Development of radioimmunoassays for human erythropoietin using recombinant erythropoietin as tracer and immunogen

Abstract

Since the estimation of erythropoietin (EPO) by radioimmunoassay (RIA) has been limited by the availability of highly purified urinary (U) human (Hu) EPO, we investigated the use of recombinant (R)-HuEPO as a replacement for U-HuEPO in the preparation of 125I-tracer and high affinity antisera. In each of two validated RIAs developed using U-HuEPO-derived reagents, dose-response lines and potency estimates for samples were compared when 125I-U-HuEPO and anti-U-HuEPO antisera were sequentially replaced to give assay variants using R-HuEPO-derived reagents. Two U-HuEPO preparations, the 2nd International Reference Preparation and CAT-1, and R-HuEPO were variously used as standards. The samples tested included clinically relevant human sera and partially purified preparations of U-HuEPO and R-HuEPO. In each RIA and for each assay variant tested, samples gave dose-response lines whose slopes did not differ significantly from that of the standard. For each of the two variant RIAs, potency estimates for any sample were consistent and, where examined, RIA potency estimates agreed with in vivo bioassay determinations. These results, obtained independently in two laboratories, indicate that RIAs having appropriate specificity and sensitivity for the estimation of EPO in clinical samples can be developed using reagents derived from R-HuEPO.