Development of a Solid-Phase Receptor-Based Assay for the Detection of Cyclic Imines Using a Microsphere-Flow Cytometry System

Abstract

Biologically active macrocycles containing a cyclic imine were isolated for the first time from aquaculture sites in Nova Scotia, Canada, during the 1990s. These compounds display a “fast-acting” toxicity in the traditional mouse bioassay for lipophilic marine toxins. Our work aimed at developing receptor-based detection method for spirolides using a microsphere/flow cytometry Luminex system. For the assay two alternatives were considered as binding proteins, the Torpedo marmorata nicotinic acetylcholine receptor (nAChR) and the Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP). A receptor-based inhibition assay was developed using the immobilization of nAChR or Ls-AChBP on the surface of carboxylated microspheres and the competition of cyclic imines with biotin-α-bungarotoxin (α-BTX) for binding to these proteins. The amount of biotin-α-BTX bound to the surface of the microspheres was quantified using phycoerythrin (PE)-labeled streptavidin and the fluorescence was analyzed in a Luminex 200 system. AChBP and nAChR bound to 13-desmethyl spirolide C efficiently; however the cross-reactivity profile of the nAChR for spirolides and gymnodimine more closely matched the relative toxic potencies reported for these toxins. The nAChR was selected for further assay development. A simple sample preparation protocol consisting of an extraction with acetone yielded a final extract with no matrix interference on the nAChR/microsphere-based assay for mussels, scallops and clams. This cyclic imine detection method allowed the detection of 13-desmethyl spirolide C in the range of 10–6000 μg/kg of shellfish meat, displaying a higher sensitivity and wider dynamic range than other receptor-based assays previously published. This microsphere-based assay provides a rapid, sensitive and easily performed screening method that could be multiplexed for the simultaneous detection of several marine toxins.

Synthesis and biology of cyclic imine toxins, an emerging class of potent, globally distributed marine toxins

“…138,139 c ) Microsphere receptor-based assay using flow cytometry was recently developed. 140 Torpedo -nAChR or AChBP from Lymnaea stagnalis –a structural surrogate of α7 nAChR – were immobilized on the surface of carboxylated microspheres. Following incubation of the coated microspheres with CI toxins and with biotinylated α-bungarotoxin, the mixture was filtered, and streptavidin-R-Phycoerythrin conjugate was added to detect the competitive binding inhibition by flow cytometry luminex.…”

“…Therefore, more than one nAChR subtype could be simultaneously analyzed. 140 d ) Microplate receptor binding assay based on the use of Torpedo electrocyte membranes rich in nAChRs, biotin-α-bungarotoxin as non-radioactive tracer, and streptavidin-HRP for calorimetric detection was developed. 130 The key step of this assay was the immobilization of Torpedo -nAChR on the surface of a 96-wells microplate providing a high throughput format for routine detection of neurotoxins targeting nAChRs in environmental samples, with minimal sample handling, high sensitivity, reduced matrix effect, and low cross-reactivity.…”

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods

“…This microsphere-based immunoassay for PLTX-like molecules performs similarly, in terms of sensitivity, to previously published immuno-detection methods using the same anti-PLTXmAb [28][29][30], and it is 100 times more sensitive than the fluorescence polarization receptorbased method [27]. This single, semi-quantitative screening tool is based on the use of a commercially available technology, Luminex, that offers the possibility of combining different single-detection assays, as those developed for some aquatic toxins [38][39][40], in multi-detection methods [33,34], which would allow saving precious amounts of sample and experimental time.…”

Spirolides and Cyclic Imines: Toxicological Profile

“…Thus, several nonradioactive ligand-binding methods have been developed (Vilariño et al 2009;Fonfría et al 2010;Otero et al 2011;Aráoz et al 2012;Rodriguez et al 2013) to replace the traditional mouse bioassay which has numerous drawbacks related to sensitivity and specificity, apart from the ethical concern of using large number of animals, as recently discussed (Daneshian et al 2013). …”