CLINICAL HEMOPOIETIN ASSAY BY RECEPTOR LIGAND BINDING

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DESCRIPTION: Current assays for clinical quantitation of cytokines such as the interleukins, colony stimulating factors, and erythropoietin (Epo) are based on monoclonal or polyclonal antibody recognition of the hormone in the form of RIAs or ELISAs. This approach requires much time and effort to develop purified, high affinity antibodies, yet these assays are relatively insensitive, and barely allow detection of the hormone in normal blood samples. They are useful only for patients with elevated Epo levels. Additionally, these assays can not 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 ligand-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, of this proposal, the Applicants prepared the murine Epo receptor and initiated an Epo binding assay. The results indicate that it is feasible to develop the assay for biologically active Epo based on its interaction with EpoR. In Phase II, they will generate recombinant human EpoR, establish its Epo binding properties and use it to develop clinical Epo assay. These studies will, in turn, establish methods which will make economically feasible to express and purify other cytokine receptors and develop clinical assays for biologically active interleukins and colony stimulating molecules.