APPLICATION OF A POLYCLONAL ANTIBODY IN THE DEVELOPMENT OF METHODS FOR DETECTING DOMOIC ACID

Bich-Thuy L. Eberhart¹, Rick Stevens², Sylvester Spencer¹, John C. Wekell¹, Scott Solberg², Clem Furlong², and Vera L. Trainer¹

¹NOAA Fisheries, Marine Biotoxins Program, Northwest Fisheries Science Center, Seattle, WA 98112, USA
²Department of Genome Sciences and Medicine, Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA

Polyclonal antiserum raised in rabbits against ovalbumin (OVA)-domoic acid conjugate was employed in the development of methods to detect domoic acid (DA), a neurotoxin produced by diatoms of the genus Pseudo-nitzschia. DA molecule was coupled to the carrier protein (OVA) through one of its three carboxyl groups using a carbodiimide reaction. This immunogen produced an anti-DA serum that is sensitive and specific to free domoic acid. We have successfully used the resulting antibody in an indirect competitive enzyme-linked immunosorbent assay (cELISA) to determine DA levels in contaminated shellfish.

This antibody is also being tested in a new surface plasmon resonance (SPR) detector system developed at the University of Washington, which uses minute changes in surface refractive index to detect analytes. Recent advances in SPR sensor technology make it possible to use our anti-DA antibody to develop a small, compact, battery-operated system for real-time monitoring of DA in the field. The SPR detector system is a competition-based assay where a low concentration of antibody is exposed to the immobilized domoate conjugate attached on the gold sensor surface. A mixture of antibody plus sample or standard is then added to the flow cell over the sensor. Rates of antibody binding to the domoate immobilized on the sensor surface are determined in the absence and presence of varying concentrations of DA in the sample or standard. Since very dilute concentrations of antibody are needed for the competition assay, many assays may be carried out with small amount of antibody. We are presently testing the prototype unit for establishing DA standard curves, examining matrix effects and determining detection limits.