title: A competitive ELISA to detect brevetoxins from Karenia brevis (formerly Gymnodinium breve) in seawater, shellfish, and mammalian body fluid.
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 microg/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.
authors.affiliation: Center for Marine Science, University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409, USA.
authors.forename: Jerome
authors.initials: J
authors.lastname: Naar
authors.forename: Andrea
authors.initials: A
authors.lastname: Bourdelais
authors.forename: Carmelo
authors.initials: C
authors.lastname: Tomas
authors.forename: Julia
authors.initials: J
authors.lastname: Kubanek
authors.forename: Philip L
authors.initials: PL
authors.lastname: Whitney
authors.forename: Leanne
authors.initials: L
authors.lastname: Flewelling
authors.forename: Karen
authors.initials: K
authors.lastname: Steidinger
authors.forename: Johnny
authors.initials: J
authors.lastname: Lancaster
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Baden
mesh_terms: D000818:Animals; D000906:Antibodies; D001681:Biological Assay; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006041:Goats; D006433:Hemocyanins / Q000276:immunology; D008387:Marine Toxins / Q000032:analysis*; D051379:Mice; D010125:Ostreidae / Q000737:chemistry*; D035964:Oxocins; D012680:Sensitivity and Specificity; D012758:Shellfish; D014867:Water / Q000737:chemistry; D000095702:Polyether Toxins
chemical_list: D000906:Antibodies; D006433:Hemocyanins; D008387:Marine Toxins; D035964:Oxocins; D014867:Water; C041017:brevetoxin T17; D000095702:Polyether Toxins; C032808:keyhole-limpet hemocyanin