title: Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.
abstract: Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.
authors.affiliation: Oncology Research, Memorial University, St. John's, Newfoundland, Canada.
authors.forename: G
authors.initials: G
authors.lastname: Tsaltas
authors.forename: C H
authors.initials: CH
authors.lastname: Ford
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000187:drug effects*; D000951:Antigens, Neoplasm / Q000276:immunology; D016477:Artifacts; D002021:Buffers; D002272:Carcinoembryonic Antigen / Q000276:immunology; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D005998:Glycine / Q000494:pharmacology*; D006801:Humans; D006851:Hydrochloric Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D002021:Buffers; D002272:Carcinoembryonic Antigen; D006851:Hydrochloric Acid; D005998:Glycine