Development of a Highly Sensitive Probe for....
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GRANT NUMBER:  NA47FD0429           NMFS NUMBER:   93-WO-038

REPORT TITLE:  Development of a Highly Sensitive Probe for Use in the Detection of Toxins Responsible for Diarrhetic Shellfish Poisoning (DSP)

AUTHOR:  Honkanen, Richard E.

PUBLISH DATE: August 5, 1995

AVAILABLE FROM:  National Marine Fisheries Service, National Program Office, 1315 East West Highway, Silver Spring, MD 20910.  PHONE: (301) 713-2358


This study investigates the potential for developing 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 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 than engineered a clone of PP2A obtained from bovine brain for the production of recombinant PP2A in E. coli.  To date, we have been able to produce a clone expressing PP2A; however, the yield is not yet optimal. We have begun to assess methods for optimizing the yield of recombinant PP2A and maintaining stability of expression with passage. In conclusion, the PP2A assay looks promising for development into a reliable method of detecting DSP-toxins in shellfish. If employed, such a test 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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