Pubblicazione scientifica

Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

FILONE TECNOLOGICO	TEMA
trasformazione e commercializzazione

RIASSUNTO

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.

DATA

Data di pubblicazione: 13/04/2016

AUTORI

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
FLORES QUINTANA HAROLD A
MORRIS JAMES A JR
LITAKER R WAYNE

ENTE DI AFFERENZA

NATL OCEANIC & ATMOSPHERIC ADMIN CTR COASTAL FISHERIES & HABITAT RSCH BEAUFORT NORTH CAROLINA USA
UNIV NORTH CAROLINA AT WILMINGTON MARBIONC AT CREST RSCH PARK WILMINGTON NORTH CAROLINA USA
SEATOX RSCH INC
INSTITUT LOUIS MALARDE ILM –UMR 241 EIO LABORATORY OF TOXIC-MICROALGAE PAPEETE TAHITI FRENCH POLYNESIA
JHT INC ORLANDO FLORIDA USA
NORTH CAROLINA STATE UNIV ENVIRONMENTAL CHEMISTRY & TOXICOLOGY LAB RALEIGH NORTH CAROLINA USA
US FOOD & DRUG ADMIN
UNIV LAGUNA SPAIN

RIVISTA

PLoS ONE, 11(4), e0153348

IMPACT FACTOR

2,870

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