The inhibitory potency was examined by the PP2A assay. As shown in Figure 1, OA, DTX1, and pal-OA after hydrolysis but not before strongly inhibited the rhPP2Ac activity in dose-dependent manners at very low concentrations from 0.001 to 5 nM. On the other hand, PTX1 and YTX, the frequent concurrent toxins, did not inhibit rhPP2Ac at concentrations below 20 nM (Figure 2). Table 1 summarizes the IC₅₀ values for the toxins tested: OA, 0.095 ± 0.007 nM; DTX1, 0.104 ± 0.006 nM; and hydrolyzate of 7-O-palmitoyl-OA, 0.135 ± 0.009 nM. These results indicate the specificity of the assay toward the OAs and the absence of interference from PTX and YTX.

To verify the suitability of rhPP2Ac for use in the routine monitoring of DSP toxins, we performed a validation study. First, the quantifiable range of the PP2A assay was assessed from the calibration curve prepared with known concentrations of OA. From the calibration curve shown in Figure 3, the quantifiable range was deduced to be in a range 0-10 ng/mL.

The limit of detection (LOD = the average of matrix blanks plus 3 SD) and the limit of quantitation (LOQ = the average of matrix blanks plus 10 SD) were calculated from 6 measurements of the unhydrolyzed and hydrolyzed matrix blanks prepared from digestive glands (DG) of mussels and scallops (Table 2). With blanks from mussels, the LOD values were 0.0424 μg/g (unhydrolyzed) and 0.0476 μg/g (hydrolyzed), and the LOQ values were 0.0725 μg/g (unhydrolyzed) and 0.0932 μg/g (hydrolyzed). With the scallop extracts the LOD values 0.0217μg/g (unhydrolyzed) and 0.0274 μg/g (hydrolyzed), and the LOQ values were 0.0372 μg/g (unhydrolyzed) and 0.0415 μg/g (hydrolyzed). The values in the hydrolyzed blanks were slightly higher than before hydrolysis but were still low enough to ensure detection and quantification of OA at the regulation level proposed by EU, 0.16μg/g whole meat [4]. The two limits were slightly higher with hydrolyzed samples, which might be due to the hydrolysis of a small amount of ester toxins contaminated in DG. Thus, the assay can detect and quantify OA in a very lower level than that of the regulation level proposed by EU (0.16 μg/g whole meat).

To evaluate the accuracy of the method, the shellfish DGs of mussels and scallops were spiked with known six different amounts (0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 μg/g DG) of standard OA. The recovery of OA in mussels and scallops was 92.4-106% and 70.8-101%, respectively (Table 3). The low recovery (70.8%) scored in scallops spiked at a low dose (0.1μg) suggested an interfering substance.

To evaluate the reproducibility of the assay method, six separate extractions were performed on each of the DG samples of mussels and scallops spiked at three levels of OA (0.1, 0.2, and 0.4 μg/g DG). Each extract was evaluated by the PP2A assay in six separate experiments. The RSD from the six separate experiments in mussels and scallops was 4.8-6.5% and 5.1-7.1%, respectively (Table 4). The result demonstrates high reproducibility of the PP2A assay. Furthermore, DG of mussels were spiked with OA (0.1, 0.2, 0.3, 0.4, 0.5, and 1.0 μg/g), and each extract was analyzed with the PP2A inhibition assay and the LC/MS method. As shown in Figure 4, a good correlation was observed between the two methods (R² = 0.996) with the regression slope of 1.097. These data confirmed the reliability of the PP2A assay using rhPP2Ac as a method for detecting and quantifying OAs in shellfish.

OA was purchased from Wako (Osaka, Japan). DTX1, YTX and PTX1 were purified as reported previously [21]. 7-O-Palmitoyl-OA (pal-OA) was synthesized from OA according to the previous method [22]. The substrate used for the PP2A inhibition assay, p-nitrophenylphosphate (p-NPP), was purchased from Sigma (MO, USA).

rhPP2Ac was synthesized in insect cells (High Five; Invitrogen, Carlsbad, CA, USA) by infection of recombinant baculoviruses encoding His_×8-tagged human PP2Acα using a baculovirus expression system and purified as described previously [16]. The purity of the recombinant human PP2Ac was confirmed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassie brilliant blue R staining for visualization. The phosphatase activity assay using p-NPP as the substrate was performed as described previously [16]. One activity unit was defined as the amount of enzyme to release 1 nmol of phosphate from p-NPP per minute at 30 °C.

The experimental condition for the PP2A assay basically followed those of the previous method, except for the use of rhPP2Ac instead of a native dimeric enzyme [15]. The assay was carried out on 96-well plates. Each well contained 50 µl of lipophilic toxin and 100 µl of p-NPP (final concentration, 7.6 mM p-NPP per well). The reaction was started by adding 100 µl of the enzyme (final concentration, 0.08 units / well) and was continued for 30 min at 36 °C. The hydrolysis of p-NPP to p-NP was recorded on a microplate reader at 405 nm against 492 nm as a reference. The measurements were done in triplicate.

The scallops (Patinopecten yessoensis) and blue mussels (Mytilus galloprovincialis) were purchased from local fish markets in Hokkaido and Aichi Prefecture in Japan.

The digestive glands (DG) were separated from other tissues, minced, and a 2-g portion was used for extraction by Polytron homogenizer in 18mL of 90% methanol at room temperature for 1 min. The homogenate was centrifuged at 2,500g for 10 min, and a 50 μL of the supernatant (extract) was diluted with 950 μL of the sample dilution buffer to make a test solution. A 50-μL portion of the test solution was used for assaying of unhydrolyzed samples.

For hydrolysis and neutralization, the extract (500 μL) was mixed with 100 μL of 1.25N NaOH in a 2-mL plastic tube equipped with a screw cap. The cap was tightened and the tube was heated for 40 min at 80 °C, using a heating block. The reaction solution was cooled down to room temperature and neutralized by adding 100 μL of l.25N HCl. A 50 μL portion of the neutralized solution was diluted with 950 μL of the sample dilution buffer, and used for the PP2A inhibition assay. The OA content in the hydrolyzed samples was calculated by using a multiplying factor of 1.4 to calibrate the volume increase resulted by addition of alkali and acid solutions.

The PP2A inhibition assay was carried out on 96-well microplates. Each well was filled with 50 μL of the sample dilution buffer (blank), OA standard solution, or a test solution of shellfish extract. Then, 100 μL of the substrate solution (19mM p-NPP) and 100 μL of the enzyme solution (0.65 unit of PP2A) were added to each well. The microplate was sealed with an adhesive film, mixed for 1 min with a microplate mixer, and incubated for 30 min at 36 °C. The color development due to hydrolysis of p-NPP to pNP (p-nitrophenol) was measured with a microplate reader at 405 nm against 490 nm as reference within 10 min from the end of incubation. OA concentrations in shellfish extracts were determined by interpolating the absorbance values of the samples from the linear portion of the standard curve.

LC-MS analysis was performed on a model 1100 liquid chromatograph (Agilent) coupled to a 4000Qtrap mass spectrometer (Applied Biosystems/MDS Sciex). Separation was carried out on a Capcellpak C18 MGII column (3 μm, ϕ2.1 x 100 mm, Shiseido, Japan) using gradient elution at 40 °C. Eluent A was water and B was acetonitrile- methanol (8:2, v/v), both containing 2 mM ammonium formate and 50 mM formic acid. Gradient elution from 40% to 100% B was performed over 20 min and then held at 100% B for 20 min for LC separation. The flow rate was 0.2 mL/min and the injection volume was 5 μL. Electrospray ionization in the negative mode was used. OA was monitored on the deprotonated [M-H]-ions at m/z 803 and the peak intensity was expressed in peak area. The OA content in sample extracts was quantified using a calibration curve prepared with reference OA. All samples and standards were injected three times in a continuous LC-MS run and the average response used.