15-Isoprostane F2t ELISA Assay Kit

$420.00

The Eagle Biosciences Isoprostane ELISA Assay kit is for the quantitative determination of  15-Isoprostane F2t in biological fluids by a microplate enzyme immunoassay (ELISA).

SKU: 15I39-K01 Categories: , ,

15-Isoprostane F2t ELISA Assay Kit

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: Biological Fluids
Sample Size: 100 µl
Species: Human

Product manufactured in the USA

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 a representative isoprostane, 15-isoprostane F2t (also known as 8-epi-PGF2a, 8-iso-PGF2a or 8-isoprostane). Levels of 15-isoprostane F2t (15-IsoP F2t) in biological fluids have been shown to be useful for assessment of oxidant stress in vivo. 15-IsoP 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.

The 15-Isoprostane F2t ELISA Assay kit can be used for the quantification of free 15-IsoP F2t in urine, plasma, serum, and tissue samples following Solid Phase Extraction (SPE) of the isoprostane-containing fraction. Instructions are also provided for the quantification of total 15-IsoP F2t following hydrolysis of phospholipids.
NOTE: Alternatively, urine samples may be analyzed without SPE using a separately available kit, ISO34-K01 – Urinary Isoprostane ELISA, also available from Eagle Biosciences, Inc.

Assay Principle

15-Isoprostane F2t ELISA Assay kit is a competitive enzyme-linked immunoassay (ELISA) for determining levels of 15-IsoP F2t in biological samples. Briefly, 15-IsoP F2t in the samples or standards competes with 15-IsoP F2t conjugated to horseradish peroxidase (HRP) for binding to a polyclonal antibody specific for 15-IsoP F2t coated on the microplate. The HRP activity results in color development when the substrate is added, with the intensity of the color proportional to the amount of 15-IsoP F2t bound and inversely proportional to the amount of unconjugated 15-IsoP F2t in the samples or standards.

  1. Add 100 µL of Sample or Standard to each well. See Scheme I for a suggested plate layout.
  2. Add 100 µL of diluted 15-IsoP F2t HRP Conjugate to each well omitting the Reagent Blank (RB) (add 100 µL of 1x Dilution Buffer in lieu of conjugate). Incubate at room temperature for 2 hours.
  3. Wash wells according to the following 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.
  • Remove the contents of each well by inversion of plate into an appropriate disposal device.
  • Repeat procedure two more times and proceed to next step.
  • 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. Incubate for 30 minutes at room temperature until an appreciable blue hue is observed for the B0.
5. Add 50 µL of 3 N H2SO4 to each well to stop the reaction. The color will change from blue to yellow.
6. Read plate at 450 nm.

Cross Reactivity Data

15-Isoprostane F2t 100.0%
9a,11b-PROSTAGLANDIN F2a 4.1%
13,14-DIHYDRO-15-KETO-F2a 3.0%
9b,11a-PROSTAGLANDIN F2a < 0.01%
PROSTAGLANDIN F2a < 0.01%
6-KETO-PROSTAGLANDIN F2a < 0.01%
PROSTAGLANDIN E2 < 0.01%
PROSTAGLANDIN D2 < 0.01%
ARACHIDONIC ACID < 0.01%

Manual

Product Manual