C. Wagener

Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non‐specific crossreacting antigen (NCA)

Immobilized carcinoembryonic antigen (CEA) and non‐specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM α‐methyl D‐mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D‐mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.

Transblot studies with biotin-labeled proteins: Electrophoretic mobilities and detection limits

Molecular weight standard proteins, mouse IgG as well as several antigens cross-reacting with the carcinoembryonic antigen (CEA), were biotin labeled, submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The bound proteins were revealed by the use of avidin-peroxidase conjugates and a suitable substrate. The ratio of N-hydrosuccinimido biotin (NHSB) to protein yielding the lowest detection limit was determined. At an optimal NHSB/protein ratio, 0.33 ng of IgG heavy chains and 0.17 ng of IgG light chains could be visualized. With the exception of human albumin and ovalbumin, the increase in apparent molecular weight after biotin labeling was less than 10% for the proteins tested. The method has proven to be a valuable addition to Western blots performed with CEA-related antigens and monoclonal anti-CEA antibodies.

The Putative Role of Members of the CEA-Gene Family (CEA, NCA an BGP) as Ligands for the Bacterial Colonization of Different Human Epithelial Tissues

Immobilized purified CEA (carcinoembryonic antigen), NCA (non-specific crossreacting antigen) and BGP I (biliary glycoprotein I) bind strains of E. coli (including EPEC) and some Salmonella species (including S. typhi, S. paratyphi A + B and S. java) while Shigella-, Yersinia- and Bacteroides- strains showed no adhesion. The binding was of high avidity, heat sensitive, dose dependent, saturable and nearly completely abolished in the presence of 10 mM alpha-methylmannoside. From inhibition studies with aromatic mannose compounds, it was suggested that in contrast to Salmonella strains E. coli strains exhibit a higher hydrophobicity in the binding region adjacent to the CEA-, NCA- and BGP-binding site. By further inhibition experiments it could be demonstrated that E. coli and Salmonella strains bind to high-mannose type oligosaccharides of these molecules via lectins on bacterial type I fimbriae. We conclude that the expression of products of this gene family on different human epithelial cells (colon-, bile canaliculi, uroepithel etc.) may function as ligands for bacterial colonization of epithelial tissues.

Characterization of biotin-labeled proteoglycans by electrophoretic separation on minigels and blotting onto nylon membranes prior and after enzymatic digestion.

Biotinylated proteoglycans were separated by sodium dodecyl sulfate electrophoresis prior and after enzymatic digestion by glycan-specific enzymes using polyacrylamide minigels. The biotin-labeled compounds were blotted onto nylon membranes either by electrophoresis or by diffusion and detected by avidin-enzyme conjugates. The method allows the nonisotopic detection of native proteoglycans and core proteins. Proteoglycans can be visualized at protein amounts as low as 0.7 ng per lane. In comparison with sensitive protein stains, compounds of enzyme preparations do not interfere with bands corresponding to core proteins. Electrophoresis, blotting, and staining of up to 12 samples per gel are accomplished in less than 3 h.

Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences

Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.

Monoclonal antibodies reveal repetitive crossreactive and single specific epitopes on the protein moiety of carcinoembryonic antigen

ConclusionsAs each of the monoclonal antibodies binds to deglycosylated CEA, the antigenic determinants reside on the protein moiety of the molecule. For this reason, homologous amino acid sequences should be responsible for the presence of repetitive epitopes on CEA. Most probably, the crossreactive determinants on NCA are also protein in nature. Assuming single epitopes on NCA and homologous repetitive epitopes on CEA residing on the protein moieties of both molecules, an ancestral NCA-related gene may have undergone a recent duplication to form a single gene with subsequent divergence of the CEA specific sequences. The extensive homology of the aminoterminal sequence of NCA with that of CEA [6] is in accordance with this conclusion.

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies.

Abstract A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.