Synthesis of Haptens and Conjugates for ELISA of Glycitein: Development and Validation of an Immunological Test

Abstract

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 Delta3-glycitein and Delta4-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 Delta4-glycitein hapten was generated with swine thyroglobulin (Thyr). Enzyme-linked immunosorbent assays (ELISAs) based on the competition between free glycitein and Delta4-glycitein-Thyr conjugates for specific antibodies were developed. The IC50 of the standard curves was 15.6 ng mL(-1) with anti-Delta3-glycitein and 62.5 ng mL(-1) with anti-Delta4-glycitein, that is, 10.9 and 44 pmol/well, respectively. With the Delta3-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.

Bioavailability of glycitein relatively to other soy isoflavones in healthy young Caucasian men

“…Concentrations of conjugates and antibodies are listed in previous articles (Le Bennetau-Pelissero et al, 2003;Shinkaruk et al, 2008). The sensitivity of the assays was improved from their original values and standard curve mid-points were 20, 10, 10 and 15.6 ng mL À1 for daidzein, genistein, equol and glycitein, respectively.…”

“…The detection limit was 5, 2.5, 2.5 and 3.9 ng mL À1 for daidzein, genistein, equol and glycitein, respectively. The antibody specificities are listed in previous articles (Bennetau-Pelissero et al, 2003;Le Houérou et al, 2000;Shinkaruk et al, 2008). Additional tests were also performed on the equol, daidzein and genistein antibodies, demonstrating cross-reactions below 0.05% with o-desmetyl-angolensin and glycitein.…”

“…The ELISA techniques used in this study are fully described in previous papers Le Houérou et al, 2000;Shinkaruk et al, 2008). These techniques are more sensitive than most physico-chemical techniques being the equivalent of LC-MS-MS. A validation of the ELISA techniques vs chromatographic techniques for genistein and daidzein is published by Bennetau-Pelissero et al (2003).…”

“…This study gives for the first time, bioavailability data of glycitein ingested at high rate as a soy-germ-based food supplement without food matrix effects. Glycitein measurements were performed in plasma and urine samples using a new ELISA technique developed by our team (Shinkaruk et al, 2008). A comparison with previous data is made to check for sex, amount ingested and matrix effect on the bioavailability of isoflavone.…”

Synthesis of Novel Haptens and Conjugates for Antibody Production against Kainoid Family

“…10 In the kainoid class of compounds, DA is the principal toxin responsible for ASP. Several immunoassays against DA have been described.…”