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Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody by Christopher H. J. Ford; Georgia C. Tsaltas; Perry A. Osborne; Karima Addetia is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

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A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDSPAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of nondegraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic

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Author

Christopher H. J. Ford; Georgia C. Tsaltas; Perry A. Osborne; Karima Addetia

Publisher

John Wiley and Sons; Wiley (John Wiley & Sons); John Wiley & Sons Inc.; John Wiley & Sons Ltd.; Wiley (ISSN 0196-4763)

Published

1996

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)