Development and evaluation of an immunoassay for biological monitoring chlorophenols in urine as potential indicators of occupational exposure.

Roger Galve, Mikaela Nichkova, Francisco Camps, Francisco Sanchez-Baeza, M-Pilar Marco

文献索引：Anal. Chem. 74(2) , 468-78, (2002)

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摘要

Trichlorophenols (TCP) eliminated by the urine can be considered as potential biomarkers of exposure of many chemicals (chlorophenols, chlorophenoxy acid herbicides, prochloraz, lindane, hexachlorobenzene, etc). High-throughput screening methods are necessary to carry out efficient monitoring programs that may help to prevent certain occupational health diseases. For this purpose, an indirect enzyme-linked immunosorbent assay (ELISA) for 2,4,6-trichlorophenol detection has been developed using polyclonal antisera raised against 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid (hapten 5) covalently coupled by the mixed anhydride (MA) method to keyhole limpet hemocyanin (KLH). The indirect ELISA uses a heterologous coating antigen prepared by conjugation of 3-(2-hydroxy-3,6-dichlorophenyl)propanoic acid (hapten 4) to bovine serum albumin (BSA) using the active ester (AE) method. The optimum hapten density for the coating antigen was found to be 3 mol of hapten/mol of protein. The assay shows a limit of detection of 0.245 +/- 0.116 microg L(-1), and it is performed on 96-well microtiter plates in about 1.5 h. The ELISA reported recognizes 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 than the corresponding chlorinated analogues (ex. 2,4,6-TBP, 710%; 2,4-DBP, 119%). With the aim of finding an explanation for this behavior, theoretical calculations have been performed for those and other halogenated phenols (2,4,6-triiodophenol and 2,4,6-trifluorophenol) to clarify which physicochemical parameter can explain better the recognition pattern observed. Finally, the assay has been adapted to the analysis of urine samples. The studies have shown that a limit of detection of 1 microg L(-1) can be accomplished on this biological matrix by combining the ELISA procedure with a C18 solid-phase extraction method.