Urinary Isoprostane ELISA Assay


The Urinary Isoprostane ELISA Assay kit is for the quantitative determination of Isoprostane in human urine by a microplate enzyme immunoassay (ELISA).

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Urinary Isoprostane ELISA Assay

The Urinary Isoprostane ELISA Assay is For Research Use Only

Size: 1×96 wells
Sensitivity: 0.05 ng/ml
Dynamic Range: 0.05 – 100 ng/ml
Incubation Time: 2.5 hour
Sample Type: Urine
Sample Size: 100 µl
Species: Human

Product manufactured in the USA

Assay Principle

This Urinary Isoprotane ELISA Assay kit is a competitive enzyme-linked immunoassay (ELISA) for determining levels of 15-isoprostane F2t (the best characterized isoprostane) in urine samples.  Briefly, urine samples are mixed with an enhanced dilution buffer that essentially eliminates interference due to non-specific binding. The 15-isoprostane F2t in the samples or standards competes with 15-isoprostane F2t conjugated to horseradish peroxidase (HRP) for binding to a polyclonal antibody specific for 15-isoprostane F2t coated on the microplate.  The HRP activity results in color development when substrate is added, with the intensity of the color proportional to the amount of 15-isoprostane F2t-HRP bound and inversely proportional to the amount of unconjugated 15-isoprostane F2t in the samples or standards.

Using the specially formulated buffers and diluents provided, one can simply dilute urine samples and perform the Isoprostane ELISA assay. No solid phase extraction is required! Results obtained using this Urinary Isoprostane ELISA kit have been extensively validated by GC/MS following solid phase extraction of separate aliquots and show a very high degree of correlation (r > 0.8).

Related Products

15-Isoprostane F2t ELISA Assay
8-Isoprostane ELISA Assay Kit
Lipoxin A4 ELISA Assay

Additional Information

Assay Background

Isoprostanes are prostaglandin-like compounds that are produced by free radical mediated peroxidation of lipoproteins. This kit is for the quantification of 15-isoprostane F2t (also known as 8-epi-PGF2a or 8-iso-PGF2a) in urine samples.  Levels of 15-isoprostane F2t in urine are useful for the non-invasive assessment of oxidant stress in vivo.  15-isoprostane F2t has also been shown to be a potent vasoconstrictor in rat kidneys and rabbit lungs, and plays a causative role in atherogenesis.  Elevated isoprostane levels are associated with hepatorenal syndrome, rheumatoid arthritis, atherosclerosis, and carcinogenesis.

Assay Procedure

  1. Add 100 µL of Standards or diluted unknowns to each well.  Recommended sample dilutions are 1:4 or 1:8 with Enhanced Dilution Buffer.  See Scheme I for a suggested plate layout.
  2. Add 100 µL of diluted 15-isoprostane F2t HRP Conjugate to each well omitting the Reagent Blank (RB) (add 100 µL of Enhanced Dilution Buffer in lieu of Conjugate).  Allow the plate to incubate for 2 hours at RT.
  3. Wash wells according to the following wash procedure:
    • Remove the contents of each well by inversion of the plate.
    • Tap out the remaining contents of the plate onto a lint free paper towel.
    • Add 300 µL of 1x Wash Buffer.
    • Let stand for 2-3 minutes.
    • Repeat procedure two more times, then proceed to the next step.
    • Remove the contents of each well by inversion of the plate into an appropriate disposal device.
    • Tap out the remaining contents of the plate onto a lint-free paper towel, then proceed to step 4.
  4. Add 200 µL of TMB Substrate to each well.
  5. Incubate for 20 – 40 minutes until an appreciable blue hue is observed for the B0.
  6. Add 50 µL of 3 M Sulfuric Acid to each well to stop the reaction.  The color will change from blue to yellow.
  7. Read the plate at 450 nm.

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