The effects of water sample treatment, preparation, and storage prior to cyanotoxin analysis for cylindrospermopsin, microcystin and saxitoxin.

Cyanobacterial harmful algal blooms occur in freshwater lakes, ponds, rivers, and reservoirs, and in brackish waters throughout the world. The wide variety of cyanotoxins and their congeners can lead to frequent exposure of humans through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and cyanobacterial scum during recreational activities, and inhalation of cyanobacterial aerosols. Cyanotoxins can also occur in the drinking water supply. In order to monitor human exposure, sensitive analytical methods such as enzyme linked immunosorbent assay and liquid chromatography-mass spectrometry are often used. Regardless of the analytical method of choice, some problems regularly occur during sample collection, treatment, storage, and preparation which cause toxin loss and therefore underestimation of the true concentration. To evaluate the potential influence of sample treatment, storage and preparation materials on surface and drinking water samples, the effects of different types of materials on toxin recovery were compared. Collection and storage materials included glass and various types of plastics. It was found that microcystin congeners LA and LF adsorbed to polystyrene, polypropylene, high density polyethylene and polycarbonate storage containers, leading to low recoveries (<70%), cylindrospermopsin and saxitoxin did not adsorb to the containers tested. Therefore, this study shows that glass or polyethylene terephthalate glycol containers are the materials of choice for collection and storage of samples containing the cyanotoxins cylindrospermopsin, microcystins, and saxitoxin. This study also demonstrated that after 15 min chlorine decreased the concentration of microcystin LR to <40%, microcystin LA and saxitoxin to <15%, therefore quenching of drinking water samples immediately upon sample collection is critical for accurate analysis. In addition, the effect of various drinking water treatment chemicals on toxin recovery and the behavior of those chemicals in the enzyme linked immunosorbent assays were also studied and are summarized.

Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors.

Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

Comparison of two ELISA-based methods for the detection of microcystins in blood serum.

Microcystins (MCs) are cyanobacterial toxins which place the public at risk via exposure to MC contaminated water, food or algal food supplements. Subsequent to the fatal intravenous exposure of dialysis patients in Caruaru, Brazil, several techniques (LC-MS, GC-MS and ELISA) were adapted to detect MCs in human serum. As patients chronically exposed to low concentrations of MCs also present with very low MC serum levels, only LC-MS methodology would appear to allow detection of these MC levels. However, LC-MS detection depends on the availability of respective MC congener standards and the levels of non-covalently bound MC in the sample. In contrast, immunological techniques, e.g. MC-ELISA potentially could detect even covalently bound MC, provided the MC-antibody was raised against an epitope found in nearly all of the MC congeners. As the Adda-side-chain moiety is present in nearly all of the MC congeners known to date, the anti-Adda antibodies, when applied in Adda-ELISAs, could represent a relatively simple and robust technique for the qualitative and quantitative determination of MC in human serum. The aim of the current study was to determine whether commercially available Adda-ELISAs and their respective sample preparation methods would allow MC quantification in human serum. The Adda-ELISA (polyclonal antibody) and the Adda-ELISA (monoclonal antibody) kit for serum (Serum-ELISA) were used for determination of the concentration-dependent recovery of MCs in MC-spiked serum. Human serum samples were spiked with varying concentrations of MCs (MC-LR, -YR, -RR, -LA, -LW, -LF and defined MC mixtures) and extracted using two different methods. MC-spiked bovine serum and standard cell culture medium containing 10% FBS served to investigate potential matrix effects. Inter-laboratory comparison was performed allowing identification of potential sources of error. The results suggest that both ELISAs are suitable tools for the analysis of MCs in human blood serum although both also displayed some weaknesses notably the time needed for sample preparation or the overestimation of some specific MC congener concentrations. Based on the ELISA detection ranges, sample concentration and/or MC spiking may be required for detection of low levels of MCs in human blood.

Pitfalls in microcystin extraction and recovery from human blood serum.

BACKGROUND: Microcystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and scum during recreational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC-extraction method from human serum with more detail. METHODS: Six MC congeners (MC-LR, -YR, -RR, -LA, -LW, -LF) and defined equimolar MC mixtures thereof were spiked into human serum, and quantified using the commercially available Adda-ELISA subsequent to standard extraction (methanol extraction with subsequent SPE). To detect the potential influence of sample storage and preparation/storage materials, different types of material such as glass, standard polypropylene and surface-treated polypropylene were compared. RESULTS: Loss of MC during preparation and storage is largely dependent on (1) the handling of the stored material, (2) the 'surface' of the storage material and (3) the hydrophobicity of the MCs. CONCLUSIONS: The pitfalls described for MC analysis with the ELISA are primarily associated with sample preparation and clean-up and thus also apply to other analytical techniques for MC detection beyond the ELISA used. It can be concluded that ELISA-based methods are suitable tools for the detection of MCs in human sera and other samples.

Development of a magnetic particle-based enzyme immunoassay for the determination of penoxsulam in water.

A competitive enzyme-linked immunoassay (ELISA) for the quantitation of Penoxsulam [2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide] in ground and surface waters was developed. This immunoassay utilizes magnetic particles as the solid phase to which polyclonal rabbit anti-Penoxsulam antibodies are attached. The ELISA has an estimated detection limit of 0.17 ppb (microg/mL) of Penoxsulam in water. Specificity studies indicate that the antibody can distinguish Penoxsulam from its major metabolites and structurally similar pesticides. Interference studies indicate that the ELISA has a wide tolerance of sample pH and salinity and for compounds commonly found in surface and ground waters. The ELISA was shown to compare favorably to LC-MS/MS on ground and surface water samples (r(2) = 0.957). The various studies performed demonstrate the usefulness of the ELISA technique as a rapid and high-throughput analytical method for the cost-effective monitoring of water samples.

Measurement of triclosan in water using a magnetic particle enzyme immunoassay.

A sensitive magnetic particle-based immunoassay to determine triclosan [5-chloro-2-(2,4-dichlorophenoxy)phenol] in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-[5-chloro-2-(2,4-dichlorophenoxy)phenoxy]hexanoic acid-keyhole limpet hemocyanin. Horseradish peroxidase was conjugated with 4-[3-bromo-4-(2,4-dibromophenoxy)phenoxy]butyric acid via N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The triclosan antibody was coupled to magnetic particles via the NHS/EDC reaction. The antibodies were able to recognize some structurally related polybrominated biphenyl ethers but did not recognize various common pollutants that were less similar to the hapten. The ELISA could detect triclosan in standard solution (25% methanol/H2O v/v) at 20 ppt and its metabolite, methyl-triclosan, at 15 ppt. Water samples from different treatment stages were prepared to contain 25% methanol and analyzed directly without any sample extraction or preconcentration. The results showed that recoveries were >80% and the % CV was <10%, demonstrating the assay was both accurate and precise. Application of the triclosan ELISA to water treatment plants showed that tap water at various purification stages had low concentrations of triclosan (<20 ppt) and required an increased sample size for appropriate detection and measurement. Application of ELISA to the wastewater treatment plants (WWTP) demonstrated high concentrations of triclosan (in general, >3000 ppt in water entering the WWTP) with the levels decreasing as the water proceeded through the processing plant (<500 ppt at outflow sewage). The ELISA measurement was shown to be equivalent to the more specific GC-MS analysis on a number of wastewater treatment samples with a high degree of correlation, with the exception of a few samples with very high triclosan concentrations (>5000 ppt). Measurement of methyl-triclosan (in WWTP) using GC-MS demonstrated the levels of this compound to be low. In summary, a rapid, sensitive, accurate, and precise magnetic particle-based immunoassay has been developed for triclosan analysis, which can serve as a cost-effective monitoring tool for various water samples.