Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.
The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.
Marine Biotoxins Program, Charleston Laboratory, U.S. National Marine Fisheries Service, SC 29412.
F M
FM
Van Dolah
E L
EL
Finley
B L
BL
Haynes
G J
GJ
Doucette
P D
PD
Moeller
J S
JS
Ramsdell
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002922:Ciguatoxins / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D008387:Marine Toxins / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D009467:Neuromuscular Depolarizing Agents / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D035964:Oxocins; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism; D011894:Rana pipiens; D051381:Rats; D017207:Rats, Sprague-Dawley; D000095702:Polyether Toxins
D002118:Calcium; D002922:Ciguatoxins; D007608:Kainic Acid; D008387:Marine Toxins; D009467:Neuromuscular Depolarizing Agents; D035964:Oxocins; C053342:brevetoxin; D000095702:Polyether Toxins; C012301:domoic acid