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Further Development of a Highly Sensitive Assay....
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GRANT NUMBER:  NA67FD0098          NMFS NUMBER:   95-WO-012

REPORT TITLE:  Further Development of a Highly Sensitive Assay for the Detection of Toxins Responsible for Diarrhetic Shellfish Poisoning (DSP)

AUTHOR:  Honkanen, Richard E.

PUBLISH DATE: May 5, 1997

AVAILABLE FROM:  National Marine Fisheries Service, Southeast Fishery Science Center, 219 Fort Johnson Road, Charleston, SC 29412.   PHONE: (803) 762-8558

ABSTRACT

This study investigated the development of an enzyme based assay (PP2A-assay) into a rapid and reliable test for the detection of DSP-toxins in shellfish. The PP2A-assay is based on a unique pharmacological property of the major DSP-toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX-1), which bind to and inhibit the activity of certain protein phosphatases (i.e. PP1 and PP2A).  Since the harmful effects of OA and DTX-1 originate from their ability to inhibit protein phosphatases, inhibition of PP2A/PP1 activity correlates well with toxicity. The initial step in developing the PP2A assay was to determine the maximal sensitivity and reliability under highly controlled conditions.   Studies with pure toxins indicate that the PP2A assay is capable of detecting as little as 10-20 pg of OA or DTX-1.  Next, the sensitivity and reliability of the PP2A assay for detecting DSP-toxins in a whole shellfish matrix was assessed with a series of spike recovery experiments.  Results from these experiments indicated that the assay was effective at detective at detecting toxins in crude methanol extracts of whole oysters. To further validate the effectiveness of the PP2A assay, a direct comparison was made with a HPLC-based method. Again, the PP2A assay proved to be sensitive and reliable. In an attempt to make the assay more cost effective, we then engineered a clone of PP2A obtained from bovine brain for the production of recombinant PP2A in E. coli. We were not satisfied with the yield so we made a chimera, by cloning the N-terminal of PP1 and the toxin binding domain of PP2A "making a hybrid" enzyme called CHRM2. This allowed us to produce a enzyme that could be expressed in large amounts (like PP1) yet still had very high sensitivity to OA (like PP2A). We then assessed methods for optimizing the yield of CHRM2, and repeated the sensitivity and reliability studies to confirm that the chimera was functioning like endogenous PP2A. In conclusion, the PP2A-assay looks promising for development into a reliable method of detecting DSP-toxins in shellfish. The development of a cost effective recombinant enzyme for use in the assay (CHRM2) was an essential step in the development the assay, and this has been accomplished. These studies suggest that further development of the PP2A-assay, if developed to the point where is can be employed, will be a sensitive and reliable test for the detection of DSP. This should benefit the shellfish industry through the increased consumer confidence provided by a reliable method of toxin monitoring and the general public through improved seafood safety.

 
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