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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.