The development of a covalent enzyme-linked immunoassay (CELIA) using lipoic acid covalently bound to modified polystyrene microplates has permitted the detection, in the sera of normal BALB/c mice, of natural antibodies reacting with lipoic acid (LA). Hybridomas producing monoclonal anti-LA antibodies were obtained from splenocytes of non-immune BALB/c mice. Two of them, of IgM isotype, recognized LA but failed to react with dihydrolipoic acid (DHLA, the reduced form of LA), suggesting that the integrity of the dithiolane ring was of importance for antibody recognition. They did not give positive reactions with other disulfide linked biological molecules such as oxidized glutathione or cystine. Anti-LA antibodies, coated on polystyrene microplates, were used for the detection of free LA in a competitive assay based on peroxidase-LA conjugate.