An enzyme immunoassay for the environmental monitoring of the herbicide bromacil

Competitive enzyme-linked immunosorbent assays (ELISAs) were devised for the environmental monitoring of the herbicide bromacil. The polyclonal antibodies used in this work were raised against two haptens. The bromacil molecule was derivatized at the N-1- and 6-methyl-positions to obtain these haptens with carboxyalkyl [(CH2)nCO2H] spacer arms. The antibodies have been examined in several immunoassay formats. Two additional haptens were also synthesized and used for the preparation of coating antigens and enzyme tracers. Some of the heterologous indirect ELISAs in a coating antigen format showed promising sensitivities and, with only a few exceptions, slight cross-reactivities with a series of bromacil metabolites and related compounds. The best sensitivity (IC50 = 0.25 ppb) and specificity were achieved with a system using antibodies derived from the hapten bearing the handle at the 6-methyl group (n = 1) and coating antigen synthesized from hapten with the bridging group at the N-1-position (n = 5). Further investigations were performed with this ELISA. Changing the pH value in the range 5-8.5 did not influence the sensitivity of the optimized assay. Human urine, however, exercised a strong effect on sensitivity, which varied from sample to sample. Organic solvents also affected assay sensitivity; nevertheless, IC50s remained below 11 ppb with solvent concentrations up to 12.5%. Water samples spiked with bromacil were analyzed by ELISA. The results showed excellent correlation to spiked amounts at levels of 0.1-160 ppb. Soil samples fortified with bromacil were extracted with 1% aqueous NaOH, and then the obtained solutions were simply diluted with the assay buffer and analyzed by ELISA. Recoveries in the concentration range 0.04-20 ppm were between 92.5 and 102.5%. The direct ELISAs in enzyme tracer formats did not perform better than the heterologous coating antigen assays. However, use of this format in homologous assays dramatically improved the sensitivity from poor inhibition to IC50s of 3-10 ppb.