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Development and Evaluation of an Immunoassay for Biological Monitoring Chlorophenols in Urine as Potential Indicators of Occupational Exposure
journal contribution
posted on 2001-12-13, 00:00 authored by Roger Galve, Mikaela Nichkova, Francisco Camps, Francisco Sanchez-Baeza, M.-Pilar MarcoTrichlorophenols (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 μg 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 μg L-1 can be
accomplished on this biological matrix by combining the
ELISA procedure with a C18 solid-phase extraction
method.
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Keywords
BPchlorinated phenolsPotential Indicators1 μ g LDBPpolyclonal antiserahapten 4hapten 5detectionbrominated phenols3 mol1.5 hTCPmethodhealth diseasesurine samplesELISA procedureMATBPhydroxyBiological Monitoring Chlorophenolsphysicochemical parameterkeyhole limpet hemocyaninOccupational Exposure Trichlorophenolschlorophenoxy acid herbicideschlorinated analoguesmonitoring programsheterologous coating antigencoating antigenBSArecognition patternassayhapten densityAEKLHmicrotiter platesserum albumin
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