Synthesis of haptens and conjugates for ELISA of glycitein: Development and validation of an immunological test

Two carboxylic acid haptens of glycitein were synthesized, with a spacer arm at the C2 position. They differed in the length of the spacer arm, with the length of the spacer arms being three or four carbon atoms, and were named Delta 3-glycitein and Delta 4-glycitein haptens, respectively. The different haptens were coupled to bovine serum albumin (BSA), and the coupling efficiency was assessed by MALDI mass spectrometry. Polyclonal antibodies were generated against the BSA conjugates. An additional conjugate of Delta 4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta 4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta 3-glycitein and 62.5 ng mL(-1) with anti-Delta 4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta 3-glycitein antibody, interassay and intra-assay variations were 12.2 and 11.5%, respectively. Specificity tests did not show any significant cross-reaction with any other soy isoflavone. This specificity is not influenced by the length of the spacer arm. The assay was validated by measurements performed on plasma samples as well as on soy-based foodstuffs and on soy-based food supplements.