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.

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications

“…In addition, due to the lack of available purified toxins, very few in vitro dose response bioassays (e.g., RBA, CBA-N2A) have been conducted for C-CTXs, and they have rarely been compared to those of P-CTXs, for which the structure-activity relationship is better known [22,24]. The only available direct comparison between CTX3C and C-CTX1 was made using a fluorescent RBA, where similar binding dose response curves were found for the toxins, suggesting nearly equivalent toxicities (CTX3C 1.3 times more toxic than C-CTX1) [52]. Based on the very few studies in the literature [19,24,37], RBA relative potencies between P-CTXs and C-CTXs indicate a roughly 1:1 equivalence between CTX3C and C-CTX1.…”

An Updated Review of Ciguatera Fish Poisoning: Clinical, Epidemiological, Environmental, and Public Health Management

“…Alternative methods for CTX detection in fish include receptor binding assays [28,117,125,161,162,163,164,165], various LC-MS methods [3,9,160,162,166,167,168,169,170,171], and other less frequently employed assay systems (see the review by Lewis, 2004 [172]). Folk methods for detecting CTXs in fish may have potential utility [173], but studies are needed to establish the validity of these approaches.…”

Scaling Up From Regional Case Studies to a Global Harmful Algal Bloom Observing System

“…Precise toxin detection and quantification is possible by LC-MS in accredited laboratories and by fluorescence-based receptor binding assay (RBA-F), Hardison et al, 2016), an alternative method used to screen fish samples for ciguatoxins in facilities that are not certified to use isotopes. However, the development of affordable, reliable, and fast detection methods available to the general population and medical community is fundamental.…”