The EPO Immunoassay is a twosite ELISA [EnzymeLinked ImmunoSorbent Assay] for the measurement of the biologically active 165 amino acid chain of EPO. A sheep polyclonal antibody to human EPO, purified by affinity chromatography, is biotinylated. A mouse monoclonal antibody to human EPO is labeled with horseradish peroxidase [HRP] for detection. In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidincoated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of EPO in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of EPO present in the controls and patient samples are determined directly from this curve. The standards have been calibrated against the World Health Organization (WHO) erythropoietin international standard which consists of recombinant DNA derived EPO. The WHO reference standard used was erythropoietin 1st international standard (87/684).