ABSTRACT

The overall scientific objective of the project is to develop from concept a generic capacitance based immunodetection analyser for GMO related proteins. CP4-synthase and Cry1(A)b-protein are employed as relevant model targets. These proteins correspond to "RR-Soy" and "Bt-Maize" GMOs, respectively.

This proposed immunodetection method bears inherent advantages and the analyser to be developed will be easily adapted to new GMOs, as the whole technology that will be developed can be used for any new GMO, provided that the corresponding antibody is available.

There are four types of project deliverables: production of monoclonal antibodies and Fab related proteins, development of capacitance based immunosensors (GMO immunochips), development of ELISA protocols and hardware/software products.

OBJECTIVES

1. preparation of immunochemicals and immunogens for immunisation of mice and development of assays.
2. development of monoclonal antibodies with high affinity to certain GMOs.
3. preparation of genetically engineered Fab fragments of the antibodies that are developed.
4. development of ELISA protocols for the detection of GMOs.
5. development of capacitance immunochips for the direct monitoring of the antibody-antigen interaction.
6. development of ultra-thin insulating layers.
7. development of high-density biolayers.
8. development of Fab fragments-based biolayers.
9. development of high performance immobilisation protocols.
10. development of a portable analyser.

Full control of the system will be provided by a powerful software.

DESCRIPTION OF THE WORK

Monoclonal antibodies will be raised against non-commercial GMOs. This will be accomplished through the immunisation of mice with appropriate immunoconjugates of peptides that correspond to the target proteins (WP1). Development of insulating layers will be achieved using various techniques such as electrochemical oxidation of the metal substrates and electropolymerisation of non-conducting polymers. Chemical modification of the metal substrates with long-chain alkanethiols will be also carried out (WP2). The immunisation of mice will result in the production of monoclonal antibodies with a high affinity against GMO. Then Fab fragments will be produced by cloning and expression of the Fab fragment encoding antibody genes (WP3). Development of immunochips. The development of high-density biolayers with a proper dielectric behaviour will be accomplished using non-conductive polymers, which will be developed over the produced antibodies and Fab fragments (WP4). Target proteins (antigens) will be labelled with an enzyme in order to be used as tracers for the development of ELISA protocols for the assay of GMOs in standard solutions. Surface plasmon resonance, quartz crystal microbalance and fluorescence polarisation experiments will further assess the quality of these antibodies (WP5). Development of an integrated, fully automated capacitance based immuno-analyser. The final model and the development of software, which will control the components of the analyser, process and evaluate the signal and provide the necessary displays, will be expected by two months before the end of the project (WP6). Validation of the capacitance immunochips and cross-laboratories normalisation study (WP7). Coordination of the project, dissemination and exploitation of the results (WP8).

DELIVERABLES

1. optimum peptides for immunogens preparation meeting evaluation criteria.
2. development of ultra-thin insulating layers.
3. production of monoclonal antibodies against GMO marker proteins.
4. final form of the immunosensors bearing Fab fragments immobilised by optimum immobilisation protocols.
5. GMO detection in ELISA format.
6. assay of GMOs in real samples, both with ELISA and immunosensors.
7. workshop and demonstration of the analyser.

CONSORTIUM