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Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.
PLoS One. 2016; 11(4):e0153348.Plos

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.

Authors+Show Affiliations

National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America. SeaTox Research Inc, UNCW CREST Research Park, Wilmington, North Carolina, United States of America.University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America.University of North Carolina at Wilmington, MARBIONC at CREST Research Park, Wilmington, North Carolina, United States of America.Institut Louis Malardé (ILM)-UMR 241 EIO, Laboratory of Toxic-Microalgae, Papeete, Tahiti, French Polynesia.Institut Louis Malardé (ILM)-UMR 241 EIO, Laboratory of Toxic-Microalgae, Papeete, Tahiti, French Polynesia.National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America. JHT, Inc., Orlando, Florida, United States of America.North Carolina State University, Environmental Chemistry and Toxicology Laboratory, Raleigh, North Carolina, United States of America.U.S. Food and Drug Administration, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, Dauphin Island, Alabama, United States of America.National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.National Oceanic and Atmospheric Administration, Center for Coastal Fisheries and Habitat Research, Beaufort, North Carolina, United States of America.

Pub Type(s)

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

Language

eng

PubMed ID

27073998

Citation

Hardison, D Ransom, et al. "Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish." PloS One, vol. 11, no. 4, 2016, pp. e0153348.
Hardison DR, Holland WC, McCall JR, et al. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. PLoS One. 2016;11(4):e0153348.
Hardison, D. R., Holland, W. C., McCall, J. R., Bourdelais, A. J., Baden, D. G., Darius, H. T., Chinain, M., Tester, P. A., Shea, D., Quintana, H. A., Morris, J. A., & Litaker, R. W. (2016). Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. PloS One, 11(4), e0153348. https://doi.org/10.1371/journal.pone.0153348
Hardison DR, et al. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. PLoS One. 2016;11(4):e0153348. PubMed PMID: 27073998.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish. AU - Hardison,D Ransom, AU - Holland,William C, AU - McCall,Jennifer R, AU - Bourdelais,Andrea J, AU - Baden,Daniel G, AU - Darius,H Taiana, AU - Chinain,Mireille, AU - Tester,Patricia A, AU - Shea,Damian, AU - Quintana,Harold A Flores, AU - Morris,James A,Jr AU - Litaker,R Wayne, Y1 - 2016/04/13/ PY - 2016/01/21/received PY - 2016/03/28/accepted PY - 2016/4/14/entrez PY - 2016/4/14/pubmed PY - 2016/9/10/medline SP - e0153348 EP - e0153348 JF - PloS one JO - PLoS One VL - 11 IS - 4 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/27073998/Fluorescent_Receptor_Binding_Assay_for_Detecting_Ciguatoxins_in_Fish_ L2 - https://dx.plos.org/10.1371/journal.pone.0153348 DB - PRIME DP - Unbound Medicine ER -