title: Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.
abstract: Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.
authors.affiliation: National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.
authors.forename: D Ransom
authors.initials: DR
authors.lastname: Hardison
authors.affiliation: National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.
authors.forename: William C
authors.initials: WC
authors.lastname: Holland
authors.affiliation: University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America.|SeaTox Research Inc, UNCW CREST Research Park, Wilmington, North Carolina, United States of America.
authors.forename: Jennifer R
authors.initials: JR
authors.lastname: McCall
authors.affiliation: University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America.
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Bourdelais
authors.affiliation: University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Baden
authors.affiliation: Institut Louis Malardé (ILM)-UMR 241 EIO, Laboratory of Toxic-Microalgae, Papeete, Tahiti, French Polynesia.
authors.forename: H Taiana
authors.initials: HT
authors.lastname: Darius
authors.affiliation: Institut Louis Malardé (ILM)-UMR 241 EIO, Laboratory of Toxic-Microalgae, Papeete, Tahiti, French Polynesia.
authors.forename: Mireille
authors.initials: M
authors.lastname: Chinain
authors.affiliation: National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.|JHT, Inc., Orlando, Florida, United States of America.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Tester
authors.affiliation: North Carolina State University, Environmental Chemistry and Toxicology Laboratory, Raleigh, North Carolina, United States of America.
authors.forename: Damian
authors.initials: D
authors.lastname: Shea
authors.affiliation: U.S. Food and Drug Administration, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, Dauphin Island, Alabama, United States of America.
authors.forename: Harold A Flores
authors.initials: HA
authors.lastname: Quintana
authors.affiliation: National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.
authors.forename: James A
authors.initials: JA
authors.lastname: Morris
authors.affiliation: National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.
authors.forename: R Wayne
authors.initials: RW
authors.lastname: Litaker
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D036841:Ciguatera Poisoning / Q000175:diagnosis*; D002922:Ciguatoxins / Q000302:isolation & purification*; D005399:Fishes / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D002922:Ciguatoxins