Protein inhibition assay

After screening for toxicity, identification and/or quantification assays may need to be carried out if the screening method is not specific for the cyanobacterial toxin(s) under investigation. Suitable assays for these purposes include the physicochemical assays, HPLC, MS, and CE, and to some extent the immunoassays and protein phosphatase inhibition assays summarized in Section 2. 

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

Indirect measurement (bioassay) Mouse bioassay Mouse bioassay Neuroblastoma assay (Truman et at, 2002) Mouse bioassay Protein-phosphatase inhibition assay (Mountfort et at, 2001) Mouse bioassay Cytotoxicity assay (Flanagan et at, 2001) Mouse bioassay Neuroblastoma assay (Matta et at, 2002)... 

Bioassay detection limit Mouse bioassay 20 pg SIX eq/100 g Mouse bioassay 20 pg STX eq/lOOg Neuroblastoma assay 2 pg STX eq/ 100 g Mouse bioassay 20 pg STX eq/100 g Protein-phosphatase inhibition assay 20 pg STX eq/100 g Unknown Unknown Neuroblastoma assay 10 ng/mL... 

Mountfort, D.O., Suzuki, T. and Truman, P., Protein phosphatase inhibition assay adapted for determination of total DSP in contaminated mussels, Toxicon, 39, 2-3, 383, 2001. 

Three of the MBD family members, namely MeCP2, MBDl, and MBD2, have been shown to act as transcriptional repressors in vitro and in cell culture assays in vivo [86-89,75,76]. Initially it was proposed that methyl-CpG binding proteins inhibit transcription directly, by binding to methylated DNA and blocking the access of transcription factors to their sites at gene promoters. It was soon... 

The oleosin fusion procedure was used for the purification of the commercially valuable plant-based blood anticoagulant hirudin in transgenic Brassica carinata and Brassica napus. Hirudin, a natural protein from the medicinal leech Hirudo medicinalis, is superior to other anticoagulants such as heparin. Recombinant hirudin was cleaved from oil-bodies using endoproteinase Factor Xa. Released hirudin was biologically active, as determined by a colorimetric thrombin inhibition assay. 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

J.S. An and W.W. Carmichael, Use of a colorimetric protein phosphatase inhibition assay and enzyme-linked immunosorbent assay for the study of microcystins and nodularins, Toxicon, 32 (1994) 1495-1507. 

T. Heresztyn and B.C. Nicholson, Determination of cyanobacterial he-patotoxins directly in water using a protein phosphatase inhibition assay, Water Res., 35 (2001) 3049-3056. 

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

Carmichael, W.W, and An, J. 1999. Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay for the detection of microcystins and nodularins. Nat Toxins 7 377-385. 

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