Optimization of intracellular microcystin-LR extraction for its analysis by protein phosphatase inhibition assay

Abstract

Microcystins are toxins produced by some strains of cyanobacteria. Several methods have been developed to allow the quantification of microcystins, which are mainly endotoxins. Among those methods, the protein phosphatase inhibition assay is a good candidate as a screening method because of its sensitivity, simplicity and specificity. In this work a method for intracellular microcystin extraction in field water samples and lab cyanobacterial cultures prior to their analysis by protein phosphatase inhibition assay has been optimized. Microcystin-LR and Microcystis aeruginosa PCC 7806 were used as reference microcystin and strain, respectively, in order to optimize the protocol. The protocol consists on filtering the sample through a nylon filter of 0.8 microm, filter extraction with methanol 80% 0.1% trifluoroacetic acid (TFA) 0.1% tween 20, extract centrifugation and supernatant dilution (1/20). The establishment of an extraction protocol was carried out determining the extraction volume, time of extraction and number of extractions. The advantages of the method developed in this work are basically its simplicity and avoiding the use of specific and expensive equipment.

Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

“…Another study by Sevilla et al (2009) employed an 80% methanol, 0.1% trifluoroacetic acid, 0.1% Tween 20 extraction followed by centrifugation and dilution of cyanobacterial cultures and water samples prior to PP2A assays. As these methods are not for dried/ processed algal supplements, it is clear that further work to evaluate these protocols is needed.…”

Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit

“…Furthermore, most of the existing rapid monitoring tools for the detection of MC, including PPIAs, were designed for the testing of drinking and source waters with calibration at either the World Health Organization (WHO) provisional guideline value of 1 μg/L MC in drinking water ( WHO, 1998 ), or at the recreational water exposure response guidelines that have been adopted by several U.S. states [range 4–20 μg/L] ( https://www.epa.gov/nutrient-policy-data/guidelines-and-recommendations ) [Accessed 3 January 2018]. One such commercially available kit is the PPIA-based MicroCystest (ZEULAB S.L., Zaragoza, Spain) ( Sevilla et al., 2009 ) which was previously evaluated for the testing of source waters through the Environmental Technology Verification (ETV) Program of the U.S. Environmental Protection Agency (EPA) ( McKernan et al., 2011 ), and is now distributed in the U.S. as the Microcystin/Nodularin PP2A Kit (Abraxis LLC, Warminster, Pennsylvania). At present, the kit as sold is designed for testing water samples, but the manufacturer has developed a modified protocol, supplied upon request, to specifically test dried BGA materials as are found in BGA dietary supplements (Elena Dominguez, ZEULAB, personnel communication).…”

Sensitive determination of total microcystins with GC-MS method by using methylchloroformate as a derivatizing reagent

“…There are several methods available for the determination of MCs, including enzyme-linked immunosorbent assay (ELISA), [9][10][11] the protein phosphatase inhibition assay, 12 reversedphase high-performance liquid chromatography (HPLC), [13][14][15][16][17][18][19] LC-MS, 20 capillary electrophoresis (CE) and CE-MS analysis. 21,22 Biochemical methods such as ELISA and the protein phosphatase inhibition assay are useful as screening methods due to their high sensitivity and high throughput, but they oen suffer from false-positive responses.…”