title: Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors.
abstract: Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.
authors.affiliation: Abraxis LLC, Warminster, PA, USA. Electronic address: frubio@abraxiskits.com.
authors.forename: Fernando
authors.initials: F
authors.lastname: Rubio
authors.affiliation: Abraxis LLC, Warminster, PA, USA.
authors.forename: Lisa
authors.initials: L
authors.lastname: Kamp
authors.affiliation: Abraxis LLC, Warminster, PA, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Carpino
authors.affiliation: Abraxis LLC, Warminster, PA, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Faltin
authors.affiliation: U.S. Geological Survey, Organic Geochemistry Research Laboratory, Kansas Water Science Center, Lawrence, KS 66049, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Loftin
authors.affiliation: Centre de recherche CNRS de Gif-sur-Yvette, Institut Fédératif de Neurobiologie Alfred Fessard FR2118, Laboratoire de Neurobiologie et Développement UPR 3294, Gif sur Yvette 91198, France.
authors.forename: Jordi
authors.initials: J
authors.lastname: Molgó
authors.affiliation: Centre de recherche CNRS de Gif-sur-Yvette, Institut Fédératif de Neurobiologie Alfred Fessard FR2118, Laboratoire de Neurobiologie et Développement UPR 3294, Gif sur Yvette 91198, France. Electronic address: araoz@inaf.cnrs-gif.fr.
authors.forename: Rómulo
authors.initials: R
authors.lastname: Aráoz
mesh_terms: D000818:Animals; D003124:Colorimetry / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D005618:Fresh Water / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008387:Marine Toxins / Q000032:analysis* / Q000633:toxicity; D011978:Receptors, Nicotinic / Q000187:drug effects*; D054712:Salinity; D012623:Seawater / Q000737:chemistry*; D014101:Torpedo
chemical_list: D008387:Marine Toxins; D011978:Receptors, Nicotinic
keywords: Anatoxin-a; Cyclic imines; Nicotinic acetylcholine receptor; Pinnatoxin; Receptor binding assay; nAChRs