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.

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.