A feasibility study into the production of a mussel matrix reference material for the cyanobacterial toxins microcystins and nodularins.

Microcystins and nodularins, produced naturally by certain species of cyanobacteria, have been found to accumulate in aquatic foodstuffs such as fish and shellfish, resulting in a risk to the health of the seafood consumer. Monitoring of toxins in such organisms for risk management purposes requires the availability of certified matrix reference materials to aid method development, validation and routine quality assurance. This study consequently targeted the preparation of a mussel tissue reference material incurred with a range of microcystin analogues and nodularins. Nine targeted analogues were incorporated into the material as confirmed through liquid chromatography with tandem mass spectrometry (LC-MS/MS), with an additional 15 analogues detected using LC coupled to non-targeted high resolution mass spectrometry (LC-HRMS). Toxins in the reference material and additional source tissues were quantified using LC-MS/MS, two different enzyme-linked immunosorbent assay (ELISA) methods and with an oxidative-cleavage method quantifying 3-methoxy-2-methyl-4-phenylbutyric acid (MMPB). Correlations between the concentrations quantified using the different methods were variable, likely relating to differences in assay cross-reactivities and differences in the abilities of each method to detect bound toxins. A consensus concentration of total soluble toxins determined from the four independent test methods was 2425 ± 575 µg/kg wet weight. A mean 43 ± 9% of bound toxins were present in addition to the freely extractable soluble form (57 ± 9%). The reference material produced was homogenous and stable when stored in the freezer for six months without any post-production stabilization applied. Consequently, a cyanotoxin shellfish reference material has been produced which demonstrates the feasibility of developing certified seafood matrix reference materials for a large range of cyanotoxins and could provide a valuable future resource for cyanotoxin risk monitoring, management and mitigation

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp³]­MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO’s maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp³]­MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO’s maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp³]­MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHO’s maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs

Development of an ELISA for the Detection of Azaspiracids

Azaspiracids (AZAs) are a group of biotoxins that cause food poisoning in humans. These toxins are produced by small marine dinoflagellates such as Azadinium spinosum and accumulate in shellfish. Ovine polyclonal antibodies were produced and used to develop an ELISA for quantitating AZAs in shellfish, algal cells, and culture supernatants. Immunizing antigens were prepared from synthetic fragments of the constant region of AZAs, while plate coating antigen was prepared from AZA-1. The ELISA provides a sensitive and rapid analytical method for screening large numbers of samples. It has a working range of 0.45–8.6 ng/mL and a limit of quantitation for total AZAs in whole shellfish at 57 μg/kg, well below the maximum permitted level set by the European Commission. The ELISA has good cross-reactivity to AZA-1–10, -33, and -34 and 37-<i>epi</i>-AZA-1. Naturally contaminated Irish mussels gave similar results whether they were cooked or uncooked, indicating that the ELISA also detects 22-carboxy-AZA metabolites (e.g., AZA-17 and AZA-19). ELISA results showed excellent correlation with LC-MS/MS analysis, both for mussel extract spiked with AZA-1 and for naturally contaminated Irish mussels. The assay is therefore well suited to screening for AZAs in shellfish samples intended for human consumption, as well as for studies on AZA metabolism