Monensin, a polyether antibiotic of molecular weight 671 Da, was converted into a hemisuccinate and covalently linked to bovine serum albumin via the mixed anhydride method. Using this immunogen, polyclonal anti-monensin antibodies were raised in rabbits and monoclonal antibodies were prepared from mice. The specificity of the anti-monensin antibodies was examined by using several structural analogues as the immunogen and by performing direct binding and competitive microELISA assays on Terasaki plates. Rabbit polyclonal antibodies had a dissociation constant (KD) of 5.5 x 10(-8) M for monensin and reacted with nigericin, an antibiotic structurally related to monensin. In contrast, a mouse monoclonal antibody, 2H8, reacted only with monensin and had a much lower KD = 3 x 10(-8) M for monensin. Monoclonal antibody 2H8 was used to develop a competitive microELISA able to detect as little as 5 ng/ml of monensin in solution which corresponds to 75 pg or 110 fmol of this hapten per Terasaki well.