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Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay
- Grates, Harry E., McGowen, Richard M., Gupta, Smiti V., Falck, John R., Brown, Thomas R., Callewaert, Denis M., Sasaki, Diane M.
- Journal of biosciences 2003 v.28 no.1 pp. 109-113
- alkaline phosphatase, arachidonic acid, blood serum, colorimetry, cross reaction, cytochrome P-450, enzyme-linked immunosorbent assay, high performance liquid chromatography, mass spectrometry, peroxidase, phosphates, urine
- Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELIS As were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a proprietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3′,5,5,-tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 01 ng/ml for the HRP assay, and 0–5 ng/ml for the AP assay, withr ₂ = 0.99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (007%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (004%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.