title: High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.
abstract: 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.
authors.affiliation: Universidad de Santiago de Compostela, Departamento de Farmacología, Facultad de Veterinaria, 27002 Lugo, Spain.
authors.forename: Laura P
authors.initials: LP
authors.lastname: Rodríguez
authors.forename: Natalia
authors.initials: N
authors.lastname: Vilariño
authors.forename: Jordi
authors.initials: J
authors.lastname: Molgó
authors.forename: Rómulo
authors.initials: R
authors.lastname: Aráoz
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000818:Animals; D001681:Biological Assay; D001710:Biotin; D002038:Bungarotoxins / Q000032:analysis; D004141:Dinoflagellida / Q000737:chemistry; D005506:Food Contamination / Q000032:analysis*; D057166:High-Throughput Screening Assays / Q000379:methods*; D057230:Limit of Detection; D049449:Luminescence; D008163:Luminescent Measurements; D008387:Marine Toxins / Q000032:analysis*; D011978:Receptors, Nicotinic / Q000378:metabolism; D012758:Shellfish; D013141:Spiro Compounds / Q000032:analysis*; D019809:Streptavidin
chemical_list: C517170:13-desmethylspirolide C; D002038:Bungarotoxins; D008387:Marine Toxins; D011978:Receptors, Nicotinic; D013141:Spiro Compounds; D001710:Biotin; D019809:Streptavidin
keywords: BSA; Bovine serum albumin; CR; CV; Competition assay; Cyclic imines; HRP; LC–MS; LoD; MBA; MWCO; Nicotinic acetylcholine receptors; PBS; SD; SEM; Single-laboratory pre-validation; coefficient of variation; cross-reactivity; horseradish peroxidase; i.p.; intraperitoneal; limit of detection; liquid chromatography–mass spectrometry; molecular-weight-cutoff; mouse bioassay; nAChR; nicotinic acetylcholine receptor; phosphate-buffered saline; standard deviation; standard error of the mean; α-BTX; α-bungarotoxin