Clinical Hemopoietin Assay By Receptor-ligand Binding

Current assays from clinical quanititation of cytokines such as the interleukins, colony stimulating factors and erythropoietin (Epo) are based on monocional or polycional antibody recognition of the hormone on the form RIA's or ELISAs. This approach requires much time and effort to develop purified, high affinity antibodies, yet these assays are relatively insensitive. Additionally, these assays cannot distinguish biologically inactive molecules from active ones. The recent cloning of many growth factor receptors in our lab and others permits a new approach to cytokine assays. Using the hormone-binding exoplasmic domains of cytokine receptors instead of antibodies in traditional assays would allow for identification and quantitation of biologically-active receptor binding molecules. In addition to the greater specificity of receptor-based assays, this approach is easily generalizable to all receptor ligands. In Phase I, we will test the feasibility of preparing a soluble receptor-based assay for erythropoietin. Our preliminary data indicate that recombinant murine soluble Epo receptor (solEpoR) binds Epo as demonstrated by growth inhibition of an Epo dependent cell line, HCD-57. In this Phase, we will construct, express and purify human EpoR, characterize its binding to Epo, and initiate development of a receptor-based Epo assay.