Development of a receptor-based assay for the detection of cyclic imines using Luminex technology

LP Rodriguez1, N Vilariño1, A Crespo1, C Bueno1, J Molgó2, R Araoz2, LM Botana1. 1Universidad de Santiago de Compostela, Facultad de Veterinaria, Farmacología y Terapéutica, 27002, Spain, 2Institut Fédératif de Neurobiologie Alfred Fessard2, Gif sur Yvette Cedex, 91198, France

Spirolides are biologically active macrocycles isolated for the first time from aquaculture sites from Nova Scotia, Canada, during 1990. These marine phycotoxins are produced by the dinoflagellate Alexandrium ostenfeldii. Although their toxicity to humans and their mode of action are still under investigation, these compounds displayed "fast-acting" toxicity in the traditional bioassay. This work was aimed at the development of a receptor-based detection method for spirolides using a solid-phase microsphere assay. Several alternatives were considered as binding proteins for the detection assay: the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata (an electric ray), the acetylcholine binding protein from Lymnaea stagnalis (AChBP), and human acetylcholine muscarinic receptors. Binding of the 13-desmethyl spirolide C to the three proteins was studied using competition assays and the results demonstrated that the nAChR and the AChBP bound the spirolides efficiently, while the affinity of the spirolide for muscarinic receptors was lower. A fluorescent receptor-based assay was developed using the immobilization of the binding protein on the surface of microspheres. An inhibition assay was designed using the competition of the spirolides with biotin-α-bungarotoxin (BTX), a toxin from the venom of the snake Bungarus mulcticinctus, for binding to nAChRs or AChBP. Finally the amount of BTX bound to the surface of the microspheres was quantified using phycoerythrin –labeled streptavidin. The fluorescence bound to the microspheres was analyzed in a Luminex 200 flow system. Both assays could detect 13-desmethyl spirolide C, but the crossreactivity profile of the nAChR for cyclic imines (spirolides and gymnodimine) matched better the relative toxicity reported for these toxins. The detection range of the nAChR assay was 0.01-500 nM of 13-desmethyl spirolide C, more sensitive than other receptor-based assays previously published. This receptor-based assay for Luminex systems provides a screening method faster, easier to perform and more sensitive than other receptor-based assays.