A method to measure carcinoembryonic antigen neosynthesis by cultured colon carcinoma cells.
A double-antibody radioimmunoprecipitation assay was used to measure the rate of incorporation of radioactive precursors into carcinoembryonic antigen (CEA) newly synthesized by cultured human colon carcinoma (LoVo) cells. Mixtures of 125I-labeled CEA and standard CEA were used to construct titration curves as a function of concentration, time of incubation, and temperature for goat anti-CEA serum and rabbit antigoat serum. In contrast to the zirconyl phosphate gel method, the double-antibody technique did not precipitate compounds with low molecular weights. Similar slopes were observed when standard CEA was used to inhibit immunoprecipitation of 125I-labeled CEA by both the zirconyl phosphate gel and the double-antibody techniques. When CEA produced by the colon carcinoma cells (CEA-LoVo) was used to inhibit immunoprecipitation of radioactive 125 I-labeled CEA, the curve was superimposed upon that elicited for standard CEA. CEA-LoVo incorporated significant amounts of [N-acetyl-3H]glucosamine, and chromatography of the 3H-labeled CEA-LoVo eluted with a peak superimposed upon that of the First British Standard for CEA. This method was subsequently used to estimate changes in the rate of CEA neosynthesis by LoVo cells as a function of culture age. The rate of incorporation of [N-acetyl-3H]glucosamine into CEA of LoVo cells in stationary phase was about three times that measured for cells in exponential growth. The technique described in this report has the advantage that labeling of CEA results from actual metabolic cellular activity. Therefore, it provides a tracer method to study the localization and kinetics of CEA synthesis by cultured colon cells.
D000117:Acetylglucosamine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D002272:Carcinoembryonic Antigen / Q000096:biosynthesis*; D002277:Carcinoma / Q000276:immunology*; D002460:Cell Line; D002846:Chromatography, Affinity; D003110:Colonic Neoplasms / Q000276:immunology*; D005643:Fucose / Q000378:metabolism; D006801:Humans; D011863:Radioimmunoassay / Q000379:methods*
D002272:Carcinoembryonic Antigen; D005643:Fucose; D001224:Aspartic Acid; D000117:Acetylglucosamine