11,12 DHET ELISA Assay Kit

$330.00$2,620.00

The Eagle Biosciences 11, 12-DHET ELISA Assay kit is intended for the quantitative determination of 11, 12-DHET in biological samples by enzyme linked immunoassay (ELISA).  The Eagle Biosciences 11, 12 DHET ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

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11,12 DHET ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 0.01 ng/ml
Dynamic Range: 0.01 – 1000 ng/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma, Tissue, Biological Fluids
Sample Size: 100 µl

Product manufactured in the USA

Additional Information

Assay Background

The level of 11, 12-DHET or 11, 12-DHET epitope has been shown to exhibit correlation with hypertension in rodents (1, 2, 3).  11, 12-DHET is a representative metabolite of cytosolic epoxide hydrolase-mediated metabolism of EETs, which are generated by arachidonic acid epoxygenase activity of cytochrome P450’s (4).  The Eagle Biosciences 11,12-DHET ELISA Assay kit can be used for the determination of 11,12-DHET in serum, plasma, cells, and tissues following proper isolation and purification.  Instructions are provided as to the proper isolation and purification in the following pages.  

The competitive Eagle Biosciences 11,12-DHET ELISA Assay kit, based on competition between 11,12-DHET epitope and 11,12-DHET-HRP conjugate for a limited number of binding sites available from the anti-11,12-DHET antibody, which is coated to the wells of the 96 well ELISA plate.  The conjugate concentration is held as a constant in each well, while the concentration of the 11,12-DHET is variable, based on the concentration of the sample or standard.  Thus the amount of the 11,12-DHET conjugate which is able to bind to each of the wells is inversely proportional to the concentration of 11,12-DHETin the standard or sample.  The amount of the conjugate which is bound to each well is then determined by the amount of color obtained, when TMB is added.  The TMB reacts with the HRP available in the well.  With the addition of sulfuric acid, the blue colored product is converted into a yellow colored product, which can be read on a plate reader at 450 nm. 

Assay Procedure

  1. Load 200 microliters of Sample Dilution Buffer into the blank (BL) wells and 100 microliters of Sample Dilution Buffer into the maximum binding (BO) wells.
  2. Load 100 microliters of each of the standards into the appropriate wells.
  3. Load 100 microliters of each of the samples into the appropriate wells.
  4. Load 100 microliters of the diluted 11,12-DHET-HRP conjugate in the BO wells, the standard wells, and the sample wells.  Do NOT add HRP conjugate into the BL wells.
  5. Incubate the plate at room temperature for two hours.
  6. Wash the plate three times with 400 microliters of the diluted Wash Buffer per well.
  7. After the last of the three wash cycles pat the plate dry onto some paper toweling
  8. Add 200 microliters of the TMB substrate to all of the wells (including BL wells).
  9. Incubate the plate at room temperature for 15-30 minutes.
  10. Add 50 micoliters of 2 N sulfuric acid to all of the wells.
  11. Read the plate at 450 nm.

Manual

Product Manual


Publications

References

  • Kim et al.  Two divisional US Patents: 6,440,682 and 6,534,282 issued on 8/27/2002 and 3/18/2003, respectively. (http://patft.uspto.gov/netahtml/srchnum.htm Pat.US: 6,440,682 and Pat.US: 6,534,282).
  • Garson et al.  Computational characterization of a series of eicosanoids.  Lett. Drug Design and Discovery 2, 322, 2005.
  • Sinal et al.  Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation. J. Biol. Chem. 275, 40504, 2000.
  • Yu et al.  Soluble epoxide hydrolase regulates hydrolysis of vascoactive epoxyeicosatrienoic acids. Circulation Research 87, 992, 2000.
  • Makita et al.  Cytochrome P450, the arachidonic acid cascade, and hypertension: new vistas for an old enzyme system. FASEB J. 10, 1456, 1996, and references therein.
  • Spiecker et al.  Risk of coronary artery disease associated with polymorphism of the cytochrome P450 epoxygenase CYP2J2. Circulation 110, 2132, 2004.
  • Wang et al.  Cytochrome P450 2J2 promotes the neoplastic phenotype of carcinoma cells and is up-regulated in human tumors. Cancer Res. 65, 4707, 2005.
  • Wang et al.  Cytochrome P-450 Epoxygenase Promotes Human Cancer Metastasis. Cancer Res. 67, 6665-6667, July 15, 2007.
  • Zeldin et al.  Cytochrome P-450 epoxygenases protect endothelial cells from apoptosis induced by tumor necrosis factor-a via MAPK and PI3K/Akt signaling pathways. Am J. Physiol Heart Circ Physiol 293: H142-H151, 2007.
  • Alkayed et al.  Polymorphisms in the Human Soluble Epoxide Hydrolase Gene EPHX2 Linked to Neuronal Survival after Ischemic Injury. J. Neurosci. 27(17): 464-4649, April 25, 2007.
  • Chen et al. Synergistic effect of cytochrome P450 epoxygenase CYP2J2*7 polymorphism with smoking on the onset of premature myocardial infraction. Atherosclerosis 195, 199-206, 2007.
  • D. Wang, T. Hirase, T. Nitto, M. Soma, K. Node. Eicosapentaenoic acid increases cytochrome P450 2J2 gene expression and epoxyeicosatrienoic acid production via peroxisome proliferator-activated receptor g in endothelial cells. J. Cardiol. 54, 368-374, 2009.