrast with the immunochemical observations, however, Parlam 4 showed slight but distinct reactivity with splenic granulocytes (NCA-1) and hepatic bile canaliculi (BGP). Relative epitope specificity of the monoclonal antibodies was tested in blocking experiments. These showed that Parlam 1 and 5 detect the same epitope on CEA as they block each others binding completely. In other combinations monoclonal antibodies block each others binding only partially, indicating that they detect different or partially overlapping epitopes. These results suggest that CEA specific epitopes may partly overlap with epitopes on crossreacting antigens. In this context, we propose that on crossreacting antigens epitopes exist that are structurally similar to epitopes on CEA or that some epitopes on CEA may consist of a spatial configuration which involves CEA specific as well as non-CEA specific structures. The antibody will show the highest affinity towards CEA, as this molecule contains the uniquely matching or complete epitope and lower affinity towards the crossreacting antigen with an imperfectly matching or only partially available epitope.     

Characterization of a species of non-specific cross-reacting antigen (NCA) exressed by human monocytic cell lines: Structure and expression during cell differentiation

It has been documented that human monocytes/macro-phages are reactive with antibodies directed to carcinoembryonic antigen (CEA) and non-specific cross-reacting antigens (NCAs), a group of glycoproteins antigenically cross-reactive with CEA, yet the molecules responsible for this antigenic activity have not been fully clarified. In the present study, among 7 myelomonocytic cell lines tested, 2 monoblastoid lines, U-937 and THP-I, were found to express NCA-50/90, a glycosyl-phosphatidylinositol-anchored cell-adhesion molecule chiefly expressed on granulocytes. The 2 cell lines showed a reaction pattern with 5 distinct anti-CEA and anti-NCA monoclonal antibodies, similar to that of CHO transfectants expressing recombinant NCA-50/90. Immunoprecipitation and SDS-PAGE analyses identified glycoproteins of about 95 and 55 kDa in U-937 and THP-1 cells, respectively. Deglycosylation of the 2 antigens with N-glycanase gave the same apparent molecular mass of about 45,000, which was also the same as that of the deglycosylated form of the recombinant NCA-50/90. Upon Northern-blot analysis, only one band of approximately 2.5 kb was detected in both cell lines with a cDNA probe for NCA-50/ 90, which has a broad specificity to the CEA gene family members. cDNA cloning demonstrated that the 2.5-kb clones encode the peptide of NCA-50/90. The expression of NCA-50/90 by U-937 and THP-1 was down-regulated at both the protein and mRNA levels during cell differentiation from monoblastoid to monocyte/macrophage-like cells induced by stimulation with phorbol 12-myristate 13-acetate. Our observations suggest that NCA-50/90 is a differentiation antigen of cells of the monocyte/macrophage lineage as well as of the granulocyte lineage.     

Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonic antigen or HCT-8R cells

A library of 18 monoclonal antibodies (MAbs) reactive with purified carcinoembryonic antigen (CEA) has been prepared. The specificity of these MAbs was tested and they have been separated into nine subgroups, each recognizing a different region of the CEA molecule. Seven MAbs from four of the groups also react with the nonspecific cross-reacting antigen. Some of the MAbs are directed against conformational determinants: three of the MAb groups bind poorly to sodium dodecyl sulfate-treated CEA, while five of the groups are not reactive with reduced and alkylated CEA. Three of the groups react with purified CEA but not with the cell surface CEA of HCT-8R cells, while the other groups react with both forms. The MAbs were tested for binding to fragments of CEA obtained by chemical cleavage and the groups of MAbs were found to react with different subsets of such fragments.     

Expression of complementary DNA and genomic clones for carcinoembryonic antigen and nonspecific cross-reacting antigen in Chinese hamster ovary and mouse fibroblast cells and characterization of the membrane-expressed products

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648-4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [3H]-ethanolamine (a component of the PI-G anchor) but not with [35S] methionine, whereas the recombinant NCA could be labeled with both [3H]ethanolamine and [35S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.