[Development of enzyme linked immunosorbent assay for erythropoietin.].

BACKGROUND

The aim of our study was to optimize and establish erythropoietin (EPO) enzyme linked immunosorbent assay (ELISA) system.

METHODS

We prepared several monoclonal and polyclonal antibodies specific to human-EPO. The best combinations of antibodies for coating and detecting antibodies were selected for the establishment of ELISA. We tested several methods such as a competitive EIA and a sandwich ELISA.

RESULTS

The best sandwich ELISA was optimized compared to competitive EIA when purified polyclonal antibody (PoAb) was used as a coating antibody and biotinylated PoAb as a detecting antibody. This sandwich ELISA easily detected EPO when PoAb pairs were used compared to the ELISA using monoclonal antibody and PoAb. There were no significant differences between the effects of various blocking solutions on the performance of sandwich ELISA using biotinylated antibody. The ELISA system using PBST containing 3% BSA as a blocking solution can sensitively detect EPO (10 mU/mL) in a broad range of EPO concentrations (10-2,000 mU/mL) and there were cross-reactions with other cytokines).

CONCLUSIONS

EPO can be easily determined by using biotinylated PoAb as a detecting antibody and another PoAb as a coating antibody.