Using Recombinant Human Erythropoietin Receptor (EPOR) in research applications enables precise investigation of erythropoietin (EPO) signaling, receptor-ligand interactions, and downstream biological effects in both physiological and pathological contexts.

Key reasons to use recombinant human EPOR include:

• Mechanistic Studies: Recombinant EPOR allows for controlled in vitro assays to dissect the molecular mechanisms of EPO-EPOR binding, receptor activation, and subsequent intracellular signaling pathways. This is essential for understanding erythropoiesis and non-hematopoietic EPO functions.

• Bioassays and ELISA Standards: Recombinant EPOR is commonly used as a standard or capture reagent in ELISA and other immunoassays to quantify EPO or anti-EPOR antibodies in biological samples, supporting both basic research and clinical diagnostics.

• Drug Screening and Development: The receptor is valuable for screening EPO analogs, agonists, or antagonists, enabling the identification and characterization of novel therapeutics targeting the EPO-EPOR axis.

• Pathophysiological Research: EPOR is implicated in diverse tissues beyond erythroid progenitors, including neural, cardiac, and tumor cells. Recombinant EPOR facilitates studies on EPO’s tissue-protective, neuroprotective, and potential pro- or anti-tumorigenic effects.

• Autoimmunity and Disease Biomarkers: Recombinant EPOR is used to detect autoantibodies against EPOR, which have clinical significance in diseases such as chronic kidney disease and diabetes.

• Cellular and Animal Models: Adding recombinant EPOR to cell cultures or animal models helps clarify receptor-specific effects, separate from endogenous receptor expression, and can be used to validate antibody specificity or receptor-blocking strategies.

In summary, recombinant human EPOR is a versatile tool for elucidating EPO biology, developing diagnostics and therapeutics, and exploring the receptor’s roles in health and disease. Its use ensures reproducibility, specificity, and scalability in experimental systems.

Yes, recombinant human erythropoietin receptor (EPO R) can be used as a standard for quantification and calibration in ELISA assays, though with important considerations regarding your specific application.

Appropriate Applications

Recombinant EPO R is well-suited for use as a standard in sandwich ELISA assays designed to quantify EPO R itself in biological samples. These assays measure the amount of target protein bound between matched antibody pairs, and recombinant standards provide reliable calibration curves for this purpose. The assay range typically spans from approximately 187.5 to 12,000 pg/mL, with sensitivity around 180 pg/mL.

Formulation Considerations

When selecting recombinant EPO R for use as a standard, you should consider the formulation type:

With Carrier Protein (BSA): This formulation is generally recommended for ELISA applications. The addition of bovine serum albumin enhances protein stability, increases shelf-life, and allows storage at more dilute concentrations. The protein is lyophilized from a 0.2 μm filtered solution in PBS with trehalose and BSA as a carrier protein.

Carrier Free: This formulation is recommended only when the presence of BSA could interfere with your specific assay or downstream applications. It should be reconstituted in sterile PBS without additional carrier proteins.

Reconstitution and Storage

Reconstitute the recombinant standard at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin (for the BSA-containing formulation). Store using a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain protein integrity and prevent loss of activity.

Important Limitation

One critical consideration: some EPO R ELISA kits are specifically designed to detect native EPO R rather than recombinant forms. Before using recombinant EPO R as your standard, verify that your ELISA kit's antibodies recognize recombinant EPO R equally well as native protein, or select kits explicitly validated for both natural and recombinant EPO R detection.

Applications of Recombinant Human Erythropoietin in Published Research

Recombinant human erythropoietin (rHuEPO) has been validated for diverse applications across both hematopoietic and non-hematopoietic contexts in published research.

Hematopoietic Applications

Anemia Treatment

The primary validated application involves treating various forms of anemia. rHuEPO is used globally for anemia caused by chronic kidney disease and chronic renal failure. The protein functions by stimulating erythroid progenitor cell survival and delaying apoptosis of hematopoietic cells, thereby increasing red blood cell production and hemoglobin concentration.

FDA-approved indications include anemia induced by cancer chemotherapy in nonmyeloid malignancies, anemia resulting from zidovudine (AZT) therapy in HIV-infected patients, and perioperative blood management in elective orthopedic surgery. rHuEPO has also been applied to accelerate erythropoiesis following chemotherapy, post-transplantation, and in surgical settings.

Non-Hematopoietic Applications

Cytoprotective and Organ-Protective Effects

Beyond hematopoietic functions, rHuEPO demonstrates validated cytoprotective properties. The protein exerts anti-apoptotic, anti-inflammatory, and antioxidant effects in non-erythroid tissues including the brain, heart, small bowel, kidney, and pancreatic islets. rHuEPO enhances activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase while reducing inflammatory factor release.

Neuroprotection and Neurological Applications

rHuEPO has been validated as a neuroprotective and neuroregenerative agent with demonstrated efficacy in ischemic stroke and chronic schizophrenia in double-blind placebo-controlled clinical studies. The protein shows therapeutic potential for neurological conditions including head trauma and epilepsy. Additional applications under investigation include treatment of multiple sclerosis, retinal diseases, and various neuropsychiatric disorders.

Surgical and Cardiovascular Applications

Clinical research demonstrates widespread use of rHuEPO in orthopedics, neurosurgery, cardiovascular disease, hepatobiliary surgery, and general surgery.

Immunogenicity Reduction Research

Recent computational protein engineering approaches have validated engineered rHuEPO variants with reduced immunogenicity while maintaining bioactivity. Five engineered mutants demonstrated decreased T cell responses in HLA-DRB1*09-positive volunteers while retaining similar or slightly reduced bioactivity compared to native rHuEPO.

Laboratory and Analytical Applications

rHuEPO has been validated for use as a bioactivity control and Western blot control in research applications.

To reconstitute and prepare Recombinant Human Erythropoietin Receptor (EPOR) protein for cell culture experiments, briefly centrifuge the vial to collect the lyophilized powder, then reconstitute in sterile deionized water or PBS to a concentration of 0.1–1.0 mg/mL. Avoid vigorous mixing; gently swirl or tap to dissolve the protein.

Step-by-step protocol:

• Centrifuge the vial briefly before opening to ensure all powder is at the bottom.
• Add sterile deionized water or PBS to achieve a final concentration of 0.1–1.0 mg/mL.
• If recommended by the manufacturer or for enhanced stability, add carrier protein (e.g., 0.1% BSA or recombinant human serum albumin) to the buffer. This helps prevent adsorption and loss of protein activity, especially at low concentrations.
• Gently mix by swirling or tapping; do not vortex or shake vigorously to avoid protein denaturation.
• Allow the protein to dissolve at room temperature for 15–30 minutes.
• Aliquot the solution into working volumes to avoid repeated freeze-thaw cycles.
• Store aliquots at ≤–20°C for long-term use, or at 4°C for short-term use (2–7 days).

Additional notes for cell culture experiments:

• For further dilution, use cell culture medium or PBS containing carrier protein (e.g., 0.1% BSA) to maintain protein stability.
• If the protein is to be used in functional assays, confirm the absence of endotoxin and verify activity in a pilot experiment.
• Avoid reconstituting at concentrations above 1 mg/mL unless specifically recommended, as this may affect solubility and activity.
• If recommended, add glycerol (5–50%) for additional stability during storage.

Summary Table: Reconstitution Conditions

This protocol ensures optimal solubility, stability, and activity of recombinant EPOR protein for cell culture applications. Always consult the specific product datasheet for any additional manufacturer recommendations.