Human Erythropoietin/EPO Quantikine ELISA Kit, RUO Summary

Product Summary

The Quantikine® Human Epo ELISA uses a monoclonal antibody and polyclonal antibody conjugate in a sandwich ELISA format. The assay is designed to measure Epo levels in serum or EDTA plasma in less than 4.5 hours, or less than 2.5 hours using the shaker protocol.

Preparation and Storage

Background: Erythropoietin/EPO

Erythropoietin (Epo), a glycoprotein (~30,400 Daltons) produced primarily by the kidney, is the principal factor regulating red blood cell production (erythropoiesis) in mammals. Renal production of Epo is regulated by changes in oxygen availability. Under conditions of hypoxia, the level of Epo in the circulation increases and this leads to increased production of red blood cells. 

The over-expression of Epo may be associated with certain pathophysiological conditions (1, 2). Polycythemia exists when there is an overproduction of red blood cells (RBCs). Primary polycythemias, such as polycythemia vera, are caused by Epo-independent growth of erythrocytic progenitors from abnormal stem cells and low to normal levels of Epo are found in the serum of affected patients. On the other hand, various types of secondary polycythemias are associated with the production of higher than normal levels of Epo. The overproduction of Epo may be an adaptive response associated with conditions that produce tissue hypoxia, such as living at high altitude, chronic obstructive pulmonary disease, cyanotic heart disease, sleep apnea, high-affinity hemoglobinopathy, smoking, or localized renal hypoxia (1, 2). In other instances, excessive Epo levels are the result of production by neoplastic cells. Cases of increased Epo production and erythrocytosis have been reported for patients with renal carcinomas (3), benign renal tumors (4), Wilms' tumors, hepatomas (5), liver carcinomas (6), cerebellar hemangioblastomas (3, 7, 8), adrenal gland tumors (9), smooth muscle tumors (3, 9), and leiomyomas (10). 

Deficient Epo production is found in conjunction with certain forms of anemias. These include anemia of renal failure and end-stage renal disease (1, 2, 11), anemias of chronic disorders [chronic infections (1), autoimmune diseases (1), rheumatoid arthritis (12), AIDS (13), malignancies (14)], anemia of prematurity (2), anemia of hypothyroidism (2), and anemia of malnutrition (2). Many of these conditions are associated with the generation of IL-1 and TNF-alpha, factors that have been shown to be inhibitors of Epo activity (1, 15). Other forms of anemias, on the other hand, are due to Epo-independent causes and affected individuals show elevated levels of Epo (2). These forms include aplastic anemias, iron deficiency anemias, thalassemias, megaloblastic anemias, pure red cell aplasias, and myelodysplastic syndromes.

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.