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