MAIIA EPO SeLect-a rapid screening kit for the detection of recombinant EPO analogues in doping control: inter-laboratory prevalidation and normative study of athlete urine and plasma samples.

Recombinant analogues of erythropoietin (EPO), epoetins, have been misused by athletes due to their performance enhancing effect since the first pharmaceutical epoetin was launched in 1987. The current methods for screening urine and plasma samples for the presence of epoetins, IEF and SAR-PAGE, have high sensitivity but are time-consuming to carry out. In an effort to ease and speed up the screening procedure for EPO, MAIIA Diagnostics has developed a combined affinity chromatography and lateral flow immunoassay, MAIIA EPO SeLect, which determines the percentage of migrated isoforms (PMI) of EPO in a sample. The reproducibility of the kit was tested by analyzing a set of negative and positive urine and plasma samples in three different laboratories. All data were analyzed with both curve fit parameters from the individual assay runs, and with lot-specific predefined curve calibration. To get a measure of endogenous variation, a normative study with athlete urine and plasma samples was conducted. The average intra-laboratory variation was 6.7% while the inter-laboratory variation for all samples was calculated to 8.8%. The athlete samples yielded an average PMI and standard deviation of 71.4 ± 7.7 for urine and 83.1 ± 10.2 for plasma, respectively. There were no signs of deviating results from tested effort urines. The results also support the use of predefined curve parameters.

Detection of recombinant EPO in blood and urine samples with EPO WGA MAIIA, IEF and SAR-PAGE after microdose injections.

The misuse of microdoses of performance enhancing drugs like erythropoietin (EPO) constitutes a major challenge in doping analysis. When injected intravenously, the half-life of recombinant human EPO (rhEPO) like epoetin alfa, beta, and zeta is only a few hours and hence, the window for direct detection of rhEPO in urine is small. In order to investigate the detection window for rhEPO directly in blood and urine with a combined affinity chromatography and lateral flow immunoassay (EPO WGA MAIIA), we recruited nine healthy people who each received six intravenously injected microdoses (7.5 IU/kg) of NeoRecormon (epoetin beta) over a period of three weeks. Blood and urine samples were collected in the days following the injections and analyzed with EPO WGA MAIIA as well as the current validated methods for rhEPO; isoelectric focusing (IEF) and sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE). For samples collected 18 h after a microdose, the sensitivity of the EPO WGA MAIIA assay was 100% in plasma and 87.5% in urine samples at the respective 98% specificity threshold levels. In comparison, the sensitivity in plasma and urine was 75% and 100%, respectively, with IEF, and 87.5% in plasma and 100% in urine when analyzed with SAR-PAGE. We conclude that EPO WGA MAIIA is a sensitive assay for the detection of rhEPO, with the potential of being a fast, supplemental screening assay for use in doping analysis.

Optic atrophy 1 is an A-kinase anchoring protein on lipid droplets that mediates adrenergic control of lipolysis.

Adrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphorylates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation-dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual-specificity A-kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolocalization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perilipin. Furthermore, by a combination of siRNA-mediated knockdown, reconstitution experiments using full-length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphorylation and lipolysis.