Limitations in the use of low pH extraction to distinguish internalized from cell surface-bound radiolabeled antibody.
Internalization by cells of radiolabeled protein ligands bound to the cell surface is frequently analyzed by extraction of the cells with low pH buffers. This treatment supposedly strips the ligands from the cell surface, and remaining molecules are considered to be internalized. However, we show herein that: (1) low molecular weight catabolic products that are trapped within lysosomes (residualizing radiolabels) are efficiently extracted by low pH buffers, under the same conditions used to remove cell surface-bound material, and (2) low pH treatment lyses the majority of the cells, as shown with both a nonadherent and an adherent cell line, with the release of most of a (51)Cr label. Still, low pH extraction was effective at demonstrating Ab internalization, as has been demonstrated many times. These effects of low pH treatment may be attributed to the fixative properties of these buffers. Regardless of the mechanism, these data must be taken into consideration in interpreting the results of such experiments.
Ong
G L
GL
Garden State Cancer Center at the Center for Molecular Medicine and Immunology, Belleville, New Jersey, USA.
Mattes
M J
MJ
D000143:Acids / Q000378:metabolism / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000915:Antibody Affinity; D002277:Carcinoma / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002860:Chromium Radioisotopes; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D016393:Lymphoma, B-Cell / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology; D008247:Lysosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014343:Trypan Blue; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000737:chemistry* / Q000378:metabolism / Q000473:pathology
D000143:Acids; D000911:Antibodies, Monoclonal; D002860:Chromium Radioisotopes; D008024:Ligands; D014343:Trypan Blue