PP2A (protein phosphatase) technique appears satisfactory for the analysis of DSP toxins in shellfish and is recommended as a rapid assay method for screening and end product testing. This is one of the conclusions raised from a study carried out within the water project http://www.nppwater.com/index.php Mussel samples from the biotoxins monitoring program carried by the Irish Marine Institute, Galway, were analysed. Samples were extracted from 3 sites (inner, middle and outer) from Killary Harbour, Ireland, in 2009 and 2010. Mussels from a DSP event were tested by Mouse Bioassay (MBA), ELISA (Abraxis), LC-MS and protein phosphatase PP2A assay (OkaTest). ELISA kits showed matrix effects, but PP2A technique (OkaTest, ZEU-INMUNOTEC) was recommended as rapid assay. More information on http://www.nppwater.com/data_access.php?id=85
The MicroCystest plate kit has been recently evaluated within the Environmental Technology Verification (ETV) Program. The E.P.A. supports the ETV program to further environmental protection by accelerating the acceptance and use of improved and cost effective technologies. Microcystest kit is a rapid enzymatic test for detection of microcystins in drinking and recreational water. Microcystins are a group of hepatotoxins produced by cyanobacteria during and after blooms in water bodies, as a result of eutrophication. They are considered the most harmful cyanotoxins due to their natural abundant and potent toxicity. Incidences of animal and human fatalities caused by microcystins have led the World Health Organization to introduce a provisional upper limit of 1 µg/L in drinking water. The toxicity of microcystins is associated with the inhibition of protein phosphatases 1 and 2A enzymes, which can lead to hepatocyte dysfunction. MicroCystest kit uses this inhibitory reaction for determination of the microcystins concentration by means of a microtiter plate and a colorimetric substrate. The enzyme hydrolyses the substrate and the product 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 microcystins and analogues in the samples, the toxin concentration can be calculated by using a standard curve. MicroCystest is the first and only commercially available kit based on the inhibition of the PP2A activity by microcystins, and therefore able to detect all potential toxic microcystins present in the sample (over 80 different varieties of MCs have been described up-to-day).
www.epa.gov/etv U.S. Environmental Protection Agency Environmental Technology Verification
Recently, two papers have been published in Talanta and Analitical Chimica Acta comparing protein phosphatases (PP) from several sources (ZEU-INMUNOTEC, Millipore and GTP) and origin (recombinant or natural). A colorimetric in vitro assay was used to compare phosphatases in solution or entrapped within either a photoploymer or an agarose. Authors showed that the phosphatase from ZEU was the most sensitive to microcystins with a detection limit of 0.0039 ug/L and an IC50 of 0.56 ug/L for MC-LR in the assay conditions. In the case of okadaic acid toxins, phosphatase from ZEU had a detection limit of 0.012 ug/L when phosphatase was used in solution. Analysis of real samples (mussels) using the ZEU´s phosphatase showed also a very good repeatability (RSD = 1-6%). These results show that phosphatase from ZEU is the best choice for the detection of okadaic acid and microcystin toxins. ZEU-INMUNOTEC uses its own phosphatase to produceOKATEST (a kit for the detection of okadaic acid toxins in molluscs, recently UE-validated) and MICROCYSTEST (a kit for the detection of microcystins in water, evaluated under the ETV program in agreement with the U.S. Environmental Protection Agency).