anti-CEA Sera Research Articles

Carcinoembryonic Antigen Continuous Epitopes Determined by the SPOT Method

Carcinoembryonic antigen (CEA) is a heavily glycosylated tumor-associated protein with an N-A1-B1-A2-B2-A3-B3 domain structure. Circulating CEA immunoassays are used for monitoring digestive cancer patients, and radiolabeled anti-CEA monoclonal antibodies (MAb) are used for the diagnosis and therapy of CEA-positive tumors. The five major nonoverlapping epitopes (Gold 1–5) have been broadly correlated with the domain organization, but there is no precise localization of the epitopes at the sequence level. In an attempt to identify the peptide sequences corresponding to the five Gold epitopes on the CEA molecule, we prepared a set of 227 overlapping fifteen-mer peptides corresponding to the complete CEA sequence with the SPOT method. Using five high affinity MAbs directed against the five CEA Gold epitopes, we demonstrated that none of these epitopes could be mimicked by a fifteen-mer peptide sequence. However, using rabbit and goat anti-CEA sera, we identified six major continuous antigenic regions. All are included in the Ig-like domains of the CEA: two in the A1 domain (residues 120–134 and 153–164), one each in the A2 (329–337) and A3 domains (508–513), one at the junction between the A3 and B3 domains (553–561) and one in the B3 domain (565–573). A very homologous sequence (common residues VSPRL) was mapped in each of the three A domains. Thus, in terms of occurrence of continuous epitopes, the Ig-like domains A1, A2, A3 and B3 seem to be the most antigenic parts of CEA. These peptide sequences should be good candidates for the future development of site-specific anti-CEA MAbs.

CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae.

Isolation and Characterization of Rat Carcinoembryonic Antigen

A rat tumor-associated antigen with properties similar to those of human carcinoembryonic antigen (CEA) has been detected with rabbit immune sera in extracts of transplantable rat colonic adenocarcinoma, RCA-1. This antigen, termed rat CEA, was also detectable by a monkey antihuman CEA serum and a rat monoclonal antibody to rat CEA. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of perchloric acid extracts of RCA-1 tumor, followed by immunoblotting with the above-mentioned anti-CEA reagents, revealed that rat CEA activity resided in components with a molecular weight of approximately 350 kD. The glycoprotein nature of these components was indicated by positive staining with periodic acid-Schiff. Sephadex G-200 chromatography, as well as Sepharose 4B chromatography with and without sodium dodecyl sulfate indicated that the 350-kD components existed in the extracts as molecular aggregates. The 350-kD material, which had been purified by an affinity column containing rat monoclonal antibodies to rat CEA, reacted with the rabbit and monkey anti-CEA sera. This provided strong evidence that serological activity of rat CEA was confined to the 350-kD components.

Purification and characterization of carcinoembryonic antigen from GW-39, a xenografted human colonic tumor system.

Carcinoembryonic antigen (CEA) was purified from GW-39 human tumor xenografts in hamsters by immunoaffinity chromatography. Binding of the antigen to immobilized monoclonal antibody provided a high degree of purification of CEA in a single step. A recovery of 79% and a 750-fold purification were obtained. The purified CEA has a molecular size of 180 kilodaltons, an isoelectric point of 4.4, and a specific activity of 0.94. About 73% of the radiolabeled GW-39 CEA reacted with goat anti-CEA serum. The amino acid and carbohydrate compositions of the GW-39 CEA were comparable to those of CEA preparations from other sources.

Localization of carcinoembryonic antigen in mature intracranial teratomas.

Carcinoembryonic antigen (CEA) in serum and cerebrospinal fluid (CSF) was measured in four patients with intracranial teratoma. The CEA levels were elevated in the CSF of two patients, but were within normal limits in the serum of all four. After surgical removal of the teratomas, which were verified as mature teratomas, CEA was localized by an immunohistochemical method. Positive reactions both to anti-CEA serum and to another anti-CEA serum absorbed with nonspecific cross-reacting antigen were seen in glandular structures, with or without goblet cells, and in some portions of stratified squamous epithelium. It is concluded that CEA, detected in CSF, may originate in mature teratomas, and CEA-positive structures (especially glandular) may differentiate into gastrointestinal tract structures. An examination of serum and CSF levels of CEA may offer additional clues to the diagnosis of intracranial germ-cell tumors.

Cervical carcinoma antigen, carcinoembryonic antigen (CEA), and nonspecific cross-reacting antigen (NCA) in appraisal of uterine cervix smears.

The distribution of cervical carcinoma antigens (AgCaCx), CEA, and NCA in different pathologic states of the uterine cervix was studied in cytologic smears by an immunofluorescence method (IF) using specific immune sera against perchloric acid (PCA) extract of cervical squamous cell carcinoma, anti-CEA, and anti-NCA. After excluding cross-reactivity with CEA and NCA, the presence of AgCaCx was demonstrated in the majority of cervical carcinomas, severe dysplasias, and only in one-fourth of squamous metaplasias, especially when accompanied by mild or moderate dysplasias. The intensity and percentage of IF-positive cells varied from case to case. The preparations of uterine cervix without pathologic changes usually were negative. Similar results were obtained with anti-CEA serum. NCA was present in the majority of smears independent of histologic diagnosis. The most intense fluorescence was observed in upper layers of the epithelium. NCA could be a differentiation antigen of stratified squamous cell epithelium.

Tumour-associated antigens in mammary and extramammary Paget's disease.

The immunoperoxidase technique was used to study the immunoreactivities of two murine monoclonal antibodies to carcinoma-associated antigens raised respectively against a human breast cancer line (MBr1) and an ovarian carcinoma (MOv2) and of a conventional anti-CEA serum in 20 cases of mammary Paget's disease of the nipple and in three cases of extramammary Paget's disease. Each of the immunoreagents stained Paget's cells in a high proportion of cases and failed to discriminate mammary from extramammary disease. The antigenic phenotypes of underlying in situ or infiltrating breast carcinomas corresponded to those of the associated Paget's disease of the nipple. The consistent immunoreactivity of eccrine and apocrine sweat glands and of normal mammary epithelia indicated an antigenic relationship between epithelia of adnexal derivation and Paget's cells.

Search for specific markers of neoplastic epithelial duct and myoepithelial cell lines established from human salivary gland and characterization of their growth in vitro.

The neoplastic epithelial duct cells human salivary gland (HSG) and myoepithelial cells human pleomorphic adenoma (HPA) established from human salivary gland were examined by the immunoperoxidase method for the presence of specific antigens such as carcinoembryonic antigen (CEA), S-100 protein, secretory component (SC), lactoferrin (LF), and myosin. Isolation of the cells and their morphologic features were reported previously. Consequently, the presence of CEA, SC, and LF in the HSG cells was demonstrated. The HPA cells were identified to express the specific antigens reactive to anti-S-100 protein, anti-myosin and anti-CEA sera in addition to the presence of oxytocin receptor. When the two cell lines were co-cultured in monolayer culture or within the sponge matrix, a large number of ductlike or tubular structures were formed in an optimal ratio of 1:2 in HSG and HPA cells, whereas the cultures of HSG cells only grew with occasional formation of ductlike structure. In addition, in HSG and HPA cells in an area with their contact in the mixed cultures, CEA staining was intensified as compared with the culture of HSG or HPA cells only and further S-100 protein was detected in HSG cells, whereas S-100 protein was not detected in the culture of HSG cells only. These findings strongly suggest that the intercalated duct and myoepithelial cells from human salivary gland propagate with their interaction together in the expression of specific antigens such as CEA and S-100 protein or in the morphogenesis of salivary gland epithelial cells.

CEA and NCA in benign and malignant breast tumors.

The involvement of Nonspecific Crossreacting Antigen (NCA) in the immunohistological demonstration of Carcino Embryonic Antigen (CEA) in 56 benign and 92 malignant lesions of the breast was analyzed. For this purpose, the authors utilized both polyclonal antisera and monoclonal antibodies. Polyclonal anti-CEA sera were used after absorption with normal tissue antigens, in order to remove crossreactivity, and without such an absorption. Ninety-three percent of breast carcinomas, 85% of mastopathic lesions not associated with a carcinoma, and 66% of fibroadenomas showed positive reactions with commercial unabsorbed polyclonal anti-CEA serum, which contained antibodies to NCA, whereas incubation with monospecific anti-CEA antiserum resulted in 42% positivity in carcinomas and negativity in mastopathic lesions and fibroadenomas. Forty-eight percent of breast cancer, 84% of mastopathic lesions, and 50% of the fibroadenomas contained NCA in different quantities. The staining pattern of carcinomas and fibroadenomas obtained with unabsorbed anti-CEA antibody and anti-NCA did not run parallel in all cases. Monoclonal antibodies against CEA and NCA confirmed the results obtained with polyclonal antiserum. This study suggests a cancer specificity of CEA in breast lesions.

Carcinoembryonic antigen in gynecologic cancers. Immunohistochemical localization and serum levels.

The presence of carcinoembryonic antigen (CEA)-dependent fluorescence was observed in almost 89% of female genital tract cancers irrespective of their histologic type. Anti-CEA serum was free of antibody to normal cross-reacting antigen. The high percentage of positive fluorescence tests did not correlate with the preoperative serum CEA levels. Double immunodiffusion tests showed different content of CEA and normal cross-reacting antigen in individual specimens of genital tract cancers.

Cross-reactivity with normal antigens in commercial anti-CEA sera, used for immunohistology. The need for tissue controls and absorptions.

The demonstration of carcinoembryonic antigen (CEA) in serum and in tissue sections may be of value in diagnosis and follow-up of several malignant tumors. Anti-CEA sera, however, cross-react with normal antigens, also present in tumors. The staining patterns of three commercially produced anti-CEA sera were investigated on sections of benign and malignant tissues, using an indirect immunoperoxidase technic. All three anti-CEA sera tested showed cross-reactions. A rapid, simple, and reproducible immunoabsorption was developed. After absorption, the reactivity of the sera with normal colon mucosa and carcinomas of the colon remained positive, whereas sections of benign tissues were negative. Moreover, four of ten breast cancers, positive with unabsorbed antisera, became negative after absorption. These findings stress the importance of immunohistochemical negative and positive controls. The use of well-absorbed commercial anti-CEA sera can lead to more uniform results, exchangeability of results, and a better understanding of the value of this tumor marker.

Carcinoembryonic antigen like antigen in granular cell myoblastomas

A series of granular cell myoblastomas (GCM) and other benign and malignant tumours of soft tissue were examined for cytoplasmic content of carcinoembryonic antigen (CEA) by the two-layer conjugated immunoperoxidase technique. Using a commercial rabbit anti-CEA serum only granular cell myoblastomas showed positive cytoplasmic reaction. Pretreatment with periodic acid made this reaction less intense, but when the commercial rabbit anti-CEA serum was absorbed with tissue powder from normal human spleen the positive reaction was totally abolished. It is concluded that the positivity of GCM for CEA using commercial rabbit anti-CEA serum is due to the content of non-specific cross-reacting antigen (NCA) and maybe other cross-reacting glycoproteins in this tumour, and not to CEA as claimed in a previous study.

Sensitiziation of human lymphocytes to carcinoembryonic antigen (CEA): neutralization experiments with anti-CEA sera

By means of the macrophage electrophoretic mobility technique we could show lymphocytes of patients suffering from cancers of the digestive system to be sensitized to carcinoembryonic antigen (CEA). These findings conflict with the common view that CEA is not immunogenic in humans. The aim of the present study was to look as to whether conventional anti-CEA sera can neutralize the activity of a CEA preparation which is responsible for the human lymphocyte response. When 60 ng CEA were preincubated with highly diluted anti-CEA serum and the resulting immune complexes were thereafter co-precipitated by protein A-sepharose, positive lymphocyte responses could no longer be obtained. This effect was observed with 3 anti-CEA sera in 3 cancer patients (colon cancer, stomach cancer, teratocarcinoma), who's lymphocytes responded to CEA by lymphokine release. Normal serum had no neutralizing effect. The anti-CEA sera did not influence the activity of another tumour-relevant extract (teratocarcinoma-derived), to which cancer patients' lymphocytes reacted regardless of the tumour site. The lymphocytes from an oesophagus carcinoma patient, though reacting to the teratocarcinoma preparation, did not respond to CEA, thus, logically, all other tests with normal serum and anti-CEA sera were negative, too. The results show that the digest system cancer-associated lymphocyte reactivity to CEA can be abrogated by conventional anti-CEA sera, which finding indicates that there exist closely CEA-associated "tumour-specific" antigenicities.

A Method To Measure Carcinoembryonic Antigen Neosynthesis by Cultured Colon Carcinoma Cells<xref ref-type="fn" rid="fn2">2</xref>

A double-antibody radioimmunoprecipitation assay was used to measure the rate of incorporation of radioactive precursors into carcinoembryonic antigen (CEA) newly synthesized by cultured human colon carcinoma (LoVo) cells. Mixtures of 125I-labeled CEA and standard CEA were used to construct titration curves as a function of concentration, time of incubation, and temperature for goat anti-CEA serum and rabbit antigoat serum. In contrast to the zirconyl phosphate gel method, the double-antibody technique did not precipitate compounds with low molecular weights. Similar slopes were observed when standard CEA was used to inhibit immunoprecipitation of 125I-labeled CEA by both the zirconyl phosphate gel and the double-antibody techniques. When CEA produced by the colon carcinoma cells (CEA-LoVo) was used to inhibit immunoprecipitation of radioactive 125 I-labeled CEA, the curve was superimposed upon that elicited for standard CEA. CEA-LoVo incorporated significant amounts of [N-acetyl-3H]glucosamine, and chromatography of the 3H-labeled CEA-LoVo eluted with a peak superimposed upon that of the First British Standard for CEA. This method was subsequently used to estimate changes in the rate of CEA neosynthesis by LoVo cells as a function of culture age. The rate of incorporation of [N-acetyl-3H]glucosamine into CEA of LoVo cells in stationary phase was about three times that measured for cells in exponential growth. The technique described in this report has the advantage that labeling of CEA results from actual metabolic cellular activity. Therefore, it provides a tracer method to study the localization and kinetics of CEA synthesis by cultured colon cells.

Immunologic and biochemical studies on the carcinoembryonic antigen-like substance in human neuroblastoma.

Several authors have observed that the plasma levels of carcinoembryonic antigen (CEA) in patients with neuroblastoma were significantly elevated. The present study was undertaken to investigate the nature of CEA activity in neuroblastoma tissue. This tumor tissue contains a small amount of CEA-like substance reacting with anti-CEA serum which is characterized by gamma-globulin electrophoretic mobility, a molecular weight that is approximately equal to that of albumin (4.6S) by gel filtration, and a glycoprotein staining with periodic acid-Schiff (PAS). According to the double immunodiffusion method, this antigen is partially identical to purified CEA of colon carcinoma, and is completely identical to nonspecific crossreacting antigen (NCA). This antigen is, therefore, referred to not as the CEA as described by Gold, but as NCA in neuroblastoma tissue. The elevation of plasma CEA activity in patients with neuroblastoma may be due to the release of NCA from tumor cells, or to the destruction tissues by metastasis, of normal which are rich in NCA, or to a combination of both.

DETERMINATION OF CARCINOEMBRYONIC ANTIGEN (CEA): COMPARISON OF TWO DIFFERENT ASSAYS

A comparison was made of CEA determination by the CEA-RIAKIT and our slightly modified CEA-Roche assays. The normal levels (mean + 2 SD) found in 63 blood donors were 2.3 microgram/1 and 3.3 microgram/1, respectively. The inter-assay reproducibility was similar in the range below 15 microgram/1, with variations from 0.3 microgram/1 (1 SD, less than 5 microgram/1) to 1.4 microgram/1 (1 SD, 10--15 microgram/1). Both methods measured CEA concentrations down to 1 microgram/1. The ability of the two assays to discriminate between positive and negative values in patients with urogenital and gastrointestinal cancer was similar. The response to recurrences of colorectal carcinoma was also similar, and the CEA fluctuation was parallel after surgery. The CEA-Roche assay generally showed good correlation between measured and theoretical values at both low and high levels. Under our test conditions, the disparity between indirect and direct values was 14 microgram/1 +/- 8 (n = 4) in the range 18--32 microgram/1, and this must be taken into consideration in interpreting the CEA-Roche test. The CEA-RIAKIT measured too low values as compared to both the CEA-Roche and the theoretical values. The discrepancy increased with increasing CEA values, and the ability to distinguish between high CEA concentrations (greater than 15 microgram/1) was poor. The explanation seems to be that the anti-CEA sera used in this assay show a lower affinity for plasma CEA than for tumour-extracted CEA. Immunochemical differences between sample CEA and CEA used for calibration of the standard curves must be considered in evaluation of CEA assays.

Chemical modifications of the protein of carcinoembryonic antigen: associated changes in immunological activity and conformation.

Chemical substitution of the exposed residues of tryptophan, tyrosine, histidine and arginine in carcinoembryonic antigen (CEA), using appropriately selective reagents, caused no significant change in the capacity of the antigen to bind to anti-CEA serum. However, treatments of CEA with 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, both in the presence of guanidine HCl, caused a large reduction in binding capacity. Measurement of the circular dichroism spectra of all of the products showed that retention of conformation of the molecular correlated well with retained antigenic activity, whereas the large losses in capacity to bind to anti-CEA sera were accompanied by a probably the result of gross conformational changes. The tyrosine residues of CEA may be classified into three categories: (i) 3 freely reacting residues, (ii) 7 or 8 moderately buried residues and (iii) 15 unreactive residues.

Isolation of CEA-like material from urinary bladder carcinoma

Carcinoembryonic antigen (CEA)-like material from urinary bladder carcinoma was purified and compared with the immunological and physico-chemical properties of CEA isolated from colon cancer. After immunoadsorbent purification, the final step in a purification procedure similar to that adopted for colon cancer CEA, two main molecular species were identified: 1) Material identical with colon cancer CEA with respect to molecular size, PCA solubility, ability to bind to Con A, and most important the ability to bind to specific monkey anti-CEA serum. 2) Material with the same chromatographic and immunological properties as NCA.

Comparison of whole plasma and perchloric acid-extracted plasma assays for carcinoembryonic antigen

A direct, paired, comparison was made of two plasma radioimmunoassays for carcinoembryonic antigen (CEA), between a perchloric acid-extracted, ammonium sulphate precipitation method; and a whole-plasma, double-antibody method. For both assays the same preparations of standard CEA, 125I-labelled CEA, and anti-CEA serum were used. Plasma samples were taken from young healthy subjects (24), cancer-free hospital inpatients (44) and cancer-proven untreated patients (94). Within-batch and between-batch variation were both greater for the extracted-plasma method. Cancer discriminatory ability was assessed by probabilistic means. With cancer-free inpatients as the reference group, no difference between the assay methods was revealed. With young healthy subjects as the reference group the whole-plasma assay was superior.

Morphologic and immunologic studies of a human colon tumor cell line (SW-48).

A permanent human tissue culture cell line (SW-48) has been established from an adenocarcinoma of the transverse colon. Cells in the center of early colonies were cuboidal and loosely bound; cells on the periphery of the same colonies were more columnar, the nuclei were displaced toward the basal region, and the free surface formed stunted microvilli. These columnar cells ofter aligned themselves to resemble normal absorptive tissue. Carcinoembryonic antigen (CEA) was identified by immunofluorescent microscopy on the surface membrane of the tumor cells. Significantly more CEA could be isolated from the culture medium than from the whole cells. On agar gel diffusion analysis with a monospecific anti-CEA serum (G.P. 32), SW-48 CEA and CEA preparations from solid tumors demonstrated complete identity.