Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

Type Article
Date 2016-04
Language English
Author(s) Hardison D. Ransom1, Holland William C.1, McCall Jennifer R.2, 3, Bourdelais Andrea J.2, Baden Daniel G.2, Darius H. Taiana4, Chinain Mireille4, Tester Patricia A.1, 5, Shea DamianORCID6, Quintana Harold A. Flores7, Morris James A., Jr.1, Litaker R. Wayne1
Affiliation(s) 1 : NOAA, Ctr Coastal Fisheries & Habitat Res, Beaufort, NC USA.
2 : Univ N Carolina, MARBIONC CREST Res Pk, Wilmington, NC 28401 USA.
3 : SeaTox Res Inc, UNCW CREST Res Pk, Wilmington, NC USA.
4 : ILM, UMR EIO 241, Lab Tox Microalgae, Papeete, Tahiti, France.
5 : JHT Inc, Orlando, FL USA.
6 : N Carolina State Univ, Environm Chem & Toxicol Lab, Raleigh, NC 27695 USA.
7 : US FDA, Div Seafood Sci & Technol, Gulf Coast Seafood Lab, Dauphin Isl, AL USA.
Source Plos One (1932-6203) (Public Library Science), 2016-04 , Vol. 11 , N. 4 , P. e0153348. (19p.)
DOI 10.1371/journal.pone.0153348
WOS© Times Cited 50

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 (R)-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 (R-2 = 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 (R)-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.

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Hardison D. Ransom, Holland William C., McCall Jennifer R., Bourdelais Andrea J., Baden Daniel G., Darius H. Taiana, Chinain Mireille, Tester Patricia A., Shea Damian, Quintana Harold A. Flores, Morris James A., Jr., Litaker R. Wayne (2016). Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. Plos One, 11(4), e0153348. (19p.). Publisher's official version : https://doi.org/10.1371/journal.pone.0153348 , Open Access version : https://archimer.ifremer.fr/doc/00664/77575/