Last data update: Jun 03, 2024. (Total: 46935 publications since 2009)
Records 1-4 (of 4 Records) |
Query Trace: Cunningham BR [original query] |
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Detection of anatoxins in human urine by liquid chromatography triple quadrupole mass spectrometry and ELISA
Cunningham BR , Lagon SR , Bragg WA , Hill D , Hamelin EI . Toxins (Basel) 2024 16 (3) Harmful cyanobacterial blooms are becoming more common and persistent around the world. When in bloom, various cyanobacterial strains can produce anatoxins in high concentrations, which, unlike other cyanobacterial toxins, may be present in clear water. Potential human and animal exposures to anatoxins occur mainly through unintentional ingestion of contaminated algal mats and water. To address this public health threat, we developed and validated an LC-MS/MS method to detect anatoxins in human urine to confirm exposures. Pooled urine was fortified with anatoxin-a and dihydroanatoxin at concentrations from 10.0 to 500 ng/mL to create calibrators and quality control samples. Samples were diluted with isotopically labeled anatoxin and solvent prior to LC-MS/MS analysis. This method can accurately quantitate anatoxin-a with inter- and intraday accuracies ranging from 98.5 to 103% and relative standard deviations < 15%, which is within analytical guidelines for mass spectrometry methods. Additionally, this method qualitatively detects a common degradation product of anatoxin, dihydroanatoxin, above 10 ng/mL. We also evaluated a commercial anatoxin-a ELISA kit for potential diagnostic use; however, numerous false positives were detected from unexposed individual human urine samples. In conclusion, we have developed a method to detect anatoxins precisely and accurately in urine samples, addressing a public health area of concern, which can be applied to future exposure events. |
Measurement of microcystin activity in human plasma using immunocapture and protein phosphatase inhibition assay
Cunningham BR , Wharton RE , Lee C , Mojica MA , Krajewski LC , Gordon SC , Schaefer AM , Johnson RC , Hamelin EI . Toxins (Basel) 2022 14 (11) Microcystins are toxic chemicals generated by certain freshwater cyanobacteria. These chemicals can accumulate to dangerous levels during harmful algal blooms. When exposed to microcystins, humans are at risk of hepatic injury, including liver failure. Here, we describe a method to detect microcystins in human plasma by using immunocapture followed by a protein phosphatase inhibition assay. At least 279 microcystins have been identified, and most of these compounds share a common amino acid, the Adda side chain. We targeted this Adda side chain using a commercial antibody and extracted microcystins from human samples for screening and analysis. To quantitate the extracted microcystins, we fortified plasma with microcystin-LR, one of the most well-studied, commonly detected, and toxic microcystin congeners. The quantitation range for the detection of microcystin in human plasma using this method is 0.030-0.50 ng/mL microcystin-LR equivalents. This method detects unconjugated and conjugated forms (cysteine and glutathione) of microcystins. Quality control sample accuracies varied between 98.9% and 114%, with a precision of 7.18-15.8%. Finally, we evaluated plasma samples from a community health surveillance project of Florida residents living or working near harmful algae blooms. |
Detection of brevetoxin in human plasma by ELISA
Cunningham BR , Coleman RM , Schaefer AM , Hamelin EI , Johnson RC . J Anal Toxicol 2021 46 (3) 322-327 Florida red tides have become more common and persistent in and around the Gulf of Mexico. When in bloom, red tides can produce brevetoxins in high concentrations, leading to human exposures primarily through contaminated food and ocean spray. The research described here includes adapting and validating a commercial brevetoxin water test kit for human plasma testing. Pooled plasma was fortified with a model brevetoxin, brevetoxin 3, at concentrations from 0.00500 to 3.00 ng/mL to generate calibration curves and quality control samples. The quantitative detection range was determined to be 0.0400-2.00 ng/mL brevetoxin 3 equivalents with inter- and intraday accuracies ranging from 94.0% to 109% and relative standard deviations <20%, which is within the US Food and Drug Administration guidelines for receptor-binding assays. Additionally, cross-reactivity was tested using 4 of the 10 known brevetoxins and 12 paralytic shellfish toxins. The cross-reactivity varied from 0.173% to 144% for the commercially available brevetoxin standards and 0% for the commercially available paralytic shellfish toxin standards. Fifty individual unexposed human plasma samples were measured to determine the limit of detection and endogenous interferences to the test. The validated method was used to test 31 plasma samples collected from humans potentially exposed to brevetoxins, detecting 11 positives. This method has been proven useful to measure human exposure to brevetoxins and can be applied to future exposure events. |
Measurement of microcystin and nodularin activity in human urine by immunocapture-protein phosphatase 2A assay
Wharton RE , Cunningham BR , Schaefer AM , Guldberg SM , Hamelin EI , Johnson RC . Toxins (Basel) 2019 11 (12) 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. |
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