Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.
One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).
Tsaltas
G
G
Oncology Research, Memorial University, St John's, Nfld., Canada.
Ford
C H
CH
Gallant
M
M
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western / Q000379:methods; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D003110:Colonic Neoplasms; D015179:Colorectal Neoplasms; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006735:Horseradish Peroxidase; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms; D016253:Microscopy, Immunoelectron / Q000379:methods; D011863:Radioimmunoassay / Q000379:methods; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D006735:Horseradish Peroxidase