{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,13]],"date-time":"2026-06-13T12:23:27Z","timestamp":1781353407304,"version":"3.54.1"},"reference-count":53,"publisher":"Public Library of Science (PLoS)","issue":"7","license":[{"start":{"date-parts":[[2021,7,20]],"date-time":"2021-07-20T00:00:00Z","timestamp":1626739200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"E.C. Noyons foundation"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>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 \u03b2-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\u2019s 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 \u03b2-thalassemia major patients from healthy controls (p = 0.03) and from each other (p&lt;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 R<jats:sup>2<\/jats:sup> = 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.<\/jats:p>","DOI":"10.1371\/journal.pone.0254851","type":"journal-article","created":{"date-parts":[[2021,7,20]],"date-time":"2021-07-20T17:54:35Z","timestamp":1626803675000},"page":"e0254851","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Differentiating iron-loading anemias using a newly developed and analytically validated ELISA for human serum erythroferrone"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5351-8696","authenticated-orcid":true,"given":"Laura","family":"Diepeveen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rian","family":"Roelofs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolai","family":"Grebenchtchikov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel","family":"van 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current therapeutic options","volume":"11","author":"G Abu-Zeinah","year":"2020","journal-title":"Journal of blood medicine"},{"key":"pone.0254851.ref016","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.freeradbiomed.2018.07.003","article-title":"Erythropoietic regulators of iron metabolism","volume":"133","author":"T Ganz","year":"2019","journal-title":"Free radical biology & medicine"},{"key":"pone.0254851.ref017","doi-asserted-by":"crossref","first-page":"523","DOI":"10.3390\/jcm8040523","article-title":"Serum erythroferrone levels associate with mortality and cardiovascular events in hemodialysis and in ckd patients: A two cohorts study","volume":"8","author":"B Spoto","year":"2019","journal-title":"Journal of clinical medicine"},{"key":"pone.0254851.ref018","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1002\/ajh.25613","article-title":"The bmp-smad pathway mediates the impaired hepatic iron metabolism associated with the erfe-a260s 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hepcidin","volume":"146","author":"N Grebenchtchikov","year":"2009","journal-title":"Br J Haematol"},{"key":"pone.0254851.ref026","doi-asserted-by":"crossref","first-page":"1570","DOI":"10.1373\/clinchem.2010.149187","article-title":"Immunochemical and mass-spectrometry-based serum hepcidin assays for iron metabolism disorders","volume":"56","author":"JJ Kroot","year":"2010","journal-title":"Clin Chem"},{"key":"pone.0254851.ref027","doi-asserted-by":"crossref","first-page":"e75518","DOI":"10.1371\/journal.pone.0075518","article-title":"Improved mass spectrometry assay for plasma hepcidin: Detection and characterization of a novel hepcidin isoform","volume":"8","author":"CMM Laarakkers","year":"2013","journal-title":"PLOS ONE"},{"key":"pone.0254851.ref028","article-title":"Toxic iron species in lower-risk myelodysplastic syndrome patients: Course of disease and effects on outcome","author":"M Hoeks","year":"2020","journal-title":"Leukemia"},{"key":"pone.0254851.ref029","doi-asserted-by":"crossref","first-page":"69","DOI":"10.3324\/haematol.2017.171884","article-title":"Labile plasma iron levels predict survival in patients with lower-risk myelodysplastic syndromes","volume":"103","author":"L de Swart","year":"2018","journal-title":"Haematologica"},{"key":"pone.0254851.ref030","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1373\/clinchem.2016.256768","article-title":"Toward worldwide hepcidin assay harmonization: Identification of a commutable secondary reference material","volume":"62","author":"LN van der Vorm","year":"2016","journal-title":"Clin Chem"},{"key":"pone.0254851.ref031","doi-asserted-by":"crossref","first-page":"864","DOI":"10.1515\/cclm-2018-0783","article-title":"Provisional standardization of hepcidin assays: Creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference 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nijmegen","volume":"5","author":"P Manders","year":"2018","journal-title":"Open Journal of Bioresources"},{"issue":"Suppl 1","key":"pone.0254851.ref035","first-page":"S49","article-title":"Limit of blank, limit of detection and limit of quantitation","volume":"29","author":"DA Armbruster","year":"2008","journal-title":"The Clinical biochemist Reviews"},{"key":"pone.0254851.ref036","doi-asserted-by":"crossref","first-page":"185","DOI":"10.4155\/bio.13.292","article-title":"Parallelism: Considerations for the development, validation and implementation of pk and biomarker ligand-binding assays","volume":"6","author":"LF Stevenson","year":"2014","journal-title":"Bioanalysis"},{"key":"pone.0254851.ref037","doi-asserted-by":"crossref","DOI":"10.3389\/fneur.2015.00179","article-title":"A practical guide to immunoassay method validation","volume":"6","author":"U Andreasson","year":"2015","journal-title":"Frontiers in Neurology"},{"key":"pone.0254851.ref038","doi-asserted-by":"crossref","first-page":"869","DOI":"10.1016\/j.jpba.2011.03.033","article-title":"Validation of immunoassay for protein biomarkers: Bioanalytical study plan implementation to support pre-clinical and clinical studies","volume":"55","author":"MA Valentin","year":"2011","journal-title":"J Pharm Biomed Anal"},{"key":"pone.0254851.ref039","doi-asserted-by":"crossref","first-page":"1272","DOI":"10.1182\/blood.V128.22.1272.1272","article-title":"A novel dual monoclonal sandwich elisa for human erythroferrone","volume":"128","author":"H Han","year":"2016","journal-title":"Blood"},{"key":"pone.0254851.ref040","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.clinbiochem.2020.02.001","article-title":"Analytical and biological assessment of circulating human erythroferrone","volume":"79","author":"S Appleby","year":"2020","journal-title":"Clinical Biochemistry"},{"key":"pone.0254851.ref041","doi-asserted-by":"crossref","first-page":"1107","DOI":"10.4155\/bio-2017-0084","article-title":"Parallelism experiments to evaluate matrix effects, selectivity and sensitivity in ligand-binding assay method development: Pros and cons","volume":"9","author":"J Tu","year":"2017","journal-title":"Bioanalysis"},{"key":"pone.0254851.ref042","article-title":"Erythroferrone structure, function, and physiology: Iron homeostasis and beyond","author":"DN Srole","year":"2020","journal-title":"J Cell Physiol"},{"key":"pone.0254851.ref043","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1208\/s12248-015-9820-y","article-title":"Recommendations for use and fit-for-purpose validation of biomarker multiplex ligand binding assays in drug development","volume":"18","author":"D Jani","year":"2016","journal-title":"Aaps j"},{"key":"pone.0254851.ref044","doi-asserted-by":"crossref","first-page":"1941","DOI":"10.4155\/bio.13.174","article-title":"What is going on with my samples? A general approach to parallelism assessment and data interpretation for biomarker ligand-binding assays","volume":"5","author":"S Ciotti","year":"2013","journal-title":"Bioanalysis"},{"key":"pone.0254851.ref045","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1515\/CCLM.2004.010","article-title":"Matrix effects in clinical immunoassays and the effect of preheating and cooling analytical samples","volume":"42","author":"H Yoshida","year":"2004","journal-title":"Clin Chem Lab Med"},{"key":"pone.0254851.ref046","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1373\/clinchem.2017.274860","article-title":"Harmonization: Its time has come","volume":"63","author":"WG Miller","year":"2017","journal-title":"Clin Chem"},{"key":"pone.0254851.ref047","doi-asserted-by":"crossref","first-page":"e13890","DOI":"10.14814\/phy2.13890","article-title":"Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice","volume":"6","author":"R Coffey","year":"2018","journal-title":"Physiological Reports"},{"key":"pone.0254851.ref048","doi-asserted-by":"crossref","first-page":"e141","DOI":"10.3324\/haematol.2017.181743","article-title":"Levels of the erythropoietin-responsive hormone erythroferrone in mice and humans with chronic kidney disease","volume":"103","author":"MR Hanudel","year":"2018","journal-title":"Haematologica"},{"key":"pone.0254851.ref049","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1111\/bjh.16498","article-title":"Regulation of iron homeostasis through the erythroferrone-hepcidin axis in sickle cell disease","volume":"189","author":"AA Mangaonkar","year":"2020","journal-title":"Br J Haematol"},{"key":"pone.0254851.ref050","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1182\/blood.2019002620","article-title":"Erythroferrone lowers hepcidin by sequestering bmp2\/6 heterodimer from binding to the bmp type i receptor alk3","volume":"135","author":"CY Wang","year":"2020","journal-title":"Blood"},{"key":"pone.0254851.ref051","doi-asserted-by":"crossref","first-page":"1463","DOI":"10.1182\/blood-2018-08-869586","article-title":"A long sought after \u201creceptor\u201d for erfe?","volume":"132","author":"A-S Zhang","year":"2018","journal-title":"Blood"},{"key":"pone.0254851.ref052","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1002\/dta.2720","article-title":"Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis","volume":"12","author":"K Ramirez Cuevas","year":"2020","journal-title":"Drug Test Anal"},{"key":"pone.0254851.ref053","doi-asserted-by":"crossref","first-page":"384","DOI":"10.3324\/haematol.2019.233874","article-title":"Induction of erythroferrone in healthy humans by micro-dose recombinant erythropoietin or high-altitude exposure","volume":"106","author":"P Robach","year":"2021","journal-title":"Haematologica"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0254851","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,7,20]],"date-time":"2021-07-20T22:09:27Z","timestamp":1626818967000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0254851"}},"subtitle":[],"editor":[{"given":"Kostas","family":"Pantopoulos","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2021,7,20]]},"references-count":53,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7,20]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0254851","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,20]]}}}