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 50k 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 50k 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.