1 research outputs found
Coupling the <i>Torpedo</i> Microplate-Receptor Binding Assay with Mass Spectrometry to Detect Cyclic Imine Neurotoxins
Cyclic imine neurotoxins constitute
an emergent family of neurotoxins
of dinoflagellate origin that are potent antagonists of nicotinic
acetylcholine receptors. We developed a target-directed functional
method based on the mechanism of action of competitive agonists/antagonists
of nicotinic acetylcholine receptors for the detection of marine cyclic
imine neurotoxins. The key step for method development was the immobilization
of <i>Torpedo</i> electrocyte membranes rich in nicotinic
acetylcholine receptors on the surface of microplate wells and the
use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins
competitively inhibit biotinylated-α-bungarotoxin binding to <i>Torpedo</i>-nicotinic acetylcholine receptors in a concentration-dependent
manner. The microplate-receptor binding assay allowed rapid detection
of nanomolar concentrations of cyclic imine neurotoxins directly in
shellfish samples. Although highly sensitive and specific for the
detection of neurotoxins targeting nicotinic acetylcholine receptors
as a class, the receptor binding assay cannot identify a given analyte.
To address the low selectivity of the microplate-receptor binding
assay, the cyclic imine neurotoxins tightly bound to the coated <i>Torpedo</i> nicotinic receptor were eluted with methanol, and
the chemical nature of the eluted ligands was identified by mass spectrometry.
The immobilization of <i>Torpedo</i> electrocyte membranes
on the surface of microplate wells proved to be a high-throughput
format for the survey of neurotoxins targeting nicotinic acetylcholine
receptors directly in shellfish matrixes with high sensitivity and
reproducibility