Characterization of carcinoembryonic antigen fractionated by concanavalin A chromatography.
A small percentage (15%) of particles morphologically distinct from carcinoembryonic antigen (CEA) cruller-like particles (averaging 9 x 40 nm) as determined by electron microscopy, and previously presumed to be impurities, has been removed from CEA preparations by concanavalin A (Con A) affinity chromatography. The first of four affinity fractions constituted 45% of the recovered weight; the last, which constituted 17% of the recovered weight, was obtained only after a 2-day soak in 20% alpha-D-mannopyranoside. The affinity fractions were of essentially equivalent specific activity, morphological appearance, and composition. They all demonstrated markedly different chemical compositions and an approximately 100-fold higher specific activity than the bulk of the nonaffinity material. Thus, the bulk of the Con A-nonbound fractions apparently is not CEA but contaminating particles of several different varieties. Compositional analyses further indicated that the mannose content of various CEA fractions was directly correlated with Con A binding affinity, amino acid content, morphological type, and CEA specific activity. This led to the conclusion that the protein chain of the various CEA preparations fractionated by us is characteristic. An attempt to fractionate very impure crude CEA by Con A affinity chromatography indicated that this scheme cannot by itself produce as pure CEA as standard methods. Thus, for the first time the effectiveness of the purification of a glycoprotein has been successfully monitored by electron microscopy, demonstrating that the cruller-like appearance of the CEA particle is closely related to antigenic specificity. Whether antisera to our affinity fractions will improve the specificity and sensitivity of the clinical assay is currently being investigated.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002272:Carcinoembryonic Antigen / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003208:Concanavalin A; D000939:Epitopes; D008358:Mannose / Q000032:analysis; D008854:Microscopy, Electron
D000596:Amino Acids; D002241:Carbohydrates; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D003208:Concanavalin A; D008358:Mannose