High Sensitivity Hepcidin-25 Bioactive Elisas: Manual and Fully Automated System for the Quantification of Hepcidin-25 in Human Serum and Plasma

Since its discovery as the master regulator of iron homeostasis, hepcidin has been an exciting area of research with far reaching clinical and therapeutic applications. The bioactive peptide Hepcidin-25 is generated predominantly in the liver by proteolytic cleavage of the C-terminal 25 amino acids of prohepcidin. Subsequent N-terminal processing of Hepcidin-25 results in smaller peptides of 20-24 amino acids that show greatly reduced activity and accumulate in the urine. Hepcidin regulates iron levels by down regulating ferroportin, the major cellular iron exporter in the membrane of macrophages, hepatocytes, and the basolateral site of enterocytes. Hepcidin-25 induces the internalization and degradation of ferroportin, resulting in increased intracellular iron stores, decreased dietary iron absorption, and decreased circulating iron concentrations. Hepatocellular hepcidin synthesis decreases under conditions of increased demand for circulating iron like iron deficiency, hypoxia, anemia, and erythropoiesis. In contrast, hepcidin synthesis is induced by inflammation and infection. Measuring serum levels of Hepcidin-25 has proven to be valuable in identifying and differentiating specific disease conditions. Hepcidin deficiency causes hereditary hemochromatosis, characterized by body iron overload that may progress to liver cirrhosis. In addition, low Hepcidin-25 concentration can be induced by iron loading anemias and chronic hepatitis C. In contrast, high Hepcidin-25 levels have been found in iron-refractory iron-deficiency anemia, during infection, chronic kidney disease. There have reported links of hepcidin dysregulation in cancer and other disease states. This exciting area of biology has many potential important applications.

To further advance the field of hepcidin research and clinical application two new assays have been developed, the manual high sensitive DRG Hepcidin-25 assay and the fully automated Hepcidin 25 (bioactive) which is available on the DRG Hybrid XL. Both assays are colorimetric solid phase enzyme-linked immunosorbent assays (ELISA) based on the competitive binding of hepcidin in the sample and biotinylated hepcidin to immobilized anti-hepcidin antibody, followed by the detection with a Streptavidin-HRP conjugate.

The DRG Hepcidin-25 assay allows the quantitative determination of Hepcidin-25 covering a measuring range form 0.15-81.0 ng/mL and the total assay time for the manual assay is 1.5 hours. Serum and plasma (EDTA, heparin, Citrate) can be used for both the manual and automated assays. The analytical sensitivity of the assay is 0.153 ng/ml. The test shows good reproducibility with an intra-assay precision of 6.97% and an inter-assay precision of 12.0%. The recovery was determined to be 97.3%. Linear dilution gave an excellent overall recovery of 97.9% (mean of 3 samples, each diluted 4-fold with dilution buffer; range from 89.1-105.2%). We found no matrix interference with haemoglobin and triglycerides (up to 7.5 mg/mL). Inter-Lot precision was 7.69% (mean of 3 samples measured with 3 lots in 6 determinations). The assay also shows a good correlation to mass spectrometry (y=1.013x +1.3865, r=0.9678). Benefits of the new assay are a very straight forward procedure with ready-to-use reagents, no shaking, high sensitivity, and a short total assay time of 1.5 hours.

In addition to the highly acclaimed manual assay a new fully automated Hepcidin assay has been developed for the DRG Hybrid XL. The fully automated Hepcidin 25 assay time is 2 hours. The dynamic range is 1.67 ng/mL - 81 ng/ml. Intra assay precision is 2.6% and inter assay precision is 13.7%. Recovery averages 100.6% and linearity 100.1%. Both assays are excellent tools for detecting bioactive Hepcdin-25 in human samples and advancing this exciting field of research.