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.