Purification and characterization of a 50,000 mol. wt glycoprotein antigen, and localization of determinants involved in cross-reactivity with carcinoembryonic antigen.
A 50,000 mol. wt glycoprotein antigen (50 k) was purified from metastatic colon tumor. The initial purification was monitored by cross-reactivity of the antigen with an unabsorbed antiserum against carcinoembryonic antigen (CEA, Mr 180,000). Six mg of antigen was obtained from 250 g of tissue, with a recovery of 14% and a relative purification of 143-fold. The amino acid composition of 50 k was similar to that of CEA. The carbohydrate content, primarily glucosamine and mannose, totaled about 25%. Immunodiffusion showed that 50 k lacked some of the CEA epitopes, and that normal spleen contained an antigen identical to 50 k. Radioimmunoassays showed that the high avidity anti-CEA antibodies in the unabsorbed antiserum were mainly cross-reactive with 50 k, probably via a similar but not identical antigenic site. The immunochemical properties of 50 k thus correspond to those of the non-specific cross-reacting antigen (NCA). The molecular size and carbohydrate composition reported for various NCA-like antigens have differed, so identity of 50 k an NCA cannot be established on this basis. Immunoreactive fragments of 50 k were prepared by digestion with each of three different proteolytic enzymes, and were purified by high performance liquid chromatography (HPLC). A polypeptide of 20,000-30,000 mol. wt containing about half of the 50 k determinants, was recovered in each case. Experiments with mixtures showed that the three purified fragments all contained the same subset of antigenic determinants. The fragment-localized subset represented most, if not all of the 50 k determinants involved in CEA cross-reactivity. Similarly, a CEA fragment was shown to contain essentially all of the CEA determinants involved in 50 k cross-reactivity. The fragments of 50 k and CEA were quite resistant to further proteolytic digestion, and comparison of a CEA fragment with a 50 k fragment by partial acid hydrolysis and HPLC peptide mapping failed to reveal structural homology to account for the cross-reactivity.
D000596:Amino Acids / Q000032:analysis; D000941:Antigens / Q000276:immunology / Q000302:isolation & purification*; D000951:Antigens, Neoplasm; D002241:Carbohydrates / Q000032:analysis; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D015815:Cell Adhesion Molecules; D002851:Chromatography, High Pressure Liquid; D003429:Cross Reactions; D000939:Epitopes / Q000032:analysis*; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D011863:Radioimmunoassay
D000596:Amino Acids; D000941:Antigens; D000951:Antigens, Neoplasm; D002241:Carbohydrates; D002272:Carcinoembryonic Antigen; D015815:Cell Adhesion Molecules; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments