Human Erythropoietin/EPO DuoSet ELISA

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Human Erythropoietin/EPO DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Range
3.1 - 200 mIU/mL
Sufficient Materials
For five 96-well plates*
Specificity
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant Erythropoietin (Epo). The suggested diluent is suitable for the analysis of most cell culture supernate, serum, and plasma samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

Product Features

  • Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
  • Development protocols are provided to guide further assay optimization
  • Assay can be customized to your specific needs
  • Economical alternative to complete kits

Kit Content

  • Capture Antibody
  • Detection Antibody
  • Recombinant Standard
  • Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)

Other Reagents Required

DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.

The components listed above may be purchased separately:

PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered

Wash Buffer: (Catalog # WA126), or 0.05% Tween® 20 in PBS, pH 7.2-7.4

Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered

Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)

Stop Solution: 2 N H2SO4 (Catalog # DY994)

Microplates: R&D Systems (Catalog # DY990)

Plate Sealers: ELISA Plate Sealers (Catalog # DY992)

Data Example

Human Erythropoietin ELISA Standard Curve

Product Datasheets

Preparation and Storage

Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

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.

Entrez Gene IDs:
2056 (Human); 13856 (Mouse); 24335 (Rat)
Alternate Names:
ECYT5; EP; EPO; epoetin; Erythropoietin; MGC138142; MVCD2

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. Dilute the Capture Antibody to the working concentration in PBS without carrier protein. Immediately coat a 96-well microplate with 100 μL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
  2. Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
  3. Block plates by adding 300 μL of Block Buffer to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

FAQs

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