Current assays for quantitation of erythropoietin (epo) in human serum are based on monoclonal or polyclonal antibody recognition of the hormone in the form of RIAs or ELISAs. While these are clinically useful for patients with elevated Epo levels, they cannot distinguish biologically active and inactive epo and they are relatively insensitive and therefore barely allow detection of the hormone in normal blood samples. In-vivo biological assays are very expensive, time consuming, labor intensive and therefore are rarely used for clinical samples. This laboratory has recently isolated erythroid cell lines, HC cells, which exhibit an absolute and exclusive dependence on epo for survival and proliferation. Since they appear the most sensitive cells thus far studied, the epo-dependent cells should provide a useful bioassay for the hormone. In Phase I of this proposal, we selected, tested and stabilized the most sensitive HC clones; we compared four quantitative assays and determined that the MTT assay was most efficacious. Finally, we tested the inhibitory effects of human serum on Epo bioassays. We found that the sensitivity of HC cells sufficient to allow detection of normal, subnormal and elevated Epo levels. We propose in Phase II to use the most sensitive lines to design and test a kit which can be used to quantitate the biological activity of Epo in clinical specimens. We will compare the bioassay to the current RIAs and ELISAs with regard to the reproducibility, specificity, sensitivity, susceptibility to interfering substances, and applicability to a variety of individuals who are hematologically normal or abnormal.
