title: Solid-phase receptor-based assay for the detection of cyclic imines by chemiluminescence, fluorescence, or colorimetry.
abstract: The spirolides and gymnodimines are marine phycotoxins included in the group of cyclic imines. The toxicity of these compounds to humans is still unknown, although their toxicity by intraperitoneal injection in rodents is very high. A receptor-based method was developed using the competition of the 13-desmethyl spirolide C with biotin-labeled α-bungarotoxin for binding to nicotinic acetylcholine receptors and the immobilization of the α-bungarotoxin-receptor complex on streptavidin-coated surfaces. The quantification of the immobilized receptor can be achieved using a specific antibody. Finally, after the addition of a secondary antibody labeled with horseradish peroxidase, three alternative substrates of this enzyme generate a chemiluminescent, fluorescent, or colorimetric signal. The assay performs well in shellfish extracts and the detection range is 5-150 nM of 13-desmethyl spirolide C in shellfish extracts, which is at least 5 times more sensitive than the existing fluorescence polarization assay. This assay can also detect gymnodimine, although with 10 times lower sensitivity than the spirolide. The detection of cyclic imines with microplate assays would be useful for screening purposes in order to reduce the number of samples to be processed by bioassays or analytical methods.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 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: Alvaro
authors.initials: A
authors.lastname: Antelo
authors.forename: Mercedes R
authors.initials: MR
authors.lastname: Vieytes
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000906:Antibodies / Q000276:immunology; D001710:Biotin / Q000737:chemistry; D002038:Bungarotoxins / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D003500:Cyclization; D005454:Fluorescence Polarization / Q000379:methods*; D006575:Heterocyclic Compounds, 3-Ring / Q000032:analysis; D006844:Hydrocarbons, Cyclic / Q000032:analysis; D007097:Imines / Q000032:analysis*; D055767:Immobilized Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D013141:Spiro Compounds / Q000032:analysis; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism
chemical_list: C517170:13-desmethylspirolide C; D000906:Antibodies; D002038:Bungarotoxins; D006575:Heterocyclic Compounds, 3-Ring; D006844:Hydrocarbons, Cyclic; D007097:Imines; D055767:Immobilized Proteins; D011978:Receptors, Nicotinic; D013141:Spiro Compounds; D001710:Biotin; C407350:gymnodimine; D019809:Streptavidin