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.