History of assay achievements
A major research focus within the Department of Laboratory Medicine of the Radboud University Medical Center is on iron metabolism regulators, and the translation of new findings into diagnostic assays that can be clinically implemented. Our initial work focused on the measurement of iron-regulatory-protein hepcidin in human urine and serum and resulted in two publications in the Blood-journal. In one of these studies, an in vivo human endotoxemia model was used to analyse the effects of endotoxemia as an upstream inflammation activator of hepcidin. These studies underscored the important role of hepcidin as the mediator of anemia of inflammation. This prompted us to develop a novel mass spectrometry (MS)-based assay for quantification of hepcidin in urine and serum. In fact, in 2005 we were the first to publish the potential of this MS approach, which paved the way for hepcidin measurements in clinical studies .
Our recent improvements quantitative serum/plasma and urine hepcidin measurements opened novel opportunities for studies on hepcidin in humans resulting in further insights in the clinical implications of hepcidin-mediated regulation of iron metabolism. Hepcidin measurements are now routinely performed by weak cation exchange chromatography in combination with time-of-flight mass spectrometry (WCX-TOF MS) on a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics).
For high throughput plasma and serum hepcidin measurements, we have developed an in-house competitive ELISA . We moreover developed a competitive bioanalytical assay for plasma and serum hepcidin using the Anticalin protein Hep-Dx as a next-generation diagnostic antibody mimetic and biotin-labeled hepcidin-25 as a tracer (Enzyme Linked antibody-Mimetic Assay, ELMA). Both these method measures the total of hepcidin isoforms and are especially appropriate for large sample numbers.
To date, we have taken two initiatives (round robins) to harmonize hepcidin methods throughout the world.