Development of a displacement immunoassay by exploiting cross-reactivity of a monoclonal antibody

An enzyme-linked immunosorbent assay-based displacement assay was developed for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Advantage was taken of the cross-reactivity of a monoclonal anti-2,4-D antibody toward 2-methyl-4-chlorophenoxyacetic acid (MCPA). MCPA was conjugated with bovine serum albumin (BSA), immobilized on the surface of a microtiter plate, and saturated with the anti-2,4-D antibody. Due to the low affinity of the antibody toward MCPA (cross-reactivity of approximately 30%), the addition of 2,4-D resulted in a displacement of the antibody. Remaining antibodies were subsequently detected using a peroxidase-labeled goat anti-mouse antibody. The detection limit was as low as 0.1 microgram/liter for 2,4-D, which complies with the European Union Drinking Water Directives. When 2,4-D-BSA was used instead of MCPA-BSA conjugates, no significant displacement of bound antibody was observed.