18 A Comparison of Assay Techniques for the Analysis of Diarrhetic Shellfish. . .
207
Toxin Extraction and Analysis
Toxins were analysed using commercially available kits. Both immunoassay kit
(DSP ELISA, Abraxis) and an enzymatic protein phosphatase (PP2A) kit (OKATEST, ZEU-Inmunotec, Spain) were used for the detection of DSP toxins in the
mussel extracts. The toxins were extracted from the shellfish homogenates using
the manufacturer’s instructions supplied with each kit. Briefly, DSP ELISA extracts
were prepared by vortex mixing 1 g of mussel flesh with 9 ml 80 % (v/v) methanol
followed by centrifugation (3,000 g for 10 min). Cleaned methanolic shellfish
extracts were used for toxin analysis after filtration through 25 mm 3 m pore
size filter (Whatman, GF/C). PP2A (OKATEST) extracts were prepared in a similar
manner by vortex mixing 5 g mussel flesh with 25 ml 100 % (v/v) methanol in
a 50 ml centrifuge tube followed by centrifugation (2,000 g for 10 min at 4
ı C).
In 2009 the performance of the DSP ELISA kit only was used on relatively fresh
extracts, a decision made on logistical grounds in the initial investigative period.
Both methods were applied in 2010.
Both protocols were modified using an additional hydrolysis step in order to
quantify the total DSP toxin content including esters and DTX-3. Extracts were
diluted using sample dilution buffer supplied. All extracts were hydrolysed as part
of the procedure and diluted accordingly.
Assays were carried out in 96-well microtitre plates supplied with the kits and
incubated according to the manufacturers’ instructions. Both assays operate on a
colour reaction, the intensity being inversely proportional to the concentration of
toxin present in the sample. Absorbance readings of the test mixtures and calibration
standards were performed at 450 nm for the DSP ELISA and 405 nm for the
DSP OKATEST using a plate reader (Biotek) with Gen5 software. Results were
expressed as the concentration of okadaic acid and its equivalents, i.e. okadaic
acid (OA) and its derivative dinophysistoxins DTX-1, DTX-2 and 7-O-acyl ester
derivatives (DTX-3). Toxin concentrations were determined by external calibration
using OA standards of known concentrations supplied with each kit.
Results
Levels of DSP toxins in mussel flesh from three monitoring sites in Killary Harbour
through the summer of 2009 are summarised in Fig. 18.2a. These results were
derived from LC-MS analysis as part of the Irish National Biotoxin Monitoring
Programme. Contamination of mussel flesh with DSP toxins appeared in mid
June and lasted through July until early August. DSP toxins levels rose to values
exceeding the EU Maximum Permitted Level (MPL) of 0.16 g OA eq g
1 on
22 June at the outer and middle sites and on 29 June at the inner site. DSP toxin
levels subsequently rapidly increased at all three sites to ca. 1.2 g OA eq g
1 on 5
July, with toxicity increasing faster at the outer and middle sites than the inner site
207
Toxin Extraction and Analysis
Toxins were analysed using commercially available kits. Both immunoassay kit
(DSP ELISA, Abraxis) and an enzymatic protein phosphatase (PP2A) kit (OKATEST, ZEU-Inmunotec, Spain) were used for the detection of DSP toxins in the
mussel extracts. The toxins were extracted from the shellfish homogenates using
the manufacturer’s instructions supplied with each kit. Briefly, DSP ELISA extracts
were prepared by vortex mixing 1 g of mussel flesh with 9 ml 80 % (v/v) methanol
followed by centrifugation (3,000 g for 10 min). Cleaned methanolic shellfish
extracts were used for toxin analysis after filtration through 25 mm 3 m pore
size filter (Whatman, GF/C). PP2A (OKATEST) extracts were prepared in a similar
manner by vortex mixing 5 g mussel flesh with 25 ml 100 % (v/v) methanol in
a 50 ml centrifuge tube followed by centrifugation (2,000 g for 10 min at 4
ı C).
In 2009 the performance of the DSP ELISA kit only was used on relatively fresh
extracts, a decision made on logistical grounds in the initial investigative period.
Both methods were applied in 2010.
Both protocols were modified using an additional hydrolysis step in order to
quantify the total DSP toxin content including esters and DTX-3. Extracts were
diluted using sample dilution buffer supplied. All extracts were hydrolysed as part
of the procedure and diluted accordingly.
Assays were carried out in 96-well microtitre plates supplied with the kits and
incubated according to the manufacturers’ instructions. Both assays operate on a
colour reaction, the intensity being inversely proportional to the concentration of
toxin present in the sample. Absorbance readings of the test mixtures and calibration
standards were performed at 450 nm for the DSP ELISA and 405 nm for the
DSP OKATEST using a plate reader (Biotek) with Gen5 software. Results were
expressed as the concentration of okadaic acid and its equivalents, i.e. okadaic
acid (OA) and its derivative dinophysistoxins DTX-1, DTX-2 and 7-O-acyl ester
derivatives (DTX-3). Toxin concentrations were determined by external calibration
using OA standards of known concentrations supplied with each kit.
Results
Levels of DSP toxins in mussel flesh from three monitoring sites in Killary Harbour
through the summer of 2009 are summarised in Fig. 18.2a. These results were
derived from LC-MS analysis as part of the Irish National Biotoxin Monitoring
Programme. Contamination of mussel flesh with DSP toxins appeared in mid
June and lasted through July until early August. DSP toxins levels rose to values
exceeding the EU Maximum Permitted Level (MPL) of 0.16 g OA eq g
1 on
22 June at the outer and middle sites and on 29 June at the inner site. DSP toxin
levels subsequently rapidly increased at all three sites to ca. 1.2 g OA eq g
1 on 5
July, with toxicity increasing faster at the outer and middle sites than the inner site