A phosphatase inhibition assay- OkaTest – as a complementary test to the reference method for detection of lipophilic toxins in molluscs.

Escrito por admin el . Posteado en Agua/Water, Biotoxinas Marinas/Marine Biotoxins

OkaTest, a phosphatase inhibition assay, was successfully validated by 16 laboratories from 11 different countries in an international collaborative study. The test fulfils the method performance stipulated by the EURLMB (European Reference Laboratory for Marine Toxins) and therefore it can be used as a complementary test to the reference method for monitoring the OA-toxin group. Okadaic acid (OA) and its analogues DTX1 and DTX2, together with their ester forms are known as the OA-group toxins. The toxicity of these toxins is directly related to their inhibitory activity against a family of structurally related serine/threonine protein phosphatase (PP) present in the cells, in particular PP1 and PP2A. OkaTest is based on this strong inhibitory property for determination of toxin content in shellfish using the enzyme PP2A and a specific colorimetric substrate. The enzyme hydrolyses the substrate, and the product obtained can be determined by an absorbance measurement at 405 nm. As the ability of the PP to hydrolyse the substrate depends on the presence of OA and analogues in the samples, the toxin concentration can be calculated by using a standard curve. OkaTest is able to quantify the OA-group toxins in shellfish species such as mussels, clams, oysters and scallops. The assay is performed in a 96-wells microtiter plate and includes five ready-to-use standards. A total of 43 samples can be tested in about 2.5 hours. The performance characteristics of the kit were evaluated by the manufacturer in a Single Laboratory Validation and according to AOAC and Eurachem guidelines. A limit of detection and quantification of 44 and 56 µg/kg were calculated.

Okatest

Within-laboratory reproducibility values below 10% for mussel and scallop and recoveries between 78 and 114% were obtained. The kit was also evaluated by the EURLMB and a small scale intralaboratory study was carried out prior to a full validation study. A total of 8 materials, in blind duplicates, including mussels, scallops, clams and cockles were analysed by 16 laboratories from 9 different countries in a collaborative study to fully validate the assay. The overall mean values assigned for OA-toxins group for the test materials were 98.8, 175.4, 242.8, 255.0 and 275.0 µg total equivalents OA/kg. The estimated reproducibility standard deviation (SR) was from 10.7 to 23.2 µg/Kg, with reproducibility relative standard deviation (RSDR) values between 7.6 % and 13.2 % (mean 9.9%). The HORRAT values, the ratio between RSDR and a theoretically calculated RSDR, obtained were between 0.4 and 0.6. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for determination of the OA-toxins group. OkaTest can be used as an alternative or complementary test to the reference method (EC. No. 15/2011) for monitoring the OA-toxin group.