96-well microplate

Erythroferrone (ERFE) Human ELISA

KT1008
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Description

Erythroferrone (ERFE) is a secreted glycoprotein, also known as CTRP15 (C1q (complement component 1q) TNF (tumor necrosis factor)-related protein 15), Myonectin or Fam132b. Erythroferrone is a member of the CTRP family of proteins (C1q/TNF-related proteins), which are the conserved paralogs of adiponectin containing collagen-like and globular C1q-like domains (1). Erythroferrone plays two different regulatory roles:

  1. i) Erythroferrone (called Myonectin for this role) is a myokine abundantly expressed in skeletal muscle tissue, upregulated by voluntary exercise that is suppressed by fasting and induced by refeeding; it participates in the regulation of systemic lipid metabolism by promoting the clearance of nonesterifed fatty acids (NEFA) from circulation (2). As a myokine, Erythroferrone acts as an endurance exercise-driven myokine, which protects the heart from ischemic injury by reducing cardiomyocyte apoptosis and macrophage inflammatory response (3);
  2. ii) Erythroferrone is produced by erythroblasts after bleeding or EPO treatment (4) and is a potent inhibitor of hepcidin expression, resulting in increased release of iron from cellular iron stores. Erythroferrone acts on hepcidin by inhibiting members of the BMP5, BMP6 and BMP7 subgroup of BMPs (5). Erythroferrone functions as erythroid modulator of iron metabolism and hemoglobin synthesis and may also be an important link between altered iron metabolism, abnormal erythropoiesis, and bone loss in β-thalassemia. (6,7).

Levels of erythroferrone measured by this ELISA kit in serum and plasma correspond to measured mouse protein levels (~ 500ng/ml). Erythroferrone is a potential biomarker for different types of anemia, for cardiovascular and metabolic diseases.

Type

Sandwich type ELISA

Other names

Myonectin, CTRP15 (C1q (complement component 1q) TNF (tumor necrosis factor)-related protein 15), Fam132b

Principle of Method

This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human Erythroferrone in cell culture supernatants, serum and plasma. A polyclonal antibody specific for Erythroferrone has been precoated onto the 96-well microtiter plate. Standards (STD) and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, Erythroferrone is recognized by the addition of a biotinylated polyclonal antibody specific for Erythroferrone (DET). After removal of excess biotinylated antibody, streptavidin-peroxidase (STREP-HRP) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450nm after acidification and is directly proportional to the concentration of Erythroferrone in the samples.

Sample Types

Serum, Plasma or Cell Culture Supernatant

Special note about sample dilution

Erythroferrone/Myonectin has been shown to be expressed in muscle (called Myonectin/CTRP15) or in erythroblasts (called Erythroferrone/ERFE). Based on extensive in-house validation and in accordance with the values detected in undiluted samples by K. Ramirez Cuevas, et al.; Drug Test. Anal. (2019), we provide the following recommendations for the usage of the Erythroferrone (human) ELISA Kit.

  1. To detect ERFE increase upon Erythropoietin (EPO) treatment or blood transfusion, use undiluted or low diluted serum/plasma (about 1/10):

> Values measured will range from 2-4 ng/ml to 15-25 ng/ml.

  1. To detect steady state levels of ERFE, use diluted serum (>1/150).

> Values will range from 300 ng/ml to >1200 ng/ml

For a possible scientific explanation of these observations and further information related to this recommendation, please visit

https://ilexlife.com/pages/erythroferrone-elisa-hypothesis-about-dilution

Species crossreactivity

This ELISA is specific for the measurement of natural and recombinant human Erythroferrone [CTRP15; Myonectin].

Sample Volume

Serum and Plasma : 0.67 µL (actual amount of sample loaded in well after dilution)

Standard Curve

Standard curve with Erythroferrone (ERFE) Human ELISA

Calibration Range: 0.3125 ng/ml – 20 ng/ml ng/ml

Limit of Detection

270 pg/ml

Intra-assay (Within-Run) C.V.

3.45%

Inter-assay (Run-to-Run) C.V.

2.95%

Resources

References to Summary

  • Is erythroferrone finally the long sought-after systemic erythroid regulator of iron? A. Lawen; J. Biol. Chem. 6, 78 (2015)
  • Myonectin (CTRP15), a novel myokine that links skeletal muscle to systemic lipid homeostasis: M.M. Seldin, et al.; J. Biol. Chem. 287, 11968 (2012)
  • Myonectin Is an Exercise-Induced Myokine That Protects the Heart from Ischemia-Reperfusion Injury: N. Otaka, et al.; Res. 123, 1326 (2018)
  • Identification of Erythroferrone as an Erythroid Regulator of Iron Metabolism: L. Kautz, et al; Genet. 46, 678 (2014)
  • Erythroferrone inhibits the induction of hepcidin by BMP6: J. Arezes, et al.; Blood 132, 1473 (2018)
  • Erythropoietic regulators of iron metabolism: T. Ganz; Free Radic. Biol. Med. 133, 69 (2019)
  • The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry: M. Castro-Mollo et al; eLife 10, e68217 (2021)