A dot assay for the erythropoietin receptor using human recombinant 125I-erythropoietin

A dot assay was developed for the detection of membrane receptor(s) for erythropoietin (Ep). A relatively homogeneous population of cells bearing the receptor for Ep was generated in the spleen of mice made anemic with phenylhydrazine and crude membrane extracts were prepared from spleen cell suspensions. Aliquots of the membrane extracts were applied to microdishes of nitrocellulose in a volume of 4 microliters. After free reactive sites were blocked, the microdishes were incubated for 2 h at 37 degrees C with 125I-labeled human recombinant Ep (125I-rEp), and nitrocellulose bound radioactivity was determined thereafter. Reproducible curves were obtained, and a significant correlation between bound radioactivity and the amount of membrane proteins applied to the nitrocellulose dishes was found. Specific binding was saturable, reaching a plateau at 2.5 nM. Binding parameters of nitrocellulose-immobilized receptor were not significantly different from the values calculated using intact cells. No appreciable binding of 125I-rEp to control membranes at low Ep-receptor content was observed. Among a panel of growth factors, only unlabeled rEp was able to compete for the binding of 125I-rEp to nitrocellulose-immobilized membrane proteins in a dose-dependent fashion. The technique described herein may be of use in the study of the Ep receptor and as an assay for its purification. Moreover, it may also be of general application in the study of receptor-ligand interactions.