Abstract
Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay's limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE.
Highlights
In 2014, the hormone erythroferrone (ERFE) was discovered as erythroid regulator of iron metabolism [1]
The validation of a new enzyme-linked immunosorbent assay (ELISA) for human serum erythroferrone this end, we aimed to develop a robust, analytically characterized and eventually standardized in-house ERFE ELISA, which will guarantee reproducible and consistent ERFE results
Catching and tagging antibodies were generated by immunizing two chickens and two rabbits, respectively, with 10 μg of recombinant Flag-tagged human ERFE immunogen per injection by Davids Biotechnology (Regensburg, Germany) and purified using affinity chromatography
Summary
In 2014, the hormone erythroferrone (ERFE) was discovered as erythroid regulator of iron metabolism [1]. Failing oxygen levels in tissues, for example during hemorrhage or hypoxia [2], result in renal synthesis of erythropoietin (EPO) [3], which, in turn, promotes expression of ERFE by erythroblasts via the JAK/STAT5 pathway [1, 4, 5]. By suppressing the key regulator of iron metabolism hepcidin, ERFE promotes duodenal iron absorption and iron mobilization from stores to meet the increased iron demand of developing erythrocytes [2, 4, 6]. The validation of a new ELISA for human serum erythroferrone
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