Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish

PLoS One. 2016 Apr 13;11(4):e0153348. doi: 10.1371/journal.pone.0153348. eCollection 2016.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Chromatography, Liquid
  • Ciguatera Poisoning / diagnosis*
  • Ciguatoxins / isolation & purification*
  • Fishes / metabolism*
  • Protein Binding
  • Rats
  • Rats, Sprague-Dawley
  • Synaptosomes / metabolism
  • Tandem Mass Spectrometry

Substances

  • Ciguatoxins

Grants and funding

Funding was provided by the National Oceanic and Atmospheric Administration's Ecology and Oceanography of Harmful Algae Blooms contribution #843 -- http://coastalscience.noaa.gov/research/habs/ecohab RWL PAT DRH WCH DS. Program funds were provided by the National Oceanic and Atmospheric Administration, National Ocean Service, Centers for Coastal Ocean Science, Center for Coastal Fisheries and Habitat Research- DRH WCH RWL PAT JAM. The UNCW partnership is in part funded through the Cooperative Institute for Ocean Exploration, Research, and Technology CIOERT, award number NA14OAR4320260, NOAA, DOC. Authors associated with this are DGB, AJB, and JRM. NOAA National Marine Fisheries, Office of International Affairs- United States Department of State- JAM. The commercial partner SeaTox Research Inc. was not responsible the design or execution of the project, nor did they provide salary support to any of the collaborators. Their role was limited to providing reagents and running the radioactive receptor binding assay using extracts prepared by DRH and WCH. JHT provided support in the form of salary for PT, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.