A non-radioactive ligand-binding assay for detection of cyanobacterial anatoxins using Torpedo electrocyte membranes.
Anatoxin-a (ANTX) and homoanatoxin-a (HANTX), neurotoxins exclusively produced by cyanobacteria (LD(50) 200-250 microg kg(-1), i.p. mouse), are agonists of the nicotinic acetylcholine receptors (nAChRs) to which they tightly bind. We have exploited the high affinity of these neurotoxins for the nicotinic receptors to develop a non-radioactive ligand-binding assay using Torpedo electrocyte membranes and biotinylated alpha-bungarotoxin (Biotin-BgTx) as tracer for detection of this class of toxins. The affinity of the Torpedo nAChRs for Biotin-BgTx was determined by chemiluminescence (K(d)=1.2 x 10(-8)M Biotin-BgTx) or color development (K(d)=3.5 x 10(-8)M Biotin-BgTx). Binding of ANTX or HANTX to the nAChRs competitively inhibits the binding of Biotin-BgTx to the receptors in a concentration-dependent manner (chemiluminescence: IC(50): 6.2 x 10(-8)M ANTX; color development: IC(50): 1.7 x 10(-8)M ANTX). The proposed method was validated by HPLC/MS with detection in the single ion recording mode. The non-radioactive ligand receptor-binding assay was successfully applied to the analysis of extracts prepared from cyanobacteria in culture and from natural habitats, as well as from aqueous samples. This method is suitable for ANTX and HANTX early survey of environmental samples since it requires minimal manipulations, is highly sensitive and gives consistent signal-to-noise ratios.
Aráoz
Rómulo
R
CNRS, Institut de Neurobiologie Alfred Fessard - FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire - UPR9040, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France. araoz@nbcm.cnrs-gif.fr
Herdman
Michael
M
Rippka
Rosmarie
R
Ledreux
Aurélie
A
Molgó
Jordi
J
Changeux
Jean-Pierre
JP
Tandeau de Marsac
Nicole
N
Nghiêm
Hoàng-Oanh
HO
D000818:Animals; D001427:Bacterial Toxins / Q000032:analysis*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000032:analysis*; D002038:Bungarotoxins / Q000378:metabolism / Q000494:pharmacology; D003116:Color; D000087522:Cyanobacteria Toxins; D008163:Luminescent Measurements; D013058:Mass Spectrometry; D011978:Receptors, Nicotinic / Q000378:metabolism; D014101:Torpedo / Q000378:metabolism*; D014326:Tropanes / Q000032:analysis*
D001427:Bacterial Toxins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; D000087522:Cyanobacteria Toxins; D011978:Receptors, Nicotinic; D014326:Tropanes; C524097:homoanatoxin-a; C509783:anatoxin a