A Highly Sensitive and Rapid ELISA for the Arylurea Herbicides Diuron, Monuron, and Linuron

A highly sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for the detection of the arylurea herbicides monuron, diuron, and linuron is described. Diuron haptens with two different polymethylene handle locations were evaluated for use as immunizing and/or tracer antigens. One handle, consisting of three or five methylene groups, located at the terminal urea nitrogen distal to the aromatic ring provided the best antibodies and enzyme-labeled haptens with a variety of selectivities. The second methylene handle attachment at the internal urea nitrogen retained the basic structure of the target molecule, but was not recognized by the antibodies produced from the terminal substituted haptens, nor was it useful as an enzyme tracer. Rabbits were immunized with monuron hapten-bovine serum albumin conjugates that contained either a C3 or C5 methylene handle at the terminal nitrogen. The resulting selectivity of the antisera was not related to the immunizing antigen used. The analyses were class or compound selective for the individual herbicides depending upon the antisera used. Widely applied urea herbicides such as diuron, monuron, and linuron demonstrated 50% inhibition values at 0.4,0.5, and 0.8 pg/L and detection limits of 0.04,0.05, and 0.08 pg/L, respectively, in buffer. The test, applied to matrices such as water and orange juice, showed limited matrix effects which could be eliminated by moderate dilution of the sample. The assays were tested for tolerance to methanol, a commonly used solvent in the extraction of diuron from environmental matrices. Up to 50% methanol in the assay had no effect on assay parameters such as the 50% inhibition values. To develop a rational approach for the selection of a hapten for use as an enzyme tracer, a variety of monuron, diuron, and linuron haptens were tested for cross reactivity. Some of these same haptens were then used as hapten enzyme conjugates. In general, haptens recognized about 100 times less well than the “best” hapten were useful as enzyme tracers. In addition, we found that cross reactivity data for esters of carboxylic acid haptens may be better predictors of the binding of the carboxylic acids when conjugated as antigens or enzyme tracers.