Purification of erythropoietin from human plasma samples using an immunoaffinity well plate.

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1-2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control.

New screening protocol for recombinant human erythropoietins based on differential elution after immunoaffinity purification.

A screening method able to differentiate recombinant human erythropoietins (rhEPOs) and analogues like CERA from human urinary erythropoietin (uhEPO) is described. The method is based on the discrimination between isoforms observed when the protein is eluted under acidic followed by basic conditions from immunoaffinity microtiter wells. From a comparison with the complex IEF protocol currently applied in anti-doping analysis, the newly developed assay procedure is amenable to wide screening application and presents good resolving power between rhEPOs and uhEPO.

Convergence and stability assessment of Newton-Kantorovich reconstruction algorithms for microwave tomography.

For newly developed iterative Newton-Kantorovitch reconstruction techniques, the quality of the final image depends on both experimental and model noise. Experimental noise is inherent to any experimental acquisition scheme, while model noise refers to the accuracy of the numerical model, used in the reconstruction process, to reproduce the experimental setup. This paper provides a systematic assessment of the major sources of experimental and model noise on the quality of the final image. This assessment is conducted from experimental data obtained with a microwave circular scanner operating at 2.33 GHz. Targets to be imaged include realistic biological structures, such as a human forearm, as well as calibrated samples for the sake of accuracy evaluation. The results provide a quantitative estimation of the effect of experimental factors, such as temperature of the immersion medium, frequency, signal-to-noise ratio, and various numerical parameters.