NOVEL FLOW CYTOMETRIC ANALYSIS OF THE PROGRESS AND ROUTE OF INTERNALIZATION OF A MONOCLONAL ANTICARCINOEMBRYONIC ANTIGEN (CEA) ANTIBODY

A now cytometric method of studying the internalization of a monoclona l antibody (Mab) directed against carcinoembryonic antigen (CEA) has b een compared with Western blotting, using three human colonic cancer c ell lines which express varying amounts of the target antigen. Cell sa mples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using now cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell Lines provi ded evidence of nondegraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min, Internalized anti-CEA was det ected in the case of high CEA expressing cell lines (LS174T, SKCO1). V ery similar results were obtained with an anti-fluorescein now cytomet ric assay. Given that these two methods consistently provided comparab le results, use of now cytometry for the detection of internalized ant ibody is suggested as a rapid alternative to most currently used metho ds for assessing antibody internalization. The question of the endocyt ic route followed by CEA-anti-CEA complexes was addressed by using hyp ertonic medium to block clathrin mediated endocytosis. (C) 1996 Wiley- Liss, Inc.