Preparation of fragments of carcinoembryonic antigen and identification of a major subset of antigenic determinants.
The detailed immunochemistry of a limited molecular domain of carcinoembryonic antigen (CEA) was examined, by using new methods to prepare and purify immunoreactive fragments. Fragments of CEA were prepared by digestion of the reduced and alkylated glycoprotein with protease V8, chymotrypsin or trypsin, or by chemical cleavage at cysteine residues with 2-nitro-5-thiocyanobenzoic acid. The products were fractionated by high performance liquid chromatography (HPLC) on a reversed phase support, and the most immunoreactive fragment was recovered from each of the four mixtures. The Mr of these purified fragments ranged from 30,000-35,000, the amino acid compositions were similar, and all contained carbohydrate. The four independently derived fragments gave similar inhibitions of CEA binding to anti-CEA antisera; maximum values were 15-55% depending on the antiserum employed. Competitive assays with pairs of fragments in different combinations showed that the four contained essentially the same subset of antigenic determinants. The fragments were quite resistant to further enzymatic digestion, but comparison of the HPLC profiles of mild acid hydrolysates of two of the fragments showed substantial similarity. These results suggest that the CEA molecule includes a region of about 30,000 mol. wt that is contained between consecutive cysteine residues, is relatively resistant to a number of residue-specific proteases, and constitutes a significant subset of antigenic determinants. Assays to identify cleavage products with different antigenic specificities were negative, suggesting that other determinants were destroyed or that the corresponding antibody populations were too small to detect.
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002272:Carcinoembryonic Antigen; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D011863:Radioimmunoassay
D000596:Amino Acids; D002241:Carbohydrates; D002272:Carcinoembryonic Antigen; D000939:Epitopes; D010446:Peptide Fragments; D010450:Endopeptidases