title: Development of antibodies for determination of alkylresorcinol metabolites in human urine and elucidation of ELISA cross-reactivity.
abstract: 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.
authors: Department of Food Science, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7051, SE-75007 Uppsala, Sweden; Department of Virology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. Electronic address: Roksana.Wierzbicka@slu.se.
authors: R
authors: R
authors: Wierzbicka
authors: Department of Virology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. Electronic address: eyer@vri.cz.
authors: L
authors: L
authors: Eyer
authors: Department of Food Science, Uppsala BioCenter, Swedish University of Agricultural Sciences, P.O. Box 7051, SE-75007 Uppsala, Sweden. Electronic address: rikard.landberg@slu.se.
authors: R
authors: R
authors: Landberg
authors: Department of Food Science, Faculty of Food and Agriculture, United Arab Emirates University, P.O. Box 17551, Al-Ain, United Arab Emirates. Electronic address: afaf.kamal@uaeu.ac.ae.
authors: A
authors: A
authors: Kamal-Eldin
authors: Department of Virology, Veterinary Research Institute, Hudcova 70, 621 00 Brno, Czech Republic. Electronic address: franek@vri.cz.
authors: M
authors: M
authors: Franek
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002138:Calibration; D003429:Cross Reactions; D004040:Dietary Carbohydrates / Q000652:urine*; D002523:Edible Grain / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D008401:Gas Chromatography-Mass Spectrometry; D006241:Haptens / Q000737:chemistry; D006801:Humans; D062385:Hydroxybenzoates / Q000652:urine*; D057230:Limit of Detection; D010667:Phenylpyruvic Acids / Q000652:urine*; D011817:Rabbits; D012118:Resorcinols / Q000652:urine*; D012434:Secale / Q000378:metabolism; D014908:Triticum / Q000378:metabolism
chemical_list: D000906:Antibodies; D004040:Dietary Carbohydrates; D006241:Haptens; D062385:Hydroxybenzoates; D010667:Phenylpyruvic Acids; D012118:Resorcinols; C076950:alpha-resorcylic acid; C019344:3,4-dihydroxyphenylpyruvic acid; D000469:Alkaline Phosphatase
keywords: Alkylresorcinol; Antibodies; Dietary biomarker; ELISA; High-resolution Orbitrap mass spectrometry