Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

Harry E. Grates, Richard M. McGowen, Smiti V. Gupta, J R Falck, Thomas R. Brown, Denis M. Callewaert, Diane M. Sasaki

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

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 ELISAs 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 0.1 ng/ml for the HRP assay, and 0.5 ng/ml for the AP assay, with r2 = 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 (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). 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.

Original languageEnglish (US)
Pages (from-to)109-113
Number of pages5
JournalJournal of Biosciences
Volume28
Issue number1
DOIs
StatePublished - Feb 2003

Keywords

  • 20-Hydroxyeicosatetraenoic acid
  • Colorimetric
  • Competitive ELISA

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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    Grates, H. E., McGowen, R. M., Gupta, S. V., Falck, J. R., Brown, T. R., Callewaert, D. M., & Sasaki, D. M. (2003). Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay. Journal of Biosciences, 28(1), 109-113. https://doi.org/10.1007/BF02970140