Background
There is strong evidence that isoprostanes are excellent molecular biomarkers for oxidant stress. Indeed, 15-F2t-Isoprostane (previously called 8-epi-PGF2α) is already widely recognized as the "gold standard" of oxidant status. This new ELISA kit has been developed for the rapid and accurate measurement of 15-F2t-Isoprostane (the best characterized isoprostane) in urine samples. Using the specially formulated buffers and diluents provided, one can simply dilute urine samples and perform the ELISA assay. No solid phase extraction is needed! Results obtained using this 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). For the analysis of isoprostane levels in serum, tissue culture supernatants or tissue extracts, use product EA84 following solid phase extraction.
Assay Principle
This kit is a competitive enzyme-linked immunoassay (ELISA) for determining levels of 15-F2t-Isoprostane (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-F2t-Isoprostane in the samples or standards competes with 15-F2t-Isoprostane conjugated to horseradish peroxidase (HRP) for binding to a polyclonal antibody specific for 15-F2t-Isoprostane 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-F2t-Isoprostane-HRP bound and inversely proportional to the amount of unconjugated 15-F2t-Isoprostane in the samples or standards.
Recommended Products
To obtain a complete picture of all 15-F2t-Isoprostane, free and esterified, in your sample, we recommend using our Glucuronidase Sample Prep Kit (GL85).
If using urine as a sample, it is recommended to run our Creatinine Assay Kit (CR01) in conjunction with your samples since urinary creatinine levels may be used to normalize the rate of excretion of other analytes.
Typical Standard Curve
Correlation of samples run using EA85 ELISA and by GC/MS.
References
1. Morrow, J., Harris, T., & Roberts, L., Anal. Biochem. 14:1-10 (1990).
2. Morrow, J. D., Hill, K. , Burke, R. F., Nammour, T. M., Badr, K. F., and Roberts, L. J., Proc. Natl. Acad. Sci. U.S.A. 87:9383-9387 (1990).
3. Morrow, et. al., The Isoprostanes: Unique Bioactive Products of Lipid Peroxidation. Prog. Lipid Res. 36:1-21, 1997.
4. Morrow, J., Awad, J. A., Boss, H. J., Blair, I. A., and Roberts, L. J., Proc. Natl Acad. Sci USA 89:10721-10725 (1992).
5. Wang, et al., Immunological characterization of urinary 8-epi-prostaglandin F2a excretion in man. J. Pharm. Exp. Ther. 275:94-100 (1995).
6. Morrow, J., Zackert, W., Yang, J., Kuhrts, E., Callewaert, D., Taber, D., Oates, J., Roberts, J., Quantitiation of the major urinary metabolite of the isoprostane 15-F2t-Isoprostane(8-iso-PGF2a) by a stable isotope dilution mass spectrometric assay, Analytical Biochem. 269: 326-331 (1999).
7. Roberts II, L.J., Morrow, J.D., Measurement of F2-isoprostanes as an index of oxidative stress in vivo, Free Radic. Biol. Med. 28:505-513 (2000).
