Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti‐carcinoembryonic antigen (CEA) antibody

Abstract

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. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded 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 medium to block clathrin mediated endocytosis.

Bispecific antibody targeting of doxorubicin to carcinoembryonic antigen-expressing colon cancer cell linesin vitro andin vivo

“…19 Anti-CEA MAb (hybridoma 11-285-14, one of the fusion partners for BsMAb 26-61-10) and non-specific Ag8 as control (P3x63Ag8, a mouse myeloma that secretes a non-specific IgG1 of the same isotype as 11-285-14) were used to assess CEA expression (both at 10 g/ml) with an EPICS XL (Coulter, Hialeah, FL). Goat anti-mouse Ig-FITC conjugate (Coulter), at a dilution of 1/100, was used for detection.…”

“…Human colon cancer cell lines LS174T, SKCO1 and COLO320DM were maintained in culture as described previously. 19 …”

A homogeneous fluorescence-based method to measure antibody internalization in tumor cells

“…The traditional method relies on an acidic buffer to dissociate the surface-bound radioisotope-labeled antibody [2]. A variation of this method uses a fluorophore for signal detection and an anti-fluorophore antibody to quench the surface-associated fluorescence [3,4]. A fluorophorelabeled secondary antibody can also be employed to measure the decrease of membrane-associated primary antibody, thereby providing an indirect estimate of the internalized fraction of primary antibody [5].…”

Combined flow cytometry and confocal laser scanning microscopy for evaluation of BR96 antibody cancer cell targeting and internalization

“…Quantitative analyses using flow cytometry (FCM) allow fast evaluation of a large number of cells which provides statistically reliable data. FCM methods for detection of one fluorescence‐conjugated mAb have previously been used to demonstrate binding and internalization (21). The so‐called “multicolor FCM” enables analysis of differently labeled markers simultaneously in a high number of cells.…”

“…The so‐called “multicolor FCM” enables analysis of differently labeled markers simultaneously in a high number of cells. Parts of the internalization process of the BR96/LeY have been revealed by means of analysis in situ at cellular and subcellular levels using CLSM and electron microscopy on a human breast cancer cell line (H3396) (1, 3, 10–12, 19–23). Among the various cell imaging techniques available for analyses of tissues and cells labeled with fluorophores and/or antibody fluorescence conjugates, CLSM is commonly used to identify and visualize different specific components simultaneously at the cellular level (24, 25).…”