A sensitive sandwich ELISA for measuring erythropoietin in human serum

A sandwich, non-competitive enzyme-linked immunosorbent assay (ELISA) for erythropoietin (EPO) is described. The ELISA utilizes a monospecific, polyclonal antibody raised in rabbits against human recombinant EPO (rhu EPO) and purified over a rhu EPO affinity chromatography column. The ELISA procedure can be summarized as follows: Anti-EPO is coated onto 96-well ELISA microtitre plates; standard EPO or sample is added and left to bind to this catching antibody; this is followed by the addition of the same antibody which has been biotinylated; finally, anti-biotin conjugated to alkaline phosphatase is added and the enzyme reaction developed and read at 405 nm. All parameters of the assay have been optimized. Recombinant human EPO was standardized against the World Health Organization 2nd International Reference Preparation for erythropoietin. The minimal detectable concentration of rhu EPO was 0.3-0.5 mU/ml, which corresponded to 1.2-2 mU/ml of EPO in serum (serum diluted 1:4). No reaction was obtained with a variety of blood components and cytokines, indicating that the anti-EPO antibody did not cross-react with those substances to produce false-positive results. The intra-assay variation ranged from 3% to 10%, while the inter-assay variation ranged from 8.5% to 24%. Serum dose-response curves were parallel to the standard dose-response curve. The assay is easy to use, rapid, reproducible, but above all quantitative, specific and sensitive to measure the EPO content in all serum samples.