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: harmful algal bloom; immunocapture; microcystins; nodularin; protein phosphatase 2A.

Figure 1

The structure of microcystin-LR (MC-LR) (a) and nodularin (NOD) (b). The boxed portion denotes the adda moiety which is conserved amongst microcystin (MC) congeners and NOD.

Figure 2

MC-LR immunocapture (IC) optimization. All optimization experiments were performed using 500 µL of 1 ng/mL singly-charged (MC-LR) congeners in pooled urine. In panel B, 500 µL of 1 ng/mL doubly-charged (MC-RR) and uncharged (MC-LF) congeners in pooled urine were also used. MC antibody titration to optimize capture of MC-LR from pooled urine (n = 3) (A). Selection of optimal elution buffer for IC of three MC congeners. Black bar (100% ACN/0.5% FA), striped bar (70% ACN/30% water/0.5% FA), dark gray bar (50% ACN/50% water/0.5% FA), light gray bar (30% ACN/70% water/0.5% FA), white bar (100% water/0.5% FA), (n = 3) (B). Capture time optimization for antibody conjugation to magnetic beads (n = 3) (C). Capture time optimization of MC-LR from pooled urine (n = 3) (D). Time optimization for eluting MC-LR from magnetic beads (n = 3) (E). Optimal conditions for removing supernatants from beads (n = 3) (F). Significance was determined by one-way ANOVA and Tukey’s multiple comparisons post-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.0005, **** p ≤ 0.0001, ns = not significant. Error bars represent the standard deviation of replicate samples. % Recovery = peak area of pre-spike sample/peak area of post-spike sample × 100%.

Figure 3

Method recovery of pooled urine samples spiked with 0.250 (black bars) or 0.080 ng/mL (gray bars) of MC-RR, MC-LR, or MC-LF prior to IC compared to post-spike controls. % Recovery = concentration of pre-spike sample/concentration of post-spike sample × 100%. Statistical significance of data was determined by one-way ANOVA and Tukey’s multiple comparisons post-test (n = 3).

Figure 4

Method interferences from known PP2A inhibitors. Pooled urine spiked with 0.500 ng/mL of PP2A inhibitors prior to IC (gray bars) were compared to 5.00 ng/mL post-spike controls (black bars). Significance was determined by unpaired t-test. * p ≤ 0.05, *** p ≤ 0.005, **** p ≤ 0.0001, n.d. = not detected. Error bars represent the standard deviation of duplicate samples.

Figure 5

Representative calibration curve of MC-LR from validation. Points at 1.00 and 0.030 ng/mL are curve anchors.

Figure 6

Individual urine samples were spiked with 0.080 and 0.250 ng/mL MC-LR and evaluated for % accuracy and precision (n = 10 for each spike level). Error bars represent the standard deviation.