Optimization and validation of a Protein Phosphatase inhibition assay for accessible microcystin detection

Abstract

The presence of cyanobacterial toxins in freshwater constitutes an increasing public healthconcern, especially affecting developing countries where the high cost of available methodsmakes monitoring programs difficult. The phosphatase inhibition assay (PPIAs) is a sensitivemethod with low instrument requirements that allows the quantification of the most frequentcyanotoxins, microcystins (MC). In this work, we implemented a PPIAs, starting from ProteinPhosphatase 1 (PP1) expression up to the validation with samples of algal blooms fromArgentina. To do this, we optimized the expression and lyophilization of PP1, and the assayconditions. Also, we included robustness and possible interfering analysis. We evaluated themost widely used cyanobacterial lysis methods and determined that heating for 15 minutes at 95°C is simple and adequate for this assay. Then, we performed MC spikes recoveryassays on water samples from three dams from Argentina, resulting in a recovery rangingfrom 77 to 115%. The limit of detection (LOD) was 0.4 μg/L and the linear range is 0.4 μg/L -5 μg/L. Finally, we evaluated 64 environmental samples where MC was measured by ELISAtest containing from 0 μg/L to 625 μg/L. The PPIA showed excellent correlation (Pearsoncorrelation coefficient = 0.967), no false negative and no false positives above the 1 μg/LWHO guideline (0.11 false positive rate). In conclusion, we optimized and validated a PPIAsto be an effective and accessible alternative to available commercial tests.