Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow cytometry system.
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 a 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.
Universidad de Santiago de Compostela, Departamento de Farmacología, Facultad de Veterinaria, 27002 Lugo, Spain.
Laura P
LP
Rodríguez
Natalia
N
Vilariño
Jordi
J
Molgó
Rómulo
R
Aráoz
M Carmen
MC
Louzao
Palmer
P
Taylor
Todd
T
Talley
Luis M
LM
Botana
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002038:Bungarotoxins / Q000032:analysis* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006575:Heterocyclic Compounds, 3-Ring / Q000032:analysis / Q000378:metabolism; D006844:Hydrocarbons, Cyclic / Q000032:analysis / Q000378:metabolism; D007097:Imines / Q000032:analysis* / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008195:Lymnaea / Q000378:metabolism; D008863:Microspheres; D010799:Phycoerythrin / Q000737:chemistry; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism; D012758:Shellfish / Q000032:analysis; D013141:Spiro Compounds / Q000032:analysis* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism
C517170:13-desmethylspirolide C; C428944:AChBP protein, Lymnaea; D002038:Bungarotoxins; D002352:Carrier Proteins; D006575:Heterocyclic Compounds, 3-Ring; D006844:Hydrocarbons, Cyclic; D007097:Imines; D055767:Immobilized Proteins; D011978:Receptors, Nicotinic; D013141:Spiro Compounds; D010799:Phycoerythrin; D001710:Biotin; C407350:gymnodimine; D019809:Streptavidin