Paralytic Shellfish Poisoning:

1
Paralytic Shellfish Poisoning Preparation of
Monoclonal Antibodies to Saxitoxins for use in
PSP Detection Thomas N. Stewart1, Randy Allen1,
R. Wayne Litaker2 and Pat Tester2 1 Mercury
Science Inc., Raleigh, NC  27607, USA 2
National Ocean Service, NOAA, Beaufort, North
Carolina 28516, USA

Congener R1 R2 R3 R4
STX H H H H
B1 H H H SO3-
GTX2 H OSO3- H H
C1 H OSO3- H SO3-
GTX3 H H OSO3- H
C2 H H OSO3- SO3-
Neo OH H H H
B2 OH H H SO3-
GTX1 OH OSO3- H H
C3 OH OSO3- H SO3-
GTX4 OH H OSO3- H
C4 OH H OSO3- SO3-
ABSTRACT Paralytic shellfish poisoning (PSP) is
a serious public health threat caused by the
consumption of shellfish contaminated with
saxitoxins (STX). Shellfish become toxic when
they consume miroalgae in the genus Alexandrium
which are the source of the STXs. Blooms of toxic
Alexandrium are spatially and temporally
variable. This makes it difficult to predict
when shellfish are likely to become contaminated.
The logical solution would be to monitor STX
levels in shellfish on a regular basis using HPLC
or LC_MS methods, but this is not practical given
the vast shoreline of Alaska which would need to
be sampled, the high cost per assay, and the
shipping and processing times involved. What is
needed to address this issue is a means of
directly testing for saxitoxins using an
inexpensive, rapid field test. Such kits
generally employ an enzyme-linked immunoassay
(ELISA) format. Developing ELISAs for STX,
however, is challenging because there are over 50
different structural forms (congeners) of
saxitoxin which cause varying degrees of
toxicity. Currently there are ELISA test kits on
the market, but these kits fail to detect a
number of toxic congeners which may be present in
shellfish. The goal of this project is to develop
monoclonal antibodies with specificities which
allow them to fully detect the toxic STX
congeners which are not detected using the
current kits. Having these antibodies should
allow development of novel ELISA assays capable
of improved detection of the STX levels in
shellfish, better protection of human health and
improved utilization of shellfish resources.
  Our approach is to prepare several STX
immu-nogens having different structures that
mimic the major classes of STX. We report here
the results of immune response to our initial STX
immunogen indicating successful immunization and
hybridoma production.
INTRODUCTION STX TOXICITY VARIES WITH STRUCTURE
The saxitoxins encompass a family of more than 50
related neurotoxins. The main difference among
congeners are how various side groups (H, OH,
SO3-, or OSO3-) are attached to the backbone
structure of the toxin. The chart on the right
illustrates the specific side group
configurations of the most common congeners found
in shellfish. Using novel immunogens, a panel
of antibodies that can detect STX congeners with
different key side groups can be produced. Once
developed and tested for cross-reactivity, the
resulting antibodies can be combined in an ELISA
assay capable of detecting the cumulative
toxicity of a wide range of STX congeners.
STX-1-BSA
hybridoma cell
INITIAL RESULTS SERA RESPONSE AND HYBRIDOMA
SELECTION
CONCLUSIONS

• A novel synthetic method to prepare STX
  immunogens and enzyme conjugates has been
  developed.
• An immune response and recognition of STX-HRP has
  been demonstrated and monoclonal antibodies to
  STX have been developed. These antibodies are
  currently being screened for cross reactivity to
  different STX congeners.
• Additional antibodies to different toxic
  congeners of STX will be produced.

ACKNOWLEDGEMENTS
This research was funded by North Pacific
Research Board Project 1118 Improved Detection
Kit for the Toxins Which Cause Paralytic
Shellfish Poisoning STX immunogens are
synthesized and generously provided by Dr.
Sherwood Hall Food Drug Administration, College
Park, MD 20740, USA
Figure 2. Hybridoma cells produced during the
fusion are assayed for recognition of STX. In
thi s photo, eight of the ninety six hybridoma
cell lines tested show binding to STX . These
hybridomas have been cloned and are currently
being evaluated for recognition of STX congeners.
Figure 1. The sera of mice immunized with
STX-1-BSA were tested for binding to STX-HRP
conjugate. In this assay, mouse 8 shows the
highest concentration of antibodies to STX. The
Control mouse has not been exposed to STX-1-BSA
and has no STX antibodies.
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