Abstract
Oligomerization of cytokine receptors including the erythropoietin (EPO) receptor has been advanced as a model for activation. If homodimerization of the EPO receptor activates it, then bivalent antibodies raised to the extracellular domain of the EPO receptor should also homodimerize and activate. Mouse monoclonal antibodies (IgG) raised to the soluble, extracellular domain of the human EPO receptor (EPOR) were found that would stimulate thymidine uptake of an human EPO-dependent cell line, UT-7/EPO. Dose response curves showed bell shapes where activity was low at low and high concentrations. Monovalent (Fab) fragments bound to the receptor but did not stimulate thymidine uptake, which indicates that two antibody binding sites are required for activation. The anti-EPOR antibodies stimulated the formation of burst forming unit erythroid colonies from human CD34(+) cells purified from peripheral blood. This indicates that homodimerization of the EPO receptor by anti-EPOR antibodies is sufficient for both proliferation and differentiation of erythroid progenitor cells and that the constraints on dimerization necessary for activation are rather loose.
Highlights
Erythropoietin (EPO)1 is a glycoprotein hormone that is the primary regulator of erythropoiesis
Isolation and Characterization of Anti-EPO Receptor Monoclonal Antibodies—Mice were immunized with a purified preparation of the 225-amino acid soluble extracellular domain of the human EPO receptor produced in CHO cells
EPO Receptor Binding with Anti-EPOR Antibodies—The hybridomas were tested to identify those antibodies that were capable of binding to EPO receptor expressed on the surface of cells
Summary
Immunization and Hybridoma Preparation—Balb/c mice (Charles Rivers Laboratories, Wilmington, MA) were immunized with subcutaneously injected soluble EPO receptor. Cells were washed with PBS/BSA and resuspended in a solution of fluorescine isothiocyanate labeled goat anti-mouse monoclonal antibody (Southern Biotech, Birmingham, AL). Cells were washed twice with PBS/BSA and incubated a final time at 4 °C with phycoerythrin-labeled goat anti-mouse monoclonal antibody (Southern Biotech, Birmingham, AL). Cells were collected by centrifugation, washed twice with PBS, and resuspended at 5 ϫ 104 cells/ml in assay medium (1 ϫ RPMI medium 1640 without L-glutamine (Life Technologies, Inc.)/1% L-glutamine/4% fetal bovine serum). The low density cells were collected from the gradient, washed with Hanks’ balanced salt solution, and resuspended in PBS supplemented with 0.5% bovine serum albumin and 5 mM EDTA at a concentration of 5 ϫ 108 cells/ml. The tubes containing cell pellets were quick frozen in a dry ice-ethanol bath, and the cell pellet was clipped and counted in a LKB 1277 gammamaster automatic ␥ counter
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