Molecular and electrical heterogeneity of circulating human erythropoietin measured by sensitive enzyme immunoassay

We set up a sensitive sandwich enzyme immunoassay (EIA) of human erythropoietin (EPO) and further investigated molecular weight and structural heterogeneity, and electrical heterogeneity of the circulating EPO in normal human plasma without any concentrating procedure.

We set up a sensitive sandwich EIA of EPO using anti-EPO rabbit Fab'-peroxidase conjugate and anti-EPO IgG coated polystyrene balls. Standard EPO and plasma samples obtained from three normal subjects were applied to gel chromatographic analysis. Plasma samples obtained from three normal subjects were subjected to preparative isoelectric focusing with a column (pH 3.5-10).

The minimal detectable quantity was 0. 15 mIU mL-1 using 100 microL sample. There was a good parallelism between diluted human plasma samples and the standard EPO in the assay. Rat plasma EPO was cross-reacted in the assay. 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 chromatographic analysis of standard EPO revealed a single peak. On the other hand, gel chromatographic analysis of unconcentrated plasma samples obtained from three normal subjects revealed at least four components of immunoreactive EPO. Preparative isoelectrical focusing revealed at least four major components and some other small peaks in normal human plasma samples.

These findings indicate the first evidence for molecular and electrical heterogeneity of circulating EPO in normal human plasma without any concentrating procedure.