Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in FishReport as inadecuate




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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 RBAR. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBAR in certain labs. A fluorescence based receptor binding assay RBAF 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 RBAF. Results for corresponding samples analyzed using the neuroblastoma cell-based assay CBA-N2a correlated well R2 = 0.71 with those of the RBAF, 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 RBAF 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 RBAF, 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 RBAF advantages include the long-term > 5 years stability of the BODIPY®- PbTx-2 and having similar results as the commonly used RBAR. The RBAF is cost-effective, allows high sample throughput, and is well-suited for routine CTX monitoring programs.



Author: D. Ransom Hardison , William C. Holland, Jennifer R. McCall, Andrea J. Bourdelais, Daniel G. Baden, H. Taiana Darius, Mireille Ch

Source: http://plos.srce.hr/



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