期刊
TOXICON
卷 75, 期 -, 页码 35-43出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.toxicon.2013.06.015
关键词
Cyclic imines; Nicotinic acetylcholine receptors; alpha-bungarotoxin; Competition assay; Single-laboratory pre-validation
资金
- FEDER
- Ministerio de Ciencia y Tecnologia, Spain [SAF2009-12581, AGL2009 13581-CO2-01, TRA2009-0189, AGL2010-17875]
- Xunta de Galicia, Spain [GRC 2010/10, PGDIT 07MMA006261PR, PGDIT (INCITE) 9MMA003261PR, 09261080PR, 2009/XA044, 10PXIB261254 PR]
- EU VIIth Frame Program [211326, 265896, 265409, 262649, 312184]
- Atlantic Area Programme (Interreg IVB Trans-national) Pharmatlantic
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-alpha-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 mu g 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. (C) 2013 Elsevier Ltd. All rights reserved.
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