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
ABSTRACT
2
National Ocean Service, NOAA, Beaufort, North Carolina 28516, USA
INTRODUCTION: STX TOXICITY VARIES WITH STRUCTURE
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.
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.
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-
METHODS: PREPARATION OF ANTIBODIES
1. IMMUNIZE:
Mice are immunized with a
derivative of saxitoxin
covalently attached to bovine
serum albumin. Since
potentially toxic metabolites can
be produced by the
immunogens, additional steps
are taken to identify
immunization conditions that
are not lethal.
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.
5. SELECT BEST
ANTIBODIES:
Sensitivity and
specificity of clones
are measured to
determine which
antibodies are most
suitable for STX assay
development.
Our approach is to prepare several STX immunogens 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.
STX-1-BSA
2. MEASURE RESPONSE:
Mice sera are tested for
immune response
producing antibodies to
saxitoxin. Several mice
are immunized since the
immune response can
vary greatly between
mice.
4. IDENTIFY CLONES:
Hybridoma cells producing antiSTX antibodies are identified and
cloned.
hybridoma cell
3. PERFORM FUSION:
A fusion is performed
on the spleen of the
mouse showing the
best response to the
immunogen and
hybridoma cells are
created. Hybridoma
cells are immortal and
produce monoclonal
antibodies.
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 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.
Learn more at www.mercuryscience.com
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.
email: info@mercuryscience.com