Correspondence of 25‐hepcidin concentrations by ELISA and mass spectrometric methods and their discrimination across healthy and ill subject groups

Background Increasing use of hepcidin as a biomarker of iron homeostasis, especially during infection and inflammation and other iron‐related disorders, calls for affordable and convenient assessment methods capable of precisely quantifying a wide range of hepcidin concentrations. Objective To evaluate two different versions of a commercial competitive enzyme‐linked immunosorbent assay (ELISA) kit for bioactive hepcidin‐25 quantitation, with similar dynamic ranges but different specificities, within a large range of hepcidin concentrations. Methods Plasma samples from 8 apparently healthy adults recruited in Sololá, Guatemala; 8 mildly sick patients attending clinical consultation at Sololá's National Hospital, and 8 end‐stage renal failure juvenile patients undergoing hemodialysis in FUNDANIER, Guatemala City, were collected to represent the widest possible range of continuous hepcidin concentrations in a human context: low (LH), middle (MH) and high (HH) levels. Hepcidin‐25 was quantified with two ELISA kits (DRG® EIA‐5258 & EIA‐5782) and the gold standard weak cation exchange time‐of‐flight mass‐spectrometry (TOFMS) method. Pearson product‐moment correlation coefficient test was applied to the 3 data‐sets to assess statistical dependence between the 3 methods to determine validity and repeatability. Results Average hepcidin concentrations (ng/ml) for LH subjects were: 9±5, 10±2, and 155±67; for MH patients: 29±12, 20±11, and 437±283; and for HH patients: 145±130, 212±295, and 3380±3486, with the TOFMS, EIA‐5258, and EIA‐5782 methods, respectively. Pearson rank correlation coefficients between TOFMS & EIA‐5258 and TOFMS & EIA‐5782 (validity) were 0.94 and 0.123, respectively, and between EIA‐5258 & ‐5782 (repeatability), 0.061. Conclusion Despite the declared improved specificity of the newest version of the kit (EIA‐5782), the older version (EIA‐5258) revealed a higher validity when compared to the gold standard, making it a better option for the assessment of a wide range of hepcidin concentrations.