Harmful algal blooms (HABs) are becoming frequent occurrences off the coasts of the United States, with specific instances in the coastal waters of the west coast, the Gulf of Mexico, and the Pacific Ocean. Historically, HABs have been associated with fish kills and marine mammal mortalities; however, their effects on human health and economic loss due to HAB contamination of seafood are becoming more prevalent, with cost estimates around $82 million annually. The toxic effects of HABs are precipitated by the production of toxins by various species of marine algae and microorganisms, which are then consumed by filter feeding shellfish and finfish. Accumulation in significant quantities can result in toxicity and death in marine animals and humans. Many of the natural toxins produce by marine phytoplankton are heat and acid stable; therefore, cooking contaminated seafood does not eliminate the risk of poisoning. Currently, US state agencies monitor for the presence of toxic phytoplankton and when the cell count reaches a set level, shellfish beds and finfish are tested for the presence of toxin. When toxin levels reaches FDA set limits, fishery resources are closed. However, current methods for detection of marine toxins most commonly associated with seafood poisonings in the US have serious drawbacks, including lengthy assay time, high cost, animal usage, low sensitivity and/or sample throughput, or small working ranges. We have developed fluorescence based receptor binding assays (FBAs) for the toxins that cause ciguatera (ciguatoxins), neurotoxic shellfish poisoning (brevetoxins), and amnesic shellfish poisoning (domoic acid). These assays will be used as a rapid test alternative to current methods, as they have the advantages of lower cost, high sensitivity and lower animal usage. The FBAs are an improvement over the radioligand receptor binding assays (RBAs) for these toxins, as the fluorescence platform does not require the use of radioactivity and is thus safer and far less expensive. In our Phase I studies, we developed the proof-of- concept FBAs by conjugating fluorophores to the toxins, showing the fluorescent toxin conjugates retain binding to the receptors, and demonstrating the fluorescent toxin conjugates can detect the presence of standard dilution curves (pure samples of toxins). In the proposed Phase II studies, we will expand our proof- of-concept FBAs to accomplish the following goals: First, we will optimize the current FBA platform to solve two pain points: the need to keep components frozen and the use of animal parts. We will preserve the receptor components to allow for stable storage at room temperature. Shipping to our prototype customers, many of whom are overseas, is expensive and risky because of the need to keep components at -80C. We will also create a new receptor preparation derived from human cell lines, which will be renewable and eliminate the need for rat brains, thus following a societal and scientific trend, as well as NIEHS imperative, to reduce reliance on animals for toxicity testing. Secondly, we will fully validate our test kits for seafood samples (which are ?dirtier? than pure toxin samples) in comparison to competitor methods (e.g. ELISAs, HPLC, and LC/MS) for use as a commercial research tool. As 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 samples, FBAs 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.
This research is relevant to public health in that it involves the development of commercial test kits that are useful for the detection and quantification of marine neurotoxins that cause ciguatera, neurotoxic shellfish poisoning, and amnesic shellfish poisoning (ciguatoxin, brevetoxin, and domoic acid, respectively). Consumption of seafood contaminated by these toxins can lead to gastrointestinal dysfunction, sensory abnormalities, numbness, drowsiness, confusion, and incoherence. Rapid assays, such as the ones proposed to be developed in this study, are important means for protecting human health by alerting the population and quickly closing fisheries when there are outbreaks of the toxins and contaminated seafood, and resulting a more targeted approach to banning only those seafood species that retain toxins while lifting the bans on those that have cleared them. Our assay kits will provide a significant cost and time savings over traditional detection methods for marine toxins, and there are no other rapid tests available for ciguatoxin. A faster, less expensive and more sensitive test that can be correlated to human toxicity is greatly needed to protect human health, monitor fishery resources, and research marine toxin poisoning.
