Development of an ELISA for urea herbicides in foods

Abstract

Four haptens analogous to phenylurea herbicides were synthesized. Immunogens and coating proteins prepared by coupling these haptens to human serum albumin and ovalbumin, respectively. Antisera raised in rabbits immunized with three of the immunogens bound strongly to microtitration plates coated with homologous hapten, such that competitive assays were developed using heterologous coating proteins. The assay systems were characterized with respect to specificity to 13 related compounds and one selected for application to the determination of monolinuron, diuron, and linuron in foods. Satisfactory recrovery data were obtained by direct analysis of methanol extracts of foods fortified with 40-160 ng/g monolinuron or 0.25-2.0 µg/g diuron. Solvent partitioning and silicic acid column chromatography were only partially successful in removing interferences in the determination of linuron.

Synthesis of Haptens and Derivation of Monoclonal Antibodies for Immunoassay of the Phenylurea Herbicide Diuron

“…These strategies were designed to provide two different presentations of diuron, or any similar arylurea, with spacer arms of virtually any length and structure. Our scheme A resembles one originally used by Newsome and Collins (1990). Scheme B was designed to preserve both the dichlorophenyl and dimethylurea moieties as potential epitopes.…”

“…Our 1H NMR was in fair agreement with the results reported by Newsome and Collins, except that we observed a downfield carboxylic acid OH peak that they did not report. Because Newsome and Collins (1990) did not report an IR spectrum, there is no evidence that their hapten had a carboxylic acid group. We speculate that their material may have cyclized, thus preventing its conjugation to the carrier protein.…”

“…Recrystallization from acetonitrile provided 1.66 g (91%) of glistening white crystals: mp 161.5-162.5 °C (dec with gas evolution) [lit. (Newsome and Collins, 1990) mp [123][124][125] °C]; TLC R/0.05 (eluant solvent A), 0.42 [eluant ethanolchloroform (5:95 v/v)]; IR (KBr) 3376 (m, NH), 1719 (s, C=0, acid), 1635 (vs, C=0, amide I), 1517 (s, C=0, amide II), 1213 (s, CO) cm-1; NMR (DMSO-d6) 12.2 (br, 1 H, OH), 8.51 (s, 1 , NH), 7.87 (d, J = 2.1 Hz, 1 H, Ar-H2), 7.5 (m, 2 H, Ar-H66), 3.31 (t, J = 7.3 Hz, 2 H, CH2N), 2.93 (s, 3 H, CH3), 2.23 (t, J = 7.3 Hz, 2 H, CH2C02), 1.73 (quin, J = 7.3 Hz, 2 H, CCH2C) (the 12.2 and 8.51 ppm peaks disappeared with added D20); 13C NMR (DMSO-de) 174.4 (acid C=0), 154.9 (amide C=0), 141.1 (Ar-Ci), 130.6 (Ar-C3), 130.1 (Ar-C5), 123.0 (Ar-C4), 120.7 (Ar-C2), 119.5 (Ar-C6), 47.5 (CH2N, C4), 34.4 (CH3), 30.9 (CH2C02, C2), 22.9 (C3); MS m/z (relative intensity) 306 (22, M+ + 2), 304 (29, M+), 189 (70, Cl2C6H3NCO + 2), 187 (100, Cl2C6H3NCO), 161 (48, Cl2C6H3NCO + 2-CO), 159 (22, Cl2C6H3NCO -CO); FAB-HRMS caled for C12Hi3C12N203 305.0461, obsd 305.0463.…”

Application of immunoassays for the herbicides, molinate and diuron, to field water analysis

“…The diuron assay described by Schneider has already proved to be more sensitive for urea herbicides than several others reported in the literature (Newsome & Collins, 1990;Liegieois et al, 1992;Karu et al, 1994a,b). In this case, we have modified Downloaded by [New York University] the microplate assay protocol to retain high sensitivity but to reduce the assay time significantly.…”

Immunodetection of Ecosystem Contaminants

“…The IA group at Health and Welfare Canada have developed ABs to the phenylurea (carbamide) herbicides (20). The coating antigens were made heterologous with respect to the immunogen so as to weaken the AB-binding reaction and to allow the analyte molecules to compete for binding sites.…”