TY - JOUR
T1 - Development of antibodies for determination of alkylresorcinol metabolites in human urine and elucidation of ELISA cross-reactivity
AU - Wierzbicka, R.
AU - Eyer, L.
AU - Landberg, R.
AU - Kamal-Eldin, A.
AU - Franek, M.
N1 - Funding Information:
This work was supported by the Swedish Research Council for the Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the project L012018 , with financial support from the MEYS of the CR under the NPU I programme. We wish to thank K. Stastny for the HR Orbitrap MS analysis and for MS data processing. We also wish to thank M. Cechova for her excellent technical assistance.
Publisher Copyright:
© 2014 Elsevier B.V..
PY - 2014/11/1
Y1 - 2014/11/1
N2 - Alkylresorcinols (ARs) are amphiphilic phenolic lipids and their two main metabolites, 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), can be used as biomarkers of whole grain wheat and rye intake. The aim of this study was to develop antibodies against DHBA and DHPPA for use in ELISA analysis. Good calibration curves were obtained for ELISA using alkaline phosphatase (AP) conjugates. The highest sensitivity for DHPPA was found using a reagent combination of anti-DHPPA-BSA and DHPAA-AP in a direct ELISA (IC50=1.5μmol/L), and for DHBA using a reagent combination of anti-DHBA-OV and DHBA-AP (IC50=1.3μmol/L). Calibration was conducted in the linear range (0.3-27.4μmol/L), with limit of detection (LOD) 0.1μmol/L. Intra and inter CVs was in the range of 0.7-7.2% and 5.1-11.5%, respectively, for DHPPA and 1.3-9.4% and 3.5-20%, respectively, for DHBA. Mean recovery was 104% for DHPPA and 102% for DHBA. The ELISA method developed was then used for analysis of 120 urine samples from free-living men and women that had previously been analysed by gas chromatography-mass spectrometry (GC-MS). ELISA produced several-fold higher values than GC-MS. Application of high-resolution Orbitrap mass spectrometry (HR Orbitrap MS) allowed several compounds, including novel putative AR metabolites, to be identified, synthesised and confirmed as compounds with high ELISA cross-reactivity.
AB - Alkylresorcinols (ARs) are amphiphilic phenolic lipids and their two main metabolites, 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), can be used as biomarkers of whole grain wheat and rye intake. The aim of this study was to develop antibodies against DHBA and DHPPA for use in ELISA analysis. Good calibration curves were obtained for ELISA using alkaline phosphatase (AP) conjugates. The highest sensitivity for DHPPA was found using a reagent combination of anti-DHPPA-BSA and DHPAA-AP in a direct ELISA (IC50=1.5μmol/L), and for DHBA using a reagent combination of anti-DHBA-OV and DHBA-AP (IC50=1.3μmol/L). Calibration was conducted in the linear range (0.3-27.4μmol/L), with limit of detection (LOD) 0.1μmol/L. Intra and inter CVs was in the range of 0.7-7.2% and 5.1-11.5%, respectively, for DHPPA and 1.3-9.4% and 3.5-20%, respectively, for DHBA. Mean recovery was 104% for DHPPA and 102% for DHBA. The ELISA method developed was then used for analysis of 120 urine samples from free-living men and women that had previously been analysed by gas chromatography-mass spectrometry (GC-MS). ELISA produced several-fold higher values than GC-MS. Application of high-resolution Orbitrap mass spectrometry (HR Orbitrap MS) allowed several compounds, including novel putative AR metabolites, to be identified, synthesised and confirmed as compounds with high ELISA cross-reactivity.
KW - Alkylresorcinol
KW - Antibodies
KW - Dietary biomarker
KW - ELISA
KW - High-resolution Orbitrap mass spectrometry
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U2 - 10.1016/j.jim.2014.07.007
DO - 10.1016/j.jim.2014.07.007
M3 - Article
C2 - 25066633
AN - SCOPUS:84909597996
SN - 0022-1759
VL - 413
SP - 12
EP - 24
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
ER -