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 health concern, especially affecting developing countries where the high cost of available methods makes monitoring programs difficult. The phosphatase inhibition assay (PPIAs) is a sensitive method with low instrument requirements that allows the quantification of the most frequent cyanotoxins, microcystins (MC). In this work, we implemented a PPIAs, starting from Protein Phosphatase 1 (PP1) expression up to the validation with samples of algal blooms from Argentina. To do this, we optimized the expression and lyophilization of PP1, and the assay conditions. Also, we included robustness and possible interfering analysis. We evaluated the most 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 recovery assays on water samples from three dams from Argentina, resulting in a recovery ranging from 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 ELISA test containing from 0 μg/L to 625 μg/L. The PPIA showed excellent correlation (Pearson correlation coefficient = 0.967), no false negative and no false positives above the 1 μg/L WHO guideline (0.11 false positive rate). In conclusion, we optimized and validated a PPIAs to be an effective and accessible alternative to available commercial tests.