Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System

Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing a...

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Bibliographic Details
Published in:	Analytical chemistry (Washington) 2013-02, Vol.85 (4), p.2340-2347
Main Authors:	Rodríguez, Laura P, Vilariño, Natalia, Molgó, Jordi, Aráoz, Rómulo, Louzao, M. Carmen, Taylor, Palmer, Talley, Todd, Botana, Luis M
Format:	Article
Language:	eng
Subjects:	Animals Biological assays Biotin - chemistry Biotin - metabolism Bungarotoxins - analysis Bungarotoxins - metabolism Carrier Proteins - chemistry Carrier Proteins - metabolism Flow cytometry Flow Cytometry - methods Heterocyclic Compounds, 3-Ring - analysis Heterocyclic Compounds, 3-Ring - metabolism Hydrocarbons, Cyclic - analysis Hydrocarbons, Cyclic - metabolism Imines - analysis Imines - metabolism Immobilized Proteins - chemistry Immobilized Proteins - metabolism Life Sciences Lymnaea - metabolism Microspheres Neurons and Cognition Phycoerythrin - chemistry Protein Binding Proteins Receptors, Nicotinic - chemistry Receptors, Nicotinic - metabolism Rodents Shellfish - analysis Spiro Compounds - analysis Spiro Compounds - metabolism Streptavidin - chemistry Streptavidin - metabolism T cell receptors Torpedo - metabolism Toxins
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Summary:	Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing a receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay, two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin, and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however, the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops, and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10–6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive, and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.
ISSN:	0003-2700 1520-6882