Design and validation of a novel immunological test for enterolactone

Abstract

Enterolactone (ENL) is produced by the gut microflora from lignans found in edible plants. ENL is estrogenic with no effect on the E-screen test and is a natural Selected Estrogen Receptor Modulator (SERM) with health interests that have to be checked in clinical studies with bioavailability assessment. Two haptens of ENL were synthesized, with a spacer arm at the C5 position having either 2 or 4 carbon atoms (ENLΔ2 and ENLΔ4, respectively). Hapten coupling to bovine serum albumin (BSA) was characterized by MALDI mass spectrometry. Polyclonal antibodies were obtained against the BSA conjugates. Additional conjugates were generated by coupling to swine thyroglobulin (Thyr). Homologous and heterologous competitive ELISAs were developed with Thyr or BSA conjugates as coating. The best assays were validated on biological samples from mice. Both antibodies exhibited the same IC50 at 1.5 ng mL(-1) with a detection limit below 0.5 ng mL(-1). Most cross-reactions with structurally related lignans were lower than 0.03%. This new assay type is faster, more specific and more reliable than existing ones.

Keywords: (BSA); (EIA); (ELISA); (ENLΔ2 and ENLΔ4); (HR-MS); (OD); (PBS); (SERM); (TR-FIA); (Thyr); (VC); Bioavailability; Bovine Serum Albumin; ELISA; Enterolactone; Enterolactone haptens with a spacer arm of 2 and 4 carbon respectively; Enzymatic Immuno-Assay; Enzyme Linkage Immuno-Sorbent Assay; Hapten; High Resolution mass spectroscopy; Optical Density; Phosphate Buffer Saline; Phytoestrogens; Plasma; Polyclonal antibodies; Selected Estrogen Receptor Modulator; Swine Thyroglobuline; Time Resolved Immunoassay; Variation Coefficients.