Bull. Environ. Contam. Toxicol. (1995)54:797-804 9 1995Springer-Verlag New York Inc. ~, Environmental ;Contamination ]and lrbxicology Determination of Domoic Acid by Two Different Versions of a Competitive Enzyme-Linked Immunosorbent Assay (ELISA) M. Osada*, L. J. Marks, J. E. Stewart Habitat Ecology Division, Biological Sciences Branch, Department of Fisheries and Oceans, Bedford Institute of Oceanography, P.O. Box 1006, Dartmouth, N.S. B2Y 4A2, Canada Received: 8 July 1994/Accepted:2 November1994 Domoic acid has become well known as the causative agent of a potent neuroexcitation (amnesic shellfish poisoning, ASP) since the intoxication episode arising (in 1987) from the consumption of cultured blue mussels which originated in Prince Edward Island and contained large concentrations of domoic acid (Wright et al. 1989; Todd 1993). Methods for determination of domoic acid have been required to monitor food and the environment and to conduct fundamental studies in the laboratory and the field. Determinations by biological assays (Iverson et al. 1989; Wright et al. 1989), liquid chromatography (LC), and high performance liquid chromatography (HPLC) (Quilliam et al. 1989; Rao et al. 1988; Wright et al. 1989; Lawrence et al. 1989; Pocklington et al. 1990) have contributed much to the surveys for and research on domoic acid. To add to the methods available, an enzyme-linked immunosorbent assay (ELISA) for domoic acid was developed by Newsome et al. (1991). They applied it to urine samples and blood sera and described the advantages of high specificity and quantitation inherent in this technique. The method, however, involved several steps and lacked the sensitivity we desired. This method, as originally described, depended on the physical immobilization of domoic acid on the microplate via a carrier protein. Recently, however, CovaLink NH microplates have been developed in which a projecting secondary amino group has been applied in ELISAs for coupling peptides, steroids, oligonucleotides, and DNA to the microplate well surface, directly and chemically (SOnderg~d-Andersen et al. 1990; Rasmussen 1990; Rasmussen et al. 1991; Yonezawa et al. 1993; Chevrier et al. 1993). The current study was undertaken to simplify and improve on the previous domoic acid ELISA method and has resulted in two different versions of the ELISA; these are based on a physical (Method-l) and a chemical (Method-2) immobilization of domoic acid. MATERIALS AND METHODS In Method-l, the microplates were coated with ovalbumin conjugated domoic acid prepared according to Newsome et al. (1991), (which depends on a physical *Permanent address: Laboratoryof AquaculturalBiology,Facultyof Agriculture, Tohoku University,Sendai981, Japan Correspondence to: J. E. Stewart 797