Quantikine ivd human erythropoietin elisa

Plasma samples were analyzed using a Quantikine IVD™ human erythropoietin ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol. This ELISA cross-reacts with endogenous erythropoietin, which makes it impossible to distinguish darbepoetin alfa from endogenous erythropoietin. Standard curves included 2.5 [lower limit of quantification (LLOQ)], 5.0, 20, 50, 100, and 200 mU/mL of erythropoietin. A weighted (1/x²) quadratic regression model was fitted to each standard curve. Each individual sample well was quantified by interpolation, after which, duplicates were averaged. Samples that were below the LLOQ were excluded from the analysis (eight concentrations, 14 %). If samples were evaluated on more than 1 day, all wells were averaged for the final sample quantitation. Duplicates in the standard curve that had an intraday coefficient of variation >10 % or samples that had intra- or inter-day coefficients of variation >10 % were excluded from the analysis. In addition, standards that did not meet the United States Food and Drug Administration guidelines (±20 % for LLOQ, ±15 % for all others) were not included in the construction of the standard curve.

In the PUUMA study, blood samples were collected in the morning between 7:00AM and 10:00AM, and frozen stored plasma at −80°C from the initial visit blood draw was utilized for EPO measurement. EPO was measured by a commercially available ELISA kit, using a mouse monoclonal antibody specific for EPO and rabbit anti-EPO polyclonal antibody conjugated to horseradish peroxidase based on the double-antibody sandwich method (Quantikine® IVD® Human Erythropoietin ELISA R&D Systems Inc. Catalog Number DEP00). In each assay plate, Quantikine® IVD® Erythropoietin Human Serum Controls with known EPO concentrations were used as quantitative controls for the determination of EPO concentrations (Human Erythropoietin Quantikine® IVD® Control 1&2; R&D Systems Inc. Catalog Number CEP01/ CEP03 Control 3). The sensitivity of the Quantikine IVD Human Epo ELISA (minimum detectable dose) is typically less than 0.6 mIU/mL.
In the ARIC study, EPO levels were measured from plasma collected at visit 5 (2011–13) using an aptamer-based platform (SomaScan version 4)^{23 (link), 24 (link)}. Plasma samples were collected using standardized protocol and stored at −80°C. The EPO levels were quantified in relative fluorescence units (RFU).