High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
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.
['Rodríguez|Laura P|LP|', 'Vilariño|Natalia|N|', 'Molgó|Jordi|J|', 'Aráoz|Rómulo|R|', 'Botana|Luis M|LM|']
D000818:Animals D001681:Biological Assay D001710:Biotin D002038:Bungarotoxins D004141:Dinoflagellida D005506:Food Contamination D057166:High-Throughput Screening Assays D057230:Limit of Detection D049449:Luminescence D008163:Luminescent Measurements D008387:Marine Toxins D011978:Receptors, Nicotinic D012758:Shellfish D013141:Spiro Compounds D019809:Streptavidin
Nicotinic acetylcholine receptors Nicotinic acetylcholine receptors nAChR horseradish peroxidase HRP coefficient of variation BSA Bovine serum albumin CR CV Competition assay Cyclic imines LC–MS LoD MBA MWCO PBS SD SEM Single-laboratory pre-validation cross-reactivity i.p. intraperitoneal limit of detection liquid chromatography–mass spectrometry molecular-weight-cutoff mouse bioassay nicotinic acetylcholine receptor phosphate-buffered saline standard deviation standard error of the mean α-BTX α-bungarotoxin