Development of an enzyme-linked immunosorbent assay for detection of Chlorpyrifos-ethyl and its metabolites 3,5,6-Trichloro-2-Pyridinol and Diethylthiophosphate

Abstract

ABSTRACT A competitive enzyme-linked immunosorbent assay (ELISA) test using polyclonal rabbit antibodies for detection of Chlorpyrifos-ethyl (CPE) and its two major metabolites of degradation [3,5,6-Trichlororpyridin-2-ol (TCP) and O,O’-diethylthiophosphoric acid (DETP)], has been developed. We have first synthetized haptens of these three molecules, with 4 and 5 carbons spacer arms having carboxyl function at their ends. Haptens with 5 carbons’ spacer arm have been coupled to Bovine Serum Albumin (BSA) then injected to rabbits for polyclonal antibodies production. Haptens with 4 carbons spacer arm have been coupled to Human Albumin (HAS) to be in well plates coating to perform the ELISA tests. After adjusting the ELISA parameters, we have plotted the B/B0 (%) values against the logarithm of analyst concentrations. The IC50 values and detection limits were obtained through fitting the curves. The IC50 value of CPE and TCP were 651 and 5.61 ppb, with detection limits about 2.3410−2 and 2.18 10−3 ppb, respectively. We report for the first time, ELISA system developed for DETP with IC50 of 76.5 and a detection limit of 8.81 10−3 ppb. To evaluate the potential interferences that may be encountered in environmental water samples, pH, ionic strength, humic acid and some heavy metals were tested upon IC50 of the developed ELISA systems. In addition, several pesticides as well as CPE metabolites were tested for cross reactivity study. In all cases, the interference of the CPE assay was negligible. TCP assay represented a high cross reactivity to CPE but no interference with the others compounds. For DETP, no cross reactivity with the TCP was detected, but it was higher with CPE and the others pesticides. The developed ELISA systems have been compared with HPLC technique. Both techniques gave values of the same order of CPE and TCP prepared concentrations with a better sensitivity for ELISA system.