title: Development of a colorimetric inhibition assay for microcystin-LR detection: comparison of the sensitivity of different protein phosphatases.
abstract: A colorimetric protein phosphatase (PP) inhibition test for the detection of microcystin-LR (MC-LR) has been developed. Three PP2As, one recombinant and two natural versions, as well as one PP1 produced by molecular engineering, were tested. First, assays were performed using the enzymes in solution to compare their sensitivity to MC-LR. The PP2A purchased from ZEU Immunotec and PP1 appeared more sensitive to the toxin than the other enzymes. With PP2A from ZEU Immunotec, the colorimetric test showed a detection limit of 0.0039 μg L(-1) and an IC(50) value of 0.21 μg L(-1). With PP1, the assay gave a detection limit of 0.05 μg L(-1) and an IC(50) value of 0.56 μg L(-1). Therefore, this assay allowed the detection of lower microcystin-LR (MC-LR) concentrations than the maximum level (1 μg L(-1)) recommended by the World Health Organisation (WHO). The main drawback of this PP-based approach in solution is poor enzyme stabilisation. To overcome this problem, enzymes were entrapped within either a photopolymer or an agarose gel. PP2A from ZEU Immunotec and PP1 were immobilised at the bottom of microwells. The agarose-based tests performed better than the photopolymer-based assay for all of the enzymes. Therefore, the agarose gel is a good candidate to replace the photopolymer, which is generally used in PP-immobilising membranes. The assays based on enzyme-entrapping agarose gels showed detection limits equal to 0.17 μg L(-1) and 0.29 μg L(-1) with immobilised PP2A from ZEU and PP1, respectively. In view of these performances, these tests can potentially be used for monitoring water quality.
authors.affiliation: Laboratoire IMAGES, bâtiment S, 52 avenue Paul Alduy, 66860 Perpignan cedex, France. audrey.sassolas@univ-perp.fr
authors.forename: Audrey
authors.initials: A
authors.lastname: Sassolas
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Catanante
authors.forename: Didier
authors.initials: D
authors.lastname: Fournier
authors.forename: Jean Louis
authors.initials: JL
authors.lastname: Marty
mesh_terms: D003124:Colorimetry; D020128:Inhibitory Concentration 50; D008387:Marine Toxins; D052998:Microcystins / Q000032:analysis* / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D012685:Sepharose
chemical_list: D008387:Marine Toxins; D052998:Microcystins; D012685:Sepharose; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR