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Jerome Naar,¹ Andrea Bourdelais,¹ Carmelo Tomas,¹ Julia Kubanek,¹ Philip L. Whitney,² Leanne Flewelling,³ Karen Steidinger,³ Johnny Lancaster,¹ and Daniel G. Baden¹

¹Center for Marine Science, University of North Carolina at Wilmington, North Carolina, USA; ²NIEHS Marine and Freshwater Biomedical Science Center, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, Florida, USA; ³Florida Marine Research Institute, St. Petersburg, Florida, USA

Abstract

We developed a competitive enzyme-linked immunosorbent assay (ELISA) to analyze brevetoxins, using goat anti-brevetoxin antibodies obtained after immunization with keyhole limpet hemocyanin-brevetoxin conjugates, in combination with a three-step signal amplification process. The procedure, which used secondary biotinylated antibodies, streptavidine-horseradish peroxidase conjugate, and chromogenic enzyme substrate, was useful in reducing nonspecific background signals commonly observed with complex matrices. This competitive ELISA detected brevetoxins in seawater, shellfish extract and homogenate, and mammalian body fluid such as urine and serum without pretreatment, dilution, or purification. We investigated the application of this technique for shellfish monitoring by spiking shellfish meat with brevetoxins and by analyzing oysters from two commercial shellfish beds in Florida that were exposed to a bloom of Karenia brevis (formerly Gymnodinium breve) . We performed brevetoxin analysis of shellfish extracts and homogenates by ELISA and compared it with the mouse bioassay and receptor binding assay. The detection limit for brevetoxins in spiked oysters was 2.5 µg/100 g shellfish meat. This assay appears to be a useful tool for neurotoxic shellfish poisoning monitoring in shellfish and seawater, and for mammalian exposure diagnostics, and significantly reduces the time required for analyses. Key words: antibody, brevetoxin, detection, ELISA, immunoassay, neurotoxic shellfish poisoning, PbTx, seawater, serum, shellfish, urine. Environ Health Perspect 110:179-185 (2002) . [Online 17 January 2002]

http://ehpnet1.niehs.nih.gov/docs/2002/110p179-185naar/ abstract.html

Address all correspondence to D.G. Baden, Center For Marine Science, University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409 USA. Telephone: (910) 962-2302. Fax: (910) 962-2410. Email: badend@uncwil.edu

We acknowledge A. Weidner (Center for Marine Science) for excellent technical assistance with the ELISA, P. Scott (Florida Marine Research Institute) for performing mouse bioassays, and S. Campbell (Center for Marine Science) for editing the manuscript.

This work was supported by grant MR190 from the State of Florida. J.K. was supported as an O'Donnell fellow of the Life Sciences Research Foundation.

Received 1 August 2000 ; accepted 20 July 2001.