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 lin uron is described. Diuron haptens with two different polymethylene han dle locations were evaluated for:use as immunizing and/or tracer antig ens. One handle, consisting of three or five methylene groups, located at the terminal urea nitrogen;distal to the aromatic ring provided th e best-antibodies and enzyme-labeled haptens with a variety of selecti vities. The second methylene handle attachment at the internal urea ni trogen retained the basic structure of the target molecule, but was no t 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 ei ther a C3 or C5 methylene handle at the terminal nitrogen. The resulti ng selectivity of the antisera was not related to the immunizing antig en used. The analyses were class or compound selective for the individ ual herbicides depending upon the antisera used. Widely applied urea h erbicides such as diuron, monuron, and linuron demonstrated 50% inhibi tion values at 0.4, 0.5, and 0.8 mu g/L and detection limits of 0.04, 0.05, and 0.08 mu g/L, respectively, in buffer. The test, applied to m atrices such as water: and orange juice, showed limited matrix effects which could be eliminated by moderate dilution of the sample. The ass ays were tested,for tolerance to methanol, a commonly used solvent in the extraction of diuron from environmental matrices. Up to 50% methan ol in the assay had no effect on assay parameters such as the 50% inhi bition values. To develop a rational approach for the selection of a h apten for use as an enzyme tracer, a variety of monuron, diuron, and l inuron haptens were tested for cross reactivity. Some of these same ha ptens were then used as hapten enzyme conjugates. In general, haptens recognized about 100 times less well than the ''best'' hapten were use ful as enzyme tracers. In addition, we found that cross reactivity dat a for esters of carboxylic acid haptens may be better predictors of th e binding of the carboxylic acids when conjugated as antigens or enzym e tracers.