A rapid hemolysis assay for the detection of sodium channel-specific marine toxins

Abstract

Current methods of detection for fish and shellfish biotoxins in monitoring and research purposes are either labor intensive, expensive, require specialized techniques or all of the above. This paper reports on the development of a fairly sensitive, rapid, and inexpensive assay which detects the presence of compounds that affect the sodium channel. It is based on the principles of the mouse neuroblastoma tissue culture assay for sodium channel specific-biotoxins using red blood cells (RBCs) from the red tilapia ( Sarotherodon mossambicus). This assay has the potential to complement the use of live animal bioassay testing for marine toxins. Veratridine, a sodium channel activator and ouabain, an inhibitor of Na +/K + ATPase, both react with the tilapia RBCs by affecting the permeability of the cell's membrane. Saxitoxin (STX), its analogs, and tetrodotoxin (TTX) can inhibit the action of veratridine and ouabain leaving the cell morphologically normal. By sequencing the addition of veratridine and ouabain, with either the extracted samples, saxitoxin, tetrodotoxin, or ciguatoxin (CTX-a sodium channel activator) to the RBCs a sodium channel antagonist or activator can be detected. Results using pure concentrations of a sodium channel-specific toxin could be detected to inhibit hemolysis at a concentration of 0.3 μg/ml STX, 3.5 μg/ml for neo-STX, 3.0 μg/ml for GTX, and 5.0 μg/ml for TTX in the presence of ouabain and veratridine. CTX was detected at a concentration of 50 μg/ml. The RBCs from the red tilapia was used due to the fish's ability to osmoregulate its internal environment to survive in both fresh and saltwater. In addition, with growing opposition to live animal testing, this assay has been designed as a non-lethal means of testing for sodium channel affecting marine toxins. No test animals are sacrificed and blood may be drawn from the same fish for continued sample testing.