ENZYME-LINKED RECEPTOR ASSAY FOR SODIUM CHANNEL TOXINS

A LARGE NUMBER OF TOXINS (SUCH AS PARALYTIC SHELLFISH TOXINS, TETRODOTOXIN, AND BACTRACHOTOXIN) ARE POTENTIALLY LETHAL CONTAMINANTS IN SEAFOOD SAMPLES. THESE TOXINS ARE KNOWN TO ACT BY ALTERING THE CONDUCTANCE PROPERTIES OF A WELL-CHARACTERIZED SODIUM CHANNEL FOUND IN MUSCLES AND NERVES. IN THIS STUDY, WE WILL PREPARE AN ENZYME-LINKED ASSAY TO DETECT THESE TOXINS: ENZYME- OR COENZYME-TOXIN CONJUGATES WILL BE COUPLED WITH PURIFIED SODIUM CHANNELS FOR USE IN DISPLACEMENT ASSAYS TO QUANTITATE THE PRESENCE OF SITE 1 SODIUM CHANNEL TOXINS IN SAMPLES. SUCH ASSAYS SHOULD HAVE A SENSITIVITY COMPARABLE TO THE RADIOIMMUNOASSAYS CURRENTLY USED TO DETECT THESE TOXINS, BUT WILL AVOID THE HAZARDS OF USING RADIOISOTOPES, AND WILL ALSO HAVE A GREATLY IMPROVED CROSS-REACTIVITY, THUS ENABLING THIS ASSAY TO DETECT ANY SITE 1 TOXIN IN A SEAFOOD SAMPLE. IN PHASE I, WE PROPOSE TO PREPARE ENZYME AND COENZYME CONJUGATES OF SAXITOXIN AND TETRODOTOXIN, AND TEST THEM IN DISPLACEMENT ASSAYS USING PARTIALLY PURIFIED SODIUM CHANNELS. IN PHASE II, ENZYME-LINKED TOXINS WILL BE COMPARED TO RADIOLABELED TOXINS FOR BINDING AFFINITY TO THE SODIUM CHANNEL AND FOR SENSITIVITY IN ASSAYS TO DETECT UNLABELED TOXINS IN SAMPLES. ALSO, A RAPID COLOR-CHANGE ASSAY WILL BE DEVELOPED TO DETECT THESE TOXINS IN SEAFOOD SAMPLES.