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

doi:10.1007/BF02970140

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

Biochemistry

Research output: Contribution to journal › Article › peer-review

16 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 language	English (US)
Pages (from-to)	109-113
Number of pages	5
Journal	Journal of Biosciences
Volume	28
Issue number	1
DOIs	https://doi.org/10.1007/BF02970140
State	Published - Feb 2003

Keywords

20-Hydroxyeicosatetraenoic acid
Colorimetric
Competitive ELISA

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology
General Agricultural and Biological Sciences

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10.1007/BF02970140

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title = "Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay",

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.",

keywords = "20-Hydroxyeicosatetraenoic acid, Colorimetric, Competitive ELISA",

author = "Grates, {Harry E.} and McGowen, {Richard M.} and Gupta, {Smiti V.} and Falck, {J R} and Brown, {Thomas R.} and Callewaert, {Denis M.} and Sasaki, {Diane M.}",

note = "Funding Information: This work was supported in part by National Institute of Environmental Health Services contract N44-ES-05449.",

year = "2003",

month = feb,

doi = "10.1007/BF02970140",

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T1 - Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

AU - Grates, Harry E.

AU - McGowen, Richard M.

AU - Gupta, Smiti V.

AU - Falck, J R

AU - Brown, Thomas R.

AU - Callewaert, Denis M.

AU - Sasaki, Diane M.

N1 - Funding Information: This work was supported in part by National Institute of Environmental Health Services contract N44-ES-05449.

PY - 2003/2

Y1 - 2003/2

N2 - 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.

AB - 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.

KW - 20-Hydroxyeicosatetraenoic acid

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KW - Competitive ELISA

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JO - Journal of Biosciences

JF - Journal of Biosciences

IS - 1

ER -

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

Abstract

Keywords

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