A radioimmunoassay for human epidermal growth factor receptor.
The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.
D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D002850:Chromatography, Gel; D003902:Detergents; D066246:ErbB Receptors; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D010920:Placenta / Q000032:analysis; D011092:Polyethylene Glycols; D011863:Radioimmunoassay; D011956:Receptors, Cell Surface / Q000032:analysis*; D012995:Solubility
D000911:Antibodies, Monoclonal; D003902:Detergents; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011092:Polyethylene Glycols; D066246:ErbB Receptors