Alvaro Coelho Irmaos, S.A. (P2)

This objectives are overcomed with the development of polyclonal (PAb) and monoclonal (MAb) antibodies, an Elisa and finally developing and validating an electrochemical inmunosensor. The minimum agreed result is an Elisa based on polyclonal antibodies with the detection limits commented before and a minimum pretreatment.
Achieve these objectives has to permit develope a quality validation system that guarantee a free TCA product, with significant economic repercussions in cork and wine industries.

Development of the inmunosensor

1. Production oh haptens and inmunoconjugated. After the simulaton of posible molecular structures with the desired characteristics, were indentified and synthesized three haptens and produced three suboptimal haptens.

2. Production and characterization of monoclonal and polyclonal antibodies. Monoclonal and polyclonal antibodies were obtained from the inmunization of rats and rabbits respectively. One of the monoclonal antibody cell line was produced en masse in a bioreactor.

3. Use of the antibodies to an ELISA development and to the production of an electrochemical inmunosensor. Monoclonal and polyclonal antibodies were used to develope and ELISA with peroxidasa (HRP) and alacalina fosfatasa (ALP). By the other hand, the haptens were modified with enzimatic tracers to permit the development of different inmunoanalysis methods. Different ELISA methods were proved to exam inmunoreaction effects on cork and wine extracts and based on the same inmunotracers, were developed electrochemical methods.

4. Validation of the inmunosensor and the ELISA and incorporation on a system able to detect TCA on ground analysis. It was developed a study of TCA extraction on cork with different times and solvents. Different sample pretreatment methods like nmunoconcentration, ionic exchange chromatography and silica gel chromatography were showed suitable for the production of a matrix free of substances that could interfere with inmunoanalysis. Reactive strips that could combine different sample pretreatment were proved. It was developed a reactive strip that improve the ELISA detection limit in an order of magnitude.

Results of the research:

The results proved the viability of an inmunodetector that could achieve the detection limits set in the Project.

From the extrapolation of these results, it can be guaranteed that, combined with a pretreatment, the ELISA developed by TECACOR permits a detection limit from 100 ppt to 1 ppb in cork extracts and the inmunosensor permits to reach levels of 10-100 ppt in the same matrix. The validation results showed that the inmunosensor identifies TCA correctly in comparison with other tradional analysis methods (CG). It's posible the development of a reactive strip that incorporates the pretreatment and the detection in one step, with a response time of about 30 minutes and a detection limit of 10-100 ppt.

A graph is made with the obtained results.