Measurement of Microcystin and Nodularin Activity in Human Urine by Immunocapture-Protein Phosphatase 2A Assay

Abstract

Microcystins (MC) and nodularin (NOD) are toxins released by cyanobacteria during harmful algal blooms. They are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A) and cause a variety of adverse symptoms in humans and animals if ingested. More than 250 chemically diverse congeners of MCs have been identified, but certified reference materials are only available for a few. A diagnostic test that does not require each reference material for detection is necessary to identify human exposures. To address this need, our lab has developed a method that uses an antibody to specifically isolate MCs and NOD from urine prior to detection via a commercially available PP2A kit. This assay quantitates the summed inhibitory activity of nearly all MCs and NOD on PP2A relative to a common MC congener, microcystin-LR (MC-LR). The quantitation range for MC-LR using this method is from 0.050-0.500 ng/mL. No background responses were detected in a convenience set of 50 individual urines. Interday and intraday % accuracies ranged from 94%-118% and relative standard deviations were 15% or less, meeting FDA guidelines for receptor binding assays. The assay detected low levels of MCs in urines from three individuals living in close proximity to harmful algal blooms (HABs) in Florida.Keywords: microcystins; nodularin; protein phosphatase 2A; immunocapture; harmful algal bloom Key Contribution: This paper describes the development and subsequent validation of an immunocapture-protein phosphatase inhibition assay to detect and measure combined inhibitory activity of MCs and NOD in human urine. Low levels of MCs were detected in urine specimens from individuals living or working in close proximity to harmful algal blooms.

A Mini-Review on Detection Methods of Microcystins

“…Since the first establishment of PPIA (by using a colorimetric PPA, which uses substrates such as p -nitrophenyl phosphate [ 112 ]), various other PPIA techniques have been constructed for MCs detection ( Table 1 , Table 2 , Table 3 and Table 4 ). A novel colorimetric immune-PPIA (CI-PPIA) where the combination of immune detection and toxicity-based PPI in the CIPPIA provides a useful addition to existing methods [ 49 ], colorimetric and fluorometric PPIA, which require an enrichment step using C 18 cartridges to achieve lower detection limit below the WHO’s provisional guideline value [ 50 ]; electrochemical MC-LR biosensor based on the inhibition of recombinant PP1α [ 70 ]; and immunocapture PPIA (IC-PPIA), which utilizes antibody to specifically isolate MCs from urine prior to detection through PP2A kit [ 113 ] effectively detected different variants of MC. In the inhibition characteristics study of three different protein phosphatases (natural PP2A, recombinant PP2A and recombinant PP1) using three MC variants (MC-RR, MC-LR and MC-YR), MC-LR displayed the highest toxicity followed by MC-YR and MC-RR.…”

Immunoaffinity Extraction and Alternative Approaches for the Analysis of Toxins in Environmental, Food or Biological Matrices

“…For this purpose, proteins A- [ 90 ] or G- [ 80 , 99 , 104 , 105 , 106 , 107 , 108 ] based sorbents or sorbents grafted with anti-IgG [ 92 , 98 ] can be used as these proteins bind a part of the constant region of Abs, allowing the orientation of the Abs with the antigen binding sites away from the surface and towards the solution. The same orientation effect can be obtained using streptavidin activated sorbent that can react with biotinylated Abs [ 72 , 79 , 109 ]. However, the resulting non-covalent binding is quite strong under physiological conditions but can be easily disrupted by decreasing the pH of the surrounding solution.…”

“…However, lower grafting yields may be obtained if steric hindrances occur during the grafting. This is why it can be interesting to optimize the number of antibodies for a given amount of sorbent as reported [ 69 , 70 , 79 ]. However, only a theoretical capacity can be calculated based on the grafting yield because the real capacity depends on the number of specific and active antibodies, which is unknown when using pAbs, and steric hindrances that could prevent the analyte from accessing the antibody recognition sites.…”

“…This dSPE mode was reported for 34% of the studies cited in Table 2 . The particles were prepared by the covalent immobilization of antibodies on NHS-activated Sepharose beads [ 70 ], or tosyl-activated magnetic beads [ 102 , 110 , 111 ] or by non-covalent immobilization on protein G- [ 80 , 99 , 104 , 105 , 106 , 107 , 108 ] or streptavidin- [ 72 , 79 , 109 ] activated magnetic beads or on amino-coated hollow glass magnetic microspheres [ 100 , 101 ].…”

“…In addition, it is necessary in this mode to optimize the extraction, the desorption times and the vortex speed. It appears that the extraction step takes from 1–10 min [ 70 , 72 , 79 , 80 , 100 , 101 , 104 , 105 ] to 1–2 h [ 99 , 102 , 107 , 108 , 110 , 111 ], the desorption step being carried out with a similar or shorter time. Most of the procedures include a washing step before desorption to ensure an optimal selectivity, but the duration of this step was never mentioned.…”