Factors Influencing the Toxicity, Detoxification and Biotransformation of Paralytic Shellfish Toxins - Reviews of Environmental Contamination and Toxicology - page 13

5.1.3 Receptor Binding Assay

The receptor binding assay was fi rst introduced in 1984 (Davio and Fontelo 1984 )
and was later developed into a Microtiter plate format (Vieytes et al. 1993 ; Doucette
et al. 1997 ). This assay is based on the interaction between the toxins and one of the
pharmacological targets (viz., site 1) of the sodium channel. This assay is a competitive
displacement assay, in which radiolabelled tritiated saxitoxin ( 3 H-STX) competes with
unlabeled STX and its derivatives for a given number of receptor sites of the voltage-
gated sodium channel in a rat membrane preparation. When binding equilibrium is
established in this assay, the radiolabelled saxitoxin is quantifi ed by liquid scintillation
counter. The percentage of receptor-bound radiolabelled saxitoxin is directly propor-
tional to the amount of unlabeled toxin present in a standard or in a sample. Receptor
binding affi nity occurs in the descending order of STX, GTX1/4, neoSTX, GTX2/3,
dcSTX, GTX5, which is similar to the order of toxicity obtained via the MBA. Because
the affi nity of a toxin for a receptor is directly proportional to its potency, this method
yields a response that is representative of the integrated potencies of all PSP toxins
present, and can thus be correlated with human response. Using the Microtiter plate
format, a detection limit of about 4 ʼ g STXeq/mL can be achieved (Doucette et al.
1997 ). However, as in the case of in vitro cell assays, this method does not provide
information on the specifi c toxin composition, and is limited by its requirement for
a scintillation counter and radioisotopic forms of the toxins being assayed.

5.2 Chemical Assay Methods

5.2.1 High Performance Liquid Chromatography (HPLC) with Fluorescence

High performance liquid chromatography (HPLC) is a chemical assay method that
relies on the separation of toxins by using ion-interaction chromatography and a
post-column reactor. In this method, the column effl uent is oxidized to produce
readily detectable derivatives for analysis by absorption or fl uorescence detectors.
HPLC with fl uorescence is the most commonly used method for PST analysis. Two
types of chromatographic methods (pre and post column derivatization) are
employed, and each involves treating (derivatizing) the columns in a way that opti-
mizes toxin separation. Using both methods requires an entire set of STX analogs to
be prepared as external reference (standards). However, standards prepared by dif-
ferent suppliers produce variability of up to 20% in STX concentration response
( Quilliam and Janecek 1993 ). Therefore, to achieve quality results, selecting a reli-
able supplier of standards is critical and the standard selected must be comparable
to certifi ed toxin standards that are commercially available from the National
Research Council Halifax, Canada. The fi nal step in this analysis is to calculate the
net toxicity (expressed in g STXeq) from the molar specifi c potencies (MU mol −1 )
of individual PSP toxins (Oshima 1995 ).