Molecular and electrical heterogeneity of circulating human erythropoietinmeasured by sensitive enzyme immunoassay

Background We set up a sensitive sandwich enzyme immunoassay (EIA) of human erythropoietin (EPO) and further investigated molecular weight and structu ral heterogeneity, and electrical heterogeneity of the circulating EPO in n ormal human plasma without any concentrating procedure. Materials and Methods We set up a sensitive sandwich EIA of EPO using anti- EPO rabbit Fab'-peroxidase conjugate and anti-EPO IgG coated polystyrene ba lls. Standard EPO and plasma samples obtained from three normal subjects we re applied to gel chromatographic analysis. Plasma samples obtained from th ree normal subjects were subjected to preparative isoelectric focusing with a column (pH 3.5-10). Results The minimal detectable quantity was 0.15 mIU mL(-1) using 100 mu L sample. There was a good parallelism between diluted human plasma samples a nd the standard EPO in the assay. Rat plasma EPO was cross-reacted in the a ssay. There was a good correlation between EPO levels determined by the EIA and those of radioimmunoassay (y = 0.897x + 0.564, r = 0.957). Gel chromat ographic analysis of standard EPO revealed a single peak. On the other hand , gel chromatographic analysis of unconcentrated plasma samples obtained fr om three normal subjects revealed at least four components of immunoreactiv e EPO. Preparative isoelectrical focusing revealed at least four major comp onents and some other small peaks in normal human plasma samples. Conclusion These findings indicate the first evidence for molecular and ele ctrical heterogeneity of circulating EPO in normal human plasma without any concentrating procedure.