Synthesis of Haptens and Conjugates for ELISAs of Phytoestrogens. Development of the Immunological Tests

Abstract

Seven carboxylic acid haptens of isoflavonoids were synthesized, with the spacer arm on the oxygen atom at the C7 position for one series, with formononetin, daidzein, equol, biochanin A, and genistein, and at the C8 position for a second series, with only formononetin and daidzein. The different haptens were coupled to bovine serum albumin (BSA) and to swine thyroglobulin (Thyr). Polyclonal antibodies were generated against the BSA conjugates. Enzyme-linked immunosorbent assays (ELISAs) were developed based on competition between free phytoestrogens and the Thyr-hapten conjugates for specific antibodies. IC(50) values of the standard curves ranged between 0.8 and 20 ng/mL that is, 0.3 and 9.2 pmol/well. The antibodies obtained should be useful for assays in vegetable matter as well as in biological fluids after a separation step. These ELISAs should be valuable also in the food industry to control phytoestrogen concentrations prior to and after processing.

Urinary phytoestrogen concentrations in the U.S. population (1999–2000)

“…Since daidzein metabolism to equol in the gut reportedly varies, individuals are classified as ''equol producers'' or not, depending upon how efficiently they metabolize daidzein to equol (Setchell et al, 2002a). Equol production is important because equol is 100 times more estrogenic than daidzein (Bennetau-Pelissero et al, 2000). We found ratio differences between sexes and among racial/ ethnic groups; however, age did not appear to be a significant variable.…”

Urinary and serum concentrations of seven phytoestrogens in a human reference population subset

“…Daidzein metabolism to equol varies considerably among people and is influenced by other dietary components, such as carbohydrate intake. High carbohydrate intake leads to higher production of equol than does low carbohydrate intake (Setchell and Cassidy, 1999;Bennetau-Pelissero et al, 2000). In the NHANES III population, variables like this could not be assessed, which could contribute additional information to the interpretation of differences between different populations and possibly add insight in the broad variability observed.…”

Improvement of Bone-Sparing Effect of Soy Isoflavones by Pre- and Probiotics in Postmenopausal women

“…During this second month, the soy group was considered as the control group. Blood sampling was performed in the morning, after an over-night fasting, at d0 and every 15 days thereafter to assess bone specific alkaline phosphatase (bALP) (Garnero and Delmas, 1999) and isofl avones (daidzein, genistein and equol) (Bennetau-Pelissero et al 2000). Twenty-four hour urine samples were obtained from the volunteers at the same time points, during the study to analyse deoxypyridinoline (DPD) (Robins, 1994).…”

“…Plasma phytoestrogen concentrations plasma genistein, daidzein and equol concentrations were measured by ELISA (Bennetau-Pelissero et al 2000). The sensitivity of the method was 35, 40 and 10 nmoles for genistein, daidzein and equol, respectively.…”