of a Highly Sensitive Probe for Use in the Detection of
Toxins Responsible for Diarrhetic Shellfish Poisoning
Honkanen, Richard E.
Marine Fisheries Service, National Program Office, 1315
East West Highway, Silver Spring, MD 20910. PHONE:
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.