Development of an enzyme immunoassay for linoleic acid diols in urine Original Research Article

An enzyme-linked immunosorbent assay for the diol derivatives of linoleic acid, cis-9,10-dihydroxyoctadec-12(Z)-enoic acid (leukotoxindiol, LTXD) and cis-12,13-dihydroxyoctadec-9-(Z)-enoic acid (iso-leukotoxindiol, iso-LTXD), was developed. Polyclonal antibodies were generated in rabbits using an isomeric LTXD and iso-LTXD mixture conjugated with keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). Coating antigens were synthesized by conjugation of LTXD/iso-LTXD, dihydroxystearic acid, ricinoleic acid (OLE), ricelaidic acid (ELA) or 12-hydroxystearic acid to BSA or ovalbumin (OVA). Various linoleic acid derivatives did not cross react significantly. Using the ovalbumin conjugate of ricinoleic acid as a coating antigen, the assay yielded an IC50 value of 8 μg/l LTXD/iso-LTXD and was applied to the analysis of urine samples. Urine samples were treated with glucuronidase to release LTXD/iso-LTXD from its glucuronic acid conjugate. An increase of the LTXD/iso-LTXD signal was clearly observed after glucuronidase incubation. Recent evidence suggests that these diols may be involved in diseases such as acute respiratory distress syndrome and cardiovascular diseases, thus this assay will be important in assessing the significance of these compounds as biomarkers for these disease states.