Development of monoclonal antibody-eased immunoassays to the N-methylcarbamate pesticide carbofuran

To produce monoclonal antibodies (MAbs) to the pesticide carbofuran, three compounds with carboxylic spacer arms of different lengths introduced at th e carbamate group of the analyte structure were synthesized, conjugated to proteins, and used as immunizing haptens in mice. MAbs were subsequently ch aracterized for affinity and specificity in the conjugate-coated format and in the antibody-coated format using newly synthesized compounds as heterol ogous assay haptens. Depending on the immunoreagent combination and assay f ormat, competitive assays with I-50 values in the 1.2-10.2 nM (0.27-2.27 ng /mL) range were obtained. LIB-BFNB67 MAb in combination with the hapten BFN H, coupled either to horseradish peroxidase or to ovalbumin, was used to de velop a direct and an indirect enzyme-linked immunosorbent assay, respectiv ely. Optimized immunoassays displayed Very similar analytical characteristi cs, with an I-50 value around 0.7 ng/mL and a limit of detection around 0.0 8 ng/mL. Both immunoassays were able to tolerate the presence of methanol u p to a 15% concentration. Compounds very similar in structure to carbofuran (benfuracarb, furathiocarb, bendiocarb, and carbofuran-hydroxy) exhibited cross-reactivity values in the 18-37% range, but major N-methylcarbamate pe sticides were not recognized by the MAb. These immunoassays should reasonab ly allow the rapid, low-cost, and sensitive determination of carbofuran in food, in soils, and in the environment at levels of regulatory and practica l importance.