Mouse Erythropoietin/EPO Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse Epo Immunoassay is a 4.5 hour solid-phase ELISA designed to measure mouse Epo in cell culture supernates, tissue homogenates, serum, and plasma. It contains antibodies raised against recombinant Epo and has been shown to accurately quantitate the recombinant factor. Results obtained using natural mouse Epo showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that the Quantikine Mouse Epo kit can be used to determine relative mass values for naturally occurring mouse Epo.

Preparation and Storage

Background: Erythropoietin/EPO

Erythropoietin (Epo) is a 34-39 kDa secreted glycoprotein that is a member of the type I cytokine superfamily. The mouse Epo gene encodes a 192 amino acid (aa) residue precursor that contains a 26 aa signal peptide and a 166 aa mature protein containing three potential N-linked glycosylation sites (1-4). Mouse Epo lacks the O-linked glycosylation site found in human Epo. Although carbohydrate chains are not required for in vitro receptor binding, they are required for in vivo Epo bioactivity. Depending on the cell source, different Epo isoforms are produced that differ in their glycan compositions and sialic acid contents (5-8). Mature mouse and rat Epo share 94% aa sequence identity. They also share from 80%-82% aa identity with mature human, porcine, rhesus monkey and feline Epo (2, 3). Epo is primarily produced by cells in the kidney (interstitial peritubular renal fibroblasts) and liver (hepatocytes and Ito cells), where its production is up-regulated by hypoxia. Other tissues and cells, including neural tissues (astrocytes and neurons), testis (Sertoli cells), uterus, placenta, and erythroid progenitors, have also been shown to produce Epo (9-14). 

Epo is best known for its role in red blood cell formation. While Epo is not a lineage commitment factor, it inhibits apoptosis and induces burst forming unit-erythroid (BFU-E) differentiation into colony forming unit-erythroid (CFU-E), and the subsequent proliferation and maturation of CFU-E into early normoblasts (10, 15, 16). Apart from its role in erythropoiesis, Epo also acts on various non-hematopoietic cells to function as a viability and proliferation factor. Epo can stimulate myoblast proliferation while suppressing its differentiation, resulting in the expansion of the progenitor cell population (17). Epo is a tissueprotective factor that protects against ischemic and toxic injuries to neuronal, cardiovascular and renal tissues (18, 19). Epo has also been shown to promote angiogenesis in various physiologic and pathologic conditions (20, 21). 

Epo binds and signals via the high-affinity preformed homodimeric Epo receptor (Epo R) that is composed of two Epo R subunits. Each Epo R subunit is a type I transmembrane glycoprotein that belongs to the type I cytokine receptor superfamily (18, 22-24). Its extracellular domain contains the characteristic two fibronectin type III domains and a WSxWS motif near the plasma membrane (24, 25). Binding of Epo to the Epo R homodimer results in conformational change and phosphorylation and activation of the non-receptor protein kinase JAK2, which activates the downstream signaling cascade (26). An alternative Epo heteromeric receptor complex that transduces cell-protective signals and containing the beta common receptor ( beta CR) subunit in addition to the Epo R subunit has been described. beta CR also belongs to the type I cytokine receptor superfamily and is a subunit that is shared by the heteromeric IL-3, IL-5 and GM-CSF receptor complexes. Epo binds with lower affinity to the heteromeric receptor consisting of a Epo R subunit and a beta CR homodimer (18).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.