A fluorimetric microplate assay for detection and quantitation of toxins causing paralytic shellfish poisoning.
Paralytic shellfish poisoning is one of the most severe forms of food poisoning. The toxins responsible for this type of poisoning are metabolic products of dinoflagellates, which block neuronal transmission by binding to the voltage-gated Na(+) channel. Accumulation of paralytic toxins in shellfish is an unpredictable phenomenon that necessitates the implementation of a widespread and thorough monitoring program for mollusk toxicity. All of these programs require periodical collection and analysis of a wide range of shellfish. Therefore, development of accurate analytical protocols for the rapid determination of toxicity levels would streamline this process. Our laboratory has developed a fluorimetric microplate bioassay that rapidly and specifically determines the presence of paralytic shellfish toxins in many seafood samples. This method is based on the pharmacological activity of toxins and involves several steps: (i) Incubation of excitable cells in 96 well microtiter plates with the fluorescent dye, bis-oxonol, the distribution of which across the membrane is potential-dependent. (ii) Cell depolarization with veratridine, a sodium channel-activating toxin. (iii) Dose-dependent inhibition of depolarization with saxitoxin or natural samples containing paralytic shellfish toxins. Measuring toxin-induced changes in membrane potential allowed for quantification and estimation of the toxic potency of the samples. This new approach offers significant advantages over classical methods and can be easily automated.
Louzao
Maria Carmen
MC
Departamento de Farmacologia, Facultad de Veterinaria de Lugo, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
Rodriguez Vieytes
Mercedes
M
Garcia Cabado
Ana
A
Vieites Baptista De Sousa
Juan Manuel
JM
Botana
Luis Miguel
LM
D000818:Animals; D001681:Biological Assay; D049872:Bivalvia / Q000737:chemistry / Q000469:parasitology*; D004141:Dinoflagellida; D005470:Fluorometry / Q000379:methods; D005506:Food Contamination / Q000032:analysis; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D008297:Male; D008387:Marine Toxins / Q000032:analysis* / Q000633:toxicity; D008564:Membrane Potentials; D051379:Mice; D010243:Paralysis / Q000139:chemically induced; D015203:Reproducibility of Results; D012530:Saxitoxin / Q000032:analysis / Q000633:toxicity; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000469:parasitology / Q000633:toxicity; D057096:Shellfish Poisoning; D015222:Sodium Channels / Q000187:drug effects; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014701:Veratridine / Q000494:pharmacology
D008387:Marine Toxins; D015222:Sodium Channels; D006096:Gramicidin; D012530:Saxitoxin; D014701:Veratridine