Protein phosphatase inhibition assays for okadaic acid detection in shellfish: matrix effects, applicability and comparison with LC-MS/MS analysis

The applicability of the protein phosphatase inhibition assay (PPIA) to the determination of okadaic acid (OA) and its acyl derivatives in shellfish samples has been investigated, using a recombinant PP2A and a commercial one. Mediterranean mussel, wedge clam, Pacific oyster and flat oyster have been chosen as model species. Shellfish matrix loading limits for the PPIA have been established, according to the shellfish species and the enzyme source. A synergistic inhibitory effect has been observed in the presence of OA and shellfish matrix, which has been overcome by the application of a correction factor (0.48). Finally, Mediterranean mussel samples obtained from Rı´a de Arousa during a DSP closure associated to Dinophysis acuminata, determined as positive by the mouse bioassay, have been analysed with the PPIAs. The OA equivalent contents provided by the PPIAs correlate satisfactorily with those obtained by liquid chromatography–tandem mass spectrometry (LC–MS/MS).

Conjugation of genetically-engineered protein phosphatases to magnetic particles for okadaic acid detection

This work presents the functional characterisation of a protein phosphatase 2A (PP2A) catalytic subunit obtained by genetic engineering and its conjugation to magnetic particles (MPs) via metal coordination chemistry for the subsequent development of assays for diarrheic lipophilic marine toxins. Colorimetric assays with free enzyme have allowed the determination of the best enzyme activity stabiliser, which is glycerol at 10%. They have also demonstrated that the recombinant enzyme can be as sensitive towards okadaic acid (OA) (LOD=2.3μg/L) and dinophysistoxin-1 (DTX-1) (LOD=15.2μg/L) as a commercial PP2A and, moreover, it has a higher operational stability, which makes possible to perform the protein phosphatase inhibition assay (PPIA) with a lower enzyme amount. Once conjugated to MPs, the PP2A catalytic subunit still retains its enzyme activity and it can also be inhibited by OA (LOD=30.1μg/L).

Prediction of crude protein and classification of the growth stage of wheat plant samples from NIR spectra

Near-infrared spectroscopy (NIRS) was used to analyse the crude protein content of dried and milled samples of wheat and to discriminate samples according to their stage of growth. A calibration set of 72 samples from three growth stages of wheat (tillering, heading and harvest) and a validation set of 28 samples was collected for this purpose. Principal components analysis (PCA) of the calibration set discriminated groups of samples according to the growth stage of the wheat. Based on these differences, a classification procedure (SIMCA) showed a very accurate classification of the validation set samples : all of them were successfully classified in each group using this procedure when both the residual and the leverage were used in the classification criteria. Looking only at the residuals all the samples were also correctly classified except one of tillering stage that was assigned to both tillering and heading stages. Finally, the determination of the crude protein content of these samples was considered in two ways: building up a global model for all the growth stages, and building up local models for each stage, separately. The best prediction results for crude protein were obtained using a global model for samples in the two first growth stages (tillering and heading), and using a local model for the harvest stage samples.