Article Dans Une Revue Toxicon Année : 2013

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

Résumé

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods.

Dates et versions

hal-00919891 , version 1 (17-12-2013)

Identifiants

Citer

Laura P Rodríguez, Natalia Vilariño, Jordi Molgó, Rómulo Aráoz, Luis M Botana. High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.. Toxicon, 2013, 75, pp.35-43. ⟨10.1016/j.toxicon.2013.06.015⟩. ⟨hal-00919891⟩
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