Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.
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.
Tsaltas
G
G
Oncology Research, Memorial University, St. John's, Newfoundland, Canada.
Ford
C H
CH
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
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000936:Antigen-Antibody Complex; D000951:Antigens, Neoplasm; D002021:Buffers; D002272:Carcinoembryonic Antigen; D006851:Hydrochloric Acid; D005998:Glycine