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Identification and Characterization of New Antigenic Fragments Related to Carcinoembryonic Antigen in Adult Feces

Abbott Laboratories, Abbott Diagnostics Division, North Chicago, Illinois 60064 [J. G. H., E. J. V., M. S. M., H. G. R., J. T. T.] and Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010 [R. J. P., J. E. S.]

Antigens in human adult feces related to carcinoembryonic antigen (CEA) were analyzed with respect to their molecular masses, CEA domain compositions, and N-terminal amino acid sequences. By avoiding perchloric acid treatment, new fecal antigens related to CEA were identified. The fecal antigens revealed by Western blot were M_r 78,000, 70,000, 60,000, 50,000, 44,000, 36,000, 33,000, and 25,000 and a species M_r 14,000. Unlike native CEA, all of the fecal antigens were very poorly soluble in perchloric acid and did not bind to concanavalin A, suggesting that they had undergone significant deglycosylation in the digestive tract. The major fecal antigens were purified by immunoaffinity chromatography and their N-terminal amino acid sequences determined. FA78, FA60, FA33, and the M_r 14,000 antigen had the N-terminal amino acid sequence of the CEA N-domain, and FA44 and FA25, the sequence of the CEA A2 domain. The CEA domain compositions of the fecal antigens were investigated by probing them with anti-CEA monoclonal antibodies of known domain specificities. The N-terminal amino acid sequences, immunoreactivities with anti-CEA monoclonal antibodies, and apparent molecular masses of the fecal antigens allowed the following domain assignments (based on CEA as N-A1B1-A2B2-A3B3): FA78, N-A1B1-A2B2-A3B3; FA60, N-A1B1-A2B2; FA44, A2B2-A3B3; FA33, N-A1B1; and FA25, A2B2. The M_r 14,000 antigen was shown to be the N-domain of CEA or nonspecific cross-reacting antigen. FA36 was assigned the N-AB domain structure of nonspecific cross-reacting antigen. The results suggested that FA78, FA60, FA44, FA33, and FA25 were degradation products (including deglycosylation and proteolysis) of CEA and that FA36 was a degradation product of nonspecific cross-reacting antigen.

¹ To whom requests for reprints should be addressed.

² Present address: Immunex Corp., 51 University Street, Seattle, WA 98101.

³ Present address: Hybritech Inc., P.O. Box 269006, San Diego, CA 92916-9006.

Received 12/ 3/91. Accepted 5/12/92.

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