Development of user-friendly fluorescence based assays for marine toxins

ABSTRACTHarmful algal bloomsHABsare becoming frequent occurrences off the coasts of the United Stateswith specific instances in the coastal waters of the west coastthe Gulf of Mexicoand the Pacific OceanHistoricallyHABs have been associated with fish kills and marine mammal mortalitieshowevertheir effects on human health and economic loss due to HAB contamination of seafood are becoming more prevalentwith cost estimates around $million annuallyThe toxic effects of HABs are precipitated by the production of toxins by various species of marine algae and microorganismswhich are then consumed by filter feeding shellfish and finfishAccumulation in significant quantities can result in toxicity and death in marine animals and humansMany of the natural toxins produce by marine phytoplankton are heat and acid stablethereforecooking contaminated seafood does not eliminate the risk of poisoningCurrentlyUS state agencies monitor for the presence of toxic phytoplankton and when the cell count reaches a set levelshellfish beds and finfish are tested for the presence of toxinWhen toxin levels reaches FDA set limitsfishery resources are closedHowevercurrent methods for detection of marine toxins most commonly associated with seafood poisonings in the US have serious drawbacksincluding lengthy assay timehigh costanimal usagelow sensitivity and or sample throughputor small working rangesWe have developed fluorescence based receptor binding assaysFBAsfor the toxins that cause ciguateraciguatoxinsneurotoxic shellfish poisoningbrevetoxinsand amnesic shellfish poisoningdomoic acidThese assays will be used as a rapid test alternative to current methodsas they have the advantages of lower costhigh sensitivity and lower animal usageThe FBAs are an improvement over the radioligand receptor binding assaysRBAsfor these toxinsas the fluorescence platform does not require the use of radioactivity and is thus safer and far less expensiveIn our Phase I studieswe developed the proof ofconcept FBAs by conjugating fluorophores to the toxinsshowing the fluorescent toxin conjugates retain binding to the receptorsand demonstrating the fluorescent toxin conjugates can detect the presence of standard dilution curvespure samples of toxinsIn the proposed Phase II studieswe will expand our proofof concept FBAs to accomplish the following goalsFirstwe will optimize the current FBA platform to solve two pain pointsthe need to keep components frozen and the use of animal partsWe will preserve the receptor components to allow for stable storage at room temperatureShipping to our prototype customersmany of whom are overseasis expensive and risky because of the need to keep components atCWe will also create a new receptor preparation derived from human cell lineswhich will be renewable and eliminate the need for rat brainsthus following a societal and scientific trendas well as NIEHS imperativeto reduce reliance on animals for toxicity testingSecondlywe will fully validate our test kits for seafood sampleswhich aredirtierthan pure toxin samplesin comparison to competitor methodse gELISAsHPLCand LC MSfor use as a commercial research toolAs RBAs have been used for a variety of sample matrices and have been shown to strongly correlate with the mouse bioassay and HPLC results in samplesFBAs are be the next progressive step in the detection of toxins in seafood or coastal water samples thereby protecting human health and aiding in the monitoring of fishery resources