ENL, GEN, DAI, and equol (EQ)—a DAI metabolite produced by the gut flora of EQ-producers-were assayed in the urine and plasma samples using specific ELISAs, as described previously [22 (link),23 (link)]. Haptens functionalized on different carbon atoms were previously synthesized [23 (link),24 (link),25 (link)]. Specific polyclonal antibodies were obtained for each PHYTO bound to bovine serum albumin and the best antibodies were retained for assay development. Standard curve preparations and sample dilutions were performed in silicone-coated glass vials. The ELISAs followed a competitive procedure with an immobilized competitor, which was the homologous hapten bound to swine thyroglobulin, except for ENL [23 (link)]. The sensitivities varied between 0.08 ng/well and 0.4 ng/well, depending on the substance assayed, and intra-assay variation was always below 7% and inter-assay variation was below 17%. For the plasma samples, final dilutions varied between 1:5 and 1:20. For urine samples, the final dilutions varied between 1:5 and 1:4000 due to various levels of natural dilution of the urine samples. To take this phenomenon into account, the measured concentrations were corrected based on a creatinine excretion measurement performed on the same urine samples.
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