Julie P. Meneely

A rapid optical immunoassay for the screening of T-2 and HT-2 toxin in cereals and maize-based baby food

A rapid surface plasmon resonance (SPR) screening assay has been developed for the combined detection of T-2 and HT-2 toxins in naturally contaminated cereals using a sensor chip coated with an HT-2 toxin derivative and a monoclonal antibody. The antibody raised against HT-2 displayed high cross-reactivity with T-2 toxin while there was no cross-reaction observed with other commonly occurring trichothecenes. A simple extraction procedure using 40% methanol was applied to baby food, breakfast cereal, and wheat samples prior to biosensor analysis. Limits of detection (LOD) for each matrix were determined as 25 microg kg(-1) for baby food and breakfast cereal and 26 microg kg(-1) for wheat. Intra-assay precision (n=6) was calculated for each matrix. The results were expressed as the relative standard deviation and determined as 2.8% (100 microg kg(-1)) and 1.8% (200 microg kg(-1)) in breakfast cereal, 4.6% (50 microg kg(-1)) and 3.6% (100 microg kg(-1)) in wheat and 0.97% (25 microg kg(-1)) and 6.3% (50 microg kg(-1)) in baby food. Between run precision (n=3) performed at the same levels yielded relative standard deviations of 6.7% and 3.9% for breakfast cereals, 3.3% and 1.6% for wheat and 6.8% and 0.08% for baby food, respectively.

Gremlin1 preferentially binds to Bone Morphogenetic Protein-2 (BMP-2) and BMP-4 over BMP-7

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during diabetic nephropathy (DN) has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. In the present study, we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK (human embryonic kidney)-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readouts, Grem1 consistently demonstrated a higher affinity for BMP-2>BMP-4>BMP-7. Cell-associated Grem1 did not inhibit BMP-2- or BMP-4-mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated.

Rapid Surface Plasmon Resonance Immunoassay for the Determination of Deoxynivalenol in Wheat, Wheat Products, and Maize-Based Baby Food

A rapid screening assay (9 min/sample) has been developed and validated for the detection of deoxynivalenol in durum wheat, wheat products, and maize-based baby foods using an SPR biosensor. Through a single laboratory validation, the limits of detection (LOD) for wheat, wheat-based breakfast cereal, and maize-based baby food were 57, 9, and 6 μg/kg, respectively. Intra-assay and interassay precisions were calculated for each matrix at the maximum and half-maximum European Union regulatory limits and expressed as the coefficient of variation (CV). All CVs fell below 10% with the exception of the between-run CV for breakfast cereal. Recoveries at the concentrations tested ranged from 92 to 115% for all matrices. Action limits of 161, 348, and 1378 μg/kg were calculated for baby food, wheat-based breakfast cereal, and wheat, respectively, and the linear range of the assay was determined as 250-2000 μg/kg.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCβ were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCβ and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L.

Simultaneous screening for T-2/HT-2 and deoxynivalenol in cereals using a surface plasmon resonance immunoassay

This manuscript describes a rapid surface plasmon resonance (SPR) immunoassay for the simultaneous determination of the sum of T-2/HT-2 toxins (T-2/HT-2) and deoxynivalenol (DON), in cereals and cereal-based products. The assay is based on an inhibition format employing a monoclonal antibody raised against HT-2 with cross reactivity to T-2 and a polyclonal antibody raised against DON, thereby enabling the detection of the three trichothecene mycotoxins (types A and B). The surface chemistry involved an equal mixture of HT-2 and DON covalently coupled onto a high capacity COOH5 sensor chip. Using the specified antibodies and a mixed toxin sensor surface, and running calibration curves (HT-2 and DON) and samples in parallel it has been proven that it is feasible to develop a multiplex assay on this SPR platform. In-house validation has shown limits of detection of 12, 1 and 29 μg/kg for DON and 31, 47 and 36 μg/kg for HT-2 in wheat, breakfast cereal and maize-based baby food, respectively. Both intra-assay ...

Development and validation of the first high performance-lateral flow immunoassay (HP-LFIA) for the rapid screening of domoic acid from shellfish extracts.

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10 ppm (version 1) to 17.5 ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15 min after sample preparation. Stability of the aqueous extracts at room temperature (22 °C) at four time points (up to 245 min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10 ppm DA, and positive results at or above 17.5 ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20 ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12 min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5 ppm and positive results at or above 17.5 ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50 °C) was 100%. Validation for both versions included comparisons with results obtained using reference LC-UV methods.

Microcystins: measuring human exposure and the impact on human health.

Abstract Context: Freshwater cyanobacterial toxins, microcystins, may be a contributing factor to the development of hepatocellular cancer and colorectal cancer. Objectives: This review summarizes the toxicity data, exposure routes and the methodologies available to determine exposure to elucidate the relationship to liver and colorectal cancer. Methods: Literature searches were conducted using Medline, PubMed and Web of Science. Results: There is evidence of human poisonings resulting from exposure to microcystins, however current methods rely on targeted approaches only suitable for acute exposure. No methods exist for the determination of chronic exposure to microcystins. Conclusions: With the growing evidence of exposure to microcystins and the possible links to cancer, methods to measure medium to long-term human exposure are needed. The identification and validation of candidate biomarkers are key to undertaking urgently required epidemiological studies.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10 min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78 ng mL(-1) and the CCβ to be 1 ng mL(-1). Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R(2)=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1 ng mL(-1), and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1 ng mL(-1). This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1 μg L(-1). This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Rapid surface plasmon resonance immunoassays for the determination of mycotoxins in cereals and cereal-based food products

In recent times surface plasmon resonance has demonstrated its applicability to the detection of a wide range of contaminants in food and feed including mycotoxins in cereals and cereal-based food products. Commercially available, laboratory-based systems have exploited high affinity polyclonal, monoclonal and recombinant antibodies and robust sensing surfaces to provide rapid, accurate and sensitive means of determining these toxins. In addition many custom-built, prototype devices have shown a great deal of potential for this particular application and have included the combination of surface plasmon resonance with enzyme-derivatised sensors, molecularly imprinted polymers, fluorescence spectroscopy and the use of gold nanoparticles for signal enhancement. Of note is the lack of available devices that allow the detection of multiple mycotoxins simultaneously and portable devices that could be used in the field, therefore future research and development should focus on these areas to deliver cost-effecti...

β-methylamino-L-alanine (BMAA) is not found in the brains of patients with confirmed Alzheimer’s disease

Controversy surrounds the proposed hypothesis that exposure to β-methylamino-L-alanine (BMAA) could play a role in various neurodegenerative conditions including Alzheimer’s disease (AD). Here we present the results of the most comprehensive scientific study on BMAA detection ever undertaken on brain samples from patients pathologically confirmed to have suffered from AD, and those from healthy volunteers. Following the full validation of a highly accurate and sensitive mass spectrometric method, no trace of BMAA was detected in the diseased brain or in the control specimens. This contradicts the findings of other reports and calls into question the significance of this compound in neurodegenerative disease. We have attempted to explain the potential causes of misidentification of BMAA in these studies.