Phosphonic acid (trans-4-phosphono-2-butenic acid; TPB) was used as a generic hapten in order to generate broad specificity antibodies against a group of organophosphorus pesticides. The polyclonal antiserum showed, in an indirect enzyme-linked immunosorbent assay (ELISA) format, preferential binding toward pesticides containing unsaturated diethyl-phosphate functionalities rather than the equivalent thiophosphate or dimethyl structures. The level of detection in the ELISA using a heterologous system was investigated and showed a 20-fold improvement when a conjugate for which the antibody had lower affinity was immobilized on the plate. Biosensor assays using parathion as a standard indicated that the antibody had a relatively high dissociation rate, and reproducible cycles of regeneration were achieved. The potential for using TPB as a generic hapten is discussed.