The EPO ELISA Kit is intended to measure Erythropoietin (EPO) levels in human serum. This assay is intended for research use, as an aid in the diagnosis of anemias and polycythemias. With the advent of the administration of recombinant erythropoietin as a biologic therapy to increase red blood cell mass, an erythropoietin assay may be used also to aid in the prediction and monitoring of response to recombinant erythropoietin treatment in persons with anemias.
Erythropoietin (EPO) is a heavily glycosylated protein with a molecular weight of about 30,000 - 34,000 Daltons. Human EPO is a polypeptide consisting of 165 amino acids, containing one O-linked and three N-linked carbohydrate chains1. The recombinant EPO is a good substitute for the native protein for use in an immunoassay2. Serum EPO levels are dependent on the rate of production and the rate of clearance of the protein. Ninety percent of EPO is produced in the peritubular cells of the adult kidney in response to a decrease in tissue oxygenation3,4. There is evidence indicating that the protein on these cells which detects oxygen saturation of the blood is a heme-containing moiety5. As the pO2 of the plasma, a function of the hematocrit decreases, EPO concentration will increase6. There are also observations suggesting that normally there is an inverse correlation between serum EPO levels and red blood cell mass7.
Quantitation of serum erythropoietin concentration serves as a diagnostic adjunct in determining the cause of anemia or erythrocytosis. Aplastic anemia, hemolytic anemia and anemia due to iron deficiency all result in serum EPO elevation. Whereas, EPO levels in patients with secondary anemia due to renal failure and other disorders such as acquired immune deficiency syndrome (AIDS) are generally inappropriately low for the degree of anemia. This is mostly likely caused by an impaired ability of the diseased kidney to produce adequate quantities of EPO8. Low concentrations of EPO may give an early warning of kidney transplant rejection10. EPO also can be used to monitor AIDS patients undergoing Zidovudine (AZT) therapy. An increased concentration of EPO verifies that anemia associated with AZT therapy is due to red cell hypoplasia or apliasia10.
Polycythemia rubra vera, or primary erythrocytosis (an increase of red blood cell mass) results from unstimulated over production of erythrocytes. Hence, the increase in the hemoglobin causes decreased production of EPO, which results in subnormal levels of serum EPO9. Secondary polycythemias, which are also characterized by an increase in the total red blood cell mass, occur as a physiological response to elevated levels of circulatory EPO caused by tissue hypoxia. The hypoxia may be due to such factors as pulmonary fibrosis, cardiovascular disease, prolonged exposure to high altitude, abnormal forms of hemoglobin or drug treatment10. Some tumors produce EPO and, in these cases, EPO may be used as a tumor marker to monitor the effectiveness of treatment.
Sample Type: serum
Sample Size: 200 uL
Standard Curve Range: 7.5 - 500 mU/mL
Sensitivity: 1.2 mU/mL
Assay Length: 2.5 hrs
EPO ELISA Insert (PDF)
|References/Citations:||How the Erythropoietin (EPO) ELISA Kit was used:|
|AAV9-mediated erythropoietin gene delivery into the brain protects nigral dopaminergic neurons in a rat model of Parkinson's disease.|
Xue YQ, et al. Gene Therapy. 2010; 17:83-94.
|Measure the concentration of EPO in cell lysates prepared from striatal tissue of adult female Sprague-Dawley rats. Cell lysates were prepared using a sonicator and a protein extraction reagent. |
|Failure of erythropoietin to render jeopardized ischemic myocardium amenable to incremental salvage by early reperfusion.|
French CJ, et al. Coron Artery Dis. 2009 Jun; 20(4):295-9.
|Measure the concentration of plasma EPO in C57BL/6 male mice (10 weeks of age). Plasma was prepared from whole blood collected with sodium citrate used as a anti-coagulant and centrifuged for 5 minutes at 16000 g.|
|Erythropoietin to augment myocardial salvage induced by coronary thrombolysis in patients with ST segment elevation acute myocardial infarction.|
Binbrek AS, et al. Am J. Cardiol. 2009 Oct; 104(8):1035-40.
|Measure the concentration of EPO in plasma obtained from male patients (humans). |
|Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma.|
Singh AK, et al. Proteomics: Clinical Appl. 2007 Jun; 1(7):626-639
|Measure the concentration of EPO in plasma obtained from horses. Plasma was prepared from heparinized blood samples.|
How It Works
The EPO Immunoassay is a two-site ELISA (Enzyme-Linked Immunosorbent Assay) for the measurement of the biologically active 165 amino acid chain of EPO. It utilizes two different mouse monoclonal antibodies to human EPO specific for well-defined regions on the EPO molecule. One mouse monoclonal antibody to human EPO, is biotinylated and the other mouse monoclonal antibody to human EPO is labeled with horseradish peroxidase (HRP) for detection.
In this assay, calibrators, controls, or patient samples are simultaneously incubated with the enzyme labeled antibody and a biotin coupled antibody in a streptavidin-coated microplate well. At the end of the assay incubation, the microwell is washed to remove unbound components and the enzyme bound to the solid phase is incubated with the substrate, tetramethylbenzidine (TMB). An acidic stopping solution is then added to stop the reaction and converts the color to yellow. The intensity of the yellow color is directly proportional to the concentration of EPO in the sample. A dose response curve of absorbance unit vs. concentration is generated using results obtained from the calibrators. Concentrations of EPO present in the controls and patient samples are determined directly from this curve. The standards have been calibrated against the World Health Organization (WHO) erythropoietin international standard that consists of recombinant DNA derived EPO. The WHO reference standard used was erythropoietin 1st international standard (87/684).