Final Activity Report Summary - 2D ALLERGY CARD (Development of a new analytical platform for allergy detection: A miniaturised, 2D allergy card)
This project is aimed to a better understanding, diagnosis and treatment orientation of allergy, a disease undergoing explosive and worrisome progress. The project proposes the development of a new miniature and automated multicriteria platform for separating allergens from food. It will be based on two complementary electrophoresis principles, first by Capillary Iso-electric-Focusing (cIEF) into an ordered stack of proteins separated according of their respective charge or iso-electric points. Then these proteins will be further separated in a Carrier Ampholyte Based Capillary Electrophoresis (CABCE). A fully innovative immunodetection step will be applied at the end of the two successive electrophoretic separations (device under construction at the Lausanne Polytechnique).
The main tasks of the researcher in this project were:
- The preliminary food sample treatment in order to extract potentially protein allergens. For this purpose an innovative sample treatment with hexapeptides (synthesised based on combinatorial chemistry) linked to polymetacrylate particles permitted the extraction of low-abundant or unknown proteins with potential allergenicity.
- Preparation and characterisation of running buffers for the second dimension of the miniature platform (CABCE separation). For this purpose different brands of carrier ampholytes (each brand composed by a chaotic mixture of unknown ampholytes) with five different pI intervals were fractionated and characterised in order to obtain the appropriate running buffer for the CABCE separation of the proteins with potential allergenicity. Regarding this part of the work we can say that for the first time the main focusing properties as well as compositional information of the commercial mixtures of carrier ampholytes have been published. This is a valuable data for a subsequent use of these carrier ampholytes as running buffers in capillary electrophoresis or in isoelectric focusing separation of proteins.
- Developing an inner silica surface conditioning protocol for a correct separation of the proteins in the first and second dimension of the proposed miniature platform for separating proteins with potential allergenicity. In this sense the capabilities of divalent cations like Ba2+ were studied obtaining very good reproducibility and efficiency on the capillary electrophoresis separation of standard peptides and proteins.
The main tasks of the researcher in this project were:
- The preliminary food sample treatment in order to extract potentially protein allergens. For this purpose an innovative sample treatment with hexapeptides (synthesised based on combinatorial chemistry) linked to polymetacrylate particles permitted the extraction of low-abundant or unknown proteins with potential allergenicity.
- Preparation and characterisation of running buffers for the second dimension of the miniature platform (CABCE separation). For this purpose different brands of carrier ampholytes (each brand composed by a chaotic mixture of unknown ampholytes) with five different pI intervals were fractionated and characterised in order to obtain the appropriate running buffer for the CABCE separation of the proteins with potential allergenicity. Regarding this part of the work we can say that for the first time the main focusing properties as well as compositional information of the commercial mixtures of carrier ampholytes have been published. This is a valuable data for a subsequent use of these carrier ampholytes as running buffers in capillary electrophoresis or in isoelectric focusing separation of proteins.
- Developing an inner silica surface conditioning protocol for a correct separation of the proteins in the first and second dimension of the proposed miniature platform for separating proteins with potential allergenicity. In this sense the capabilities of divalent cations like Ba2+ were studied obtaining very good reproducibility and efficiency on the capillary electrophoresis separation of standard peptides and proteins.