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Final Report Summary - BIOTOXMARIN (Development of novel analytic tools for the detection of marine biotoxins)

The contamination of seafood with algal toxins can cause severe neuronal and gastrointestinal disorders but also allergies in human. Sporadic outbreaks of poisoning by ingestion of shellfish which have accumulated marine biotoxins have become a world-wide problem. The economic consequences caused by the production of marine biotoxins during algal blooms in the coastal regions are enormous. In this project, fast, simple and cost-effective detection methods for marine biotoxins in seafood as well as patient sera were developed, based on the application of high-affinity capture antibodies and novel artificial receptor mimics against the toxins.

The new tools for the detection (and quantification) of marine biotoxins developed in the project BIOTOXMARIN were based on the application of the new polymer instruction technology and the highly sensitive 'Integrated optical grating coupler' (IOGC) biosensor technology, and the use of high-affinity antibodies for sensitive ELISA und Western blotting techniques. User-friendly chip / dip-stick assay methods as well as new bioassays based on interaction of okadaic acid with phosphoprotein phosphatase 2A (microtitre-plate based PP2A inhibition assay) or the activation / phosphorylation of MAP kinase p38 were developed. The developed technologies were compared with existing techniques for evidence of improved efficiency and accuracy. Prototype kits were manufactured by the companies.

There are four forms of poisoning caused by consumption of contaminated seafood, which markedly differ in course and symptoms: 'Paralytic shellfish poisoning' (PSP), 'Neurotoxic shellfish poisoning' (NSP), 'Amnesic shellfish poisoning' (ASP) and 'Diarrhetic shellfish poisoning' (DSP). In addition, 'Ciguatera fish poisoning' (CFP) is a disorder associated with dinoflagellate toxins that accumulate in tropical fish meat. ASP can be a life threatening syndrome. It is characterised by gastrointestinal symptoms such as nausea, vomiting, abdominal cramps and diarrhoea as well as neurological symptoms. DSP causes gastrointestinal symptoms only. This disease is not fatal and characterised by severe diarrhoea, nausea, vomiting, abdominal cramps and shivers. In contrast, NSP causes an intoxication syndrome with both neurological and gastrointestinal symptoms. NSP toxins can also cause respiratory asthma-like symptoms by formation of toxic aerosols through wave movements. PSP is a life-threatening syndrome like ASP. The symptoms are purely neurological and the onset is fast. The main PSP toxins are saxitoxins and gonyautoxins, NSP toxins are brevetoxins, ASP toxins are domoic acid and DSP toxins are okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids and yessotoxins. The toxic dinoflagellates producing them can be isolated from both natural and anthropogenic algal blooms.

The main achievements and breakthroughs can be summarised as follows.
- Isolation and characterisation of two new analogues of yessotoxin (YTX): 1- desulfo carboxyhomoYTX and 1-desulfo carboxyhomoYTX (Breakthrough: discovery of new biotoxins).
- Isolation and characterisation of novel analogues of oxazinin-1: oxazinin-4, -5, -6, and -7 (Breakthrough: discovery of new biotoxins).
- Method for larger-scale production and preparation of highly pure samples of yessotoxin (Commercial application: production of standards).
- Identification of novel spirolides: 27-hydroxy-13,19-didesmethylC and others (Breakthrough: discovery of new biotoxins).
- Development of a new LC-MS method for sensitive, specific and direct determination of palytoxin (P-PTX) (Breakthrough: discovery of new biotoxins).
- Development of a new method (HILIC/MS) for determination of domoic acid (DA) (Breakthrough: novel sensitive, specific and direct method for detection of marine biotoxins).
- Demonstration of apoptotic effect (increase in [Ca2+]i level and caspase activities) of isolated marine biotoxins in neuronal cells (Yessotoxin, okadaic acid and others) in neuronal cells (Breakthrough: Elucidation of new bioactive effects of marine biotoxins).
- Preparation of high affinity monoclonal and polyclonal antibodies against marine biotoxins (yessotoxin, okadaic acid, domoic acid and others) (Commercial application: component of novel assays for detection of marine biotoxins).
- Development of a new competitive ELISA based on these antibodies (Commercial application: new assays for detection of marine biotoxins).
- Development of instructed polymer receptor phases with 'molecular memory' for the isolation, identification and binding of marine biotoxins (Breakthrough: application of a novel principle for the detection and isolation of marine biotoxins).
- Development of cheap, rapid and user-friendly lateral flow assays for marine biotoxins (dip-stick / chip assay) for detection of biotoxins (Breakthrough: application of a novel principle for the detection of marine biotoxins).
- Development of a bioassay for detection of activation / phosphorylation of p38 MAP kinase (Breakthrough: assay based on a general marker for biotoxin exposure).
- Development of a highly sensitive immunosensor for marine biotoxins (detection range between 0.001-10 ng/ml) (Commercial application: assay for detection of marine biotoxins based on novel principle).
- Development of an optical waveguide sensor based on immobilised receptor phases and OWLS technology (Commercial application: novel principle for detection of marine biotoxins).
- Improvement of the sensitivity of the microtitre-plate based PP2A inhibition assay (Commercial application: routine assay for detection of marine biotoxins).

The products and techniques developed in frame of this project are considered to be of high commercial value. This has been evaluated in a market analysis performed by the SME partners. The best commercial possibilities of the techniques examined as part of the BIOTOXMARIN project appeared to be the PP2A assay, the OWLS Biosensor and the dip stick system as these gave opportunities for the development of associated products or kits. For LCMS, the only commercial opportunities are in the production of standards (which is essential for its success) and actually offering testing services.

The SME partners involved in this project will also play the leading role in the future exploitation - dissemination activities. We expect that the tests developed in this project will contribute to the replacement of animals in tests (mouse bioassay for the detection of marine biotoxins) by non-animal tests (tests based on antibodies and instructed synthetic polymeric receptors).

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