Abstract
| - Trichlorophenols (TCP) eliminated by the urine can beconsidered as potential biomarkers of exposure of manychemicals (chlorophenols, chlorophenoxy acid herbicides,prochloraz, lindane, hexachlorobenzene, etc). High-throughput screening methods are necessary to carry outefficient monitoring programs that may help to preventcertain occupational health diseases. For this purpose,an indirect enzyme-linked immunosorbent assay (ELISA)for 2,4,6-trichlorophenol detection has been developedusing polyclonal antisera raised against 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid (hapten 5) covalently coupled by the mixed anhydride (MA) method tokeyhole limpet hemocyanin (KLH). The indirect ELISAuses a heterologous coating antigen prepared by conjugation of 3-(2-hydroxy-3,6-dichlorophenyl)propanoic acid(hapten 4) to bovine serum albumin (BSA) using theactive ester (AE) method. The optimum hapten densityfor the coating antigen was found to be 3 mol of hapten/mol of protein. The assay shows a limit of detection of0.245 ± 0.116 μg L-1, and it is performed on 96-wellmicrotiter plates in about 1.5 h. The ELISA reportedrecognizes on a much less extent other chlorinated phenols, such as 2,3,4,6-tetrachlorophenol (2,3,4,6-TtCP,21%), 2,4,5-TCP (12%) and 2,3,5-TCP (15%); however,brominated phenols (BP) are even more recognized thanthe corresponding chlorinated analogues (ex. 2,4,6-TBP,710%; 2,4-DBP, 119%). With the aim of finding anexplanation for this behavior, theoretical calculations havebeen performed for those and other halogenated phenols(2,4,6-triiodophenol and 2,4,6-trifluorophenol) to clarifywhich physicochemical parameter can explain better therecognition pattern observed. Finally, the assay has beenadapted to the analysis of urine samples. The studies haveshown that a limit of detection of 1 μg L-1 can beaccomplished on this biological matrix by combining theELISA procedure with a C18 solid-phase extractionmethod.
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