Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

FP6

DETECTOX Sintesi della relazione

Project ID: 514055
Finanziato nell'ambito di: FP6-FOOD
Paese: Spain

Final Report Summary - DETECTOX (Development of an SPR-based biosensor for the detection of lipophilic phycotoxins in shellfish residues)

The overall objective of the DETECTOX project was to develop a surface plasmon resonance (SPR) biosensor to detect lipophilic toxins: diarrheic shellfish toxins (DSP), yessotoxins (YTXs), pectenotoxins (PTXs), azaspiracids (AZAs) and gymnodimine (GYM).

The work performed can be summarised as follows:

-Work package (WP) 2 - Production of reagents
The first objective of the project was the acquisition and purification of toxins to be used as reference materials to study the mechanism of action and to develop the biosensor assay. Even though some of these toxins were commercially available, due to the large amount required and to their high price, in DETECTOX purification protocols of each toxin from contaminated material were developed by USC.
The second task in WP2 was the identification of molecular targets of phycotoxins to be used as antibody alternatives to develop sensor chips. DSP, YTX and PTX molecular targets had been already described, but no information was available with regard to AZA. Several experiments were done at USC to clarify the mechanism of action of AZAs. Early and long-term responses in the presence of AZAs in different cellular models (human lymphocytes, human BE(2)-M17 neuroblastoma cells, human Caco-2 intestinal cells, human NCI-H460 lung carcinoma cells, human mast cells HMC-1 and primary cultures of cerebellar granule cells) were checked. An important effect in actin cytoskeleton disorganisation was evidenced in the presence of AZA-1.

In addition AZA-1 inhibited cellular proliferation and induced caspases activation after 48 hours cellular treatment. This effect was related with the molecular structure since fifteen different molecules (kindly provided by Dr. K.C. Nicolaou, Scripps Institute) were tested and only ABCD,C20-epi-AZA-1 had a similar effect to AZA-1. These effects were reported as irreversible and activated after a short incubation period in the presence of the toxin.
The last objective in WP2 was the binding reagent production. The technical specifications and possible synthetic routes for reagent production have been researched, and the assay development discussed between QUB and Xenosense. Immunogens for all the toxins have been successfully studied and prepared by Xenosense.

-WP 3 - Assay developers
The antibodies obtained were used to develop the inhibition test. For this matter, other key step in the project was the development of sensor chip surfaces with immobilised toxins (task 3.1) to then develop the inhibition assay (task 3.2). Different strategies have been used depending on the toxin studied. The OA-N-hydroxy succinimide ester was immobilised onto an amine sensor chip and after some modifications and using the polyclonal antibody before described, a rapid and specific biosensor immunoassay has been developed and published. From this assay an OA prototype kit was produced. This assay showed cross reactivity with DTX1.

Later on when the monoclonal antibody was ready other assay was developed. In this case the cross reactivity with DTX-1 was 100% and 60% with DTX-2, and no cross reactivity with DTX-3. These data correlate exactly with the intrinsic toxic potency of the OA-group of toxins and point to the monoclonal antibody as an excellent candidate for full biosensor.
Several polyclonal antibodies to GYM fragment were produced by QUB and even though very good titres of antibodies against the fragments were obtained, no inhibition with the parent toxin was observed. The immunogens and the sensor surface chips were developed by Xenosense after they got the natural toxin. However, several rabbits and mice were immunised but no antibodies were produced. The cellular GYM receptor produced by CNRS was checked as an alternative to develop the chip surface at the USC. These experiments started by the study of toxinreceptor interactions in solution. After accurate results were obtained an inhibition assay was developed by immobilisation of receptor in the sensor surface, however this assay design cannot be used for GYM detection probably due to the size of receptor.

-WP 4 - Validation and comparison
Pre-validation and comparison of biosensor with current state of art was also included in the project. The DSP detection kit developed in this project showed very good sensitivity and an excellent correlation with other analytical methods. Four kits were supplied to four laboratories and a pre-validation study has been done. The biosensor assay was determined to be accurate, reliable, fast and more cost effective in terms of toxin usage than the commonly used competitive and antigen coated ELISA techniques. These international laboratories found the assay simple and straight forward to perform. The results were reproducible between laboratories.

-WP 5 - Demonstration and dissemination
Demonstration and dissemination activities were carried out. A webpage (see http://www.detectox.eu online) has been opened and updated along the project. The exchange of information through this space between partners was very fluent. A workshop to show biosensor methods for the detection of lipophilic toxins was organised by the CRLMB in Cyprus, 19 October 2007. The workshop was a tutorial session on the 'Principles of SPR' followed by a tutorial illustrating schematically the extraction procedures and sample preparation for lipophilic shellfish toxins. A questionnaire designed to obtain feedback from the workshop indicates that it was an extremely successful event. The workshop was concluded with a question and answer session. The workshop provided a forum for discussion of the projects' developments and their application between the scientists performing the research and the regulators for marine biotoxins within the EU.

A second workshop is going to be organised at Vigo, in October 2008. A virtual workshop was done. In addition 10 papers (6 already published) are directly related with results from this project, and several meeting presentations have been done.

Informazioni correlate

Reported by

UNIVERSIDADE SANTIAGO DE COMPOSTELA
SANTIAGO DE COMPOSTELA
Spain